NZ789137A

NZ789137A - Bioinformatics systems, apparatus, and methods for performing secondary and/or tertiary processing

Info

Publication number: NZ789137A
Application number: NZ789137A
Authority: NZ
Inventors: Mark Hahm; Rami Mehio; Eric Ojard; Amnon Ptashek; Michael Ruehle; Gavin Stone; Rooyen Pieter Van
Original assignee: Illumina Inc
Priority date: 2016-06-07
Filing date: 2017-06-07
Publication date: 2022-07-01

Abstract

system, method and apparatus for executing a bioinformatics analysis on genetic sequence data is provided. Particularly, a genomics analysis platform for executing a sequence analysis pipeline is provided. The genomics analysis platform includes one or more of a first integrated circuit, where each first integrated circuit forms a central processing unit(CPU) that is responsive to one or more software algorithms that are configured to instruct the CPU to perform a first set of genomic processing steps of the sequence analysis pipeline. Additionally, a second integrated circuit is also provided, where each second integrated circuit forming a field programmable gate array (FPGA), the FPGA being configured by firmware to arrange a set of hardwired digital logic circuits that are interconnected by a plurality of physical interconnects to perform a second set of genomic processing steps of the sequence analysis pipeline, the set of hardwired digital logic circuits of each FPGA being arranged as a set of processing engines to perform the second set of genomic processing steps. A shared memory is also provided. h first integrated circuit forms a central processing unit(CPU) that is responsive to one or more software algorithms that are configured to instruct the CPU to perform a first set of genomic processing steps of the sequence analysis pipeline. Additionally, a second integrated circuit is also provided, where each second integrated circuit forming a field programmable gate array (FPGA), the FPGA being configured by firmware to arrange a set of hardwired digital logic circuits that are interconnected by a plurality of physical interconnects to perform a second set of genomic processing steps of the sequence analysis pipeline, the set of hardwired digital logic circuits of each FPGA being arranged as a set of processing engines to perform the second set of genomic processing steps. A shared memory is also provided.

Description

A system, method and apparatus for executing a bioinformatics analysis on genetic sequence data is provided. Particularly, a genomics is rm for executing a sequence is pipeline is provided. The genomics analysis platform includes one or more of a first integrated circuit, where each first integrated circuit forms a central processing unit(CPU) that is sive to one or more software algorithms that are configured to instruct the CPU to perform a first set of genomic processing steps of the sequence analysis pipeline. Additionally, a second integrated circuit is also provided, where each second integrated circuit forming a field programmable gate array (FPGA), the FPGA being configured by firmware to arrange a set of hardwired l logic circuits that are interconnected by a plurality of physical interconnects to perform a second set of genomic sing steps of the ce analysis pipeline, the set of hardwired digital logic circuits of each FPGA being arranged as a set of processing engines to perform the second set of genomic processing steps. A shared memory is also provided.

NZ 789137 BIOINFORMATICS SYSTEMS, APPARATUSES, AND METHODS FOR PERFORMING ARY AND/OR TERTIARY PROCESSING Cross-Reference to Related Application The current application claims priority to U.S. Application No. 62/347,080, filed June 7, 2016, U.S. Application No. 62/399,582, filed September 26, 2016, U.S.

Application No. ,637, filed October 28, 2016, U.S. Application No. 15/404,146, filed January 11, 2017, U.S. Application No. 62/462,869, filed February 23, 2017, U.S.

Application No. 62/469,442, filed March 9, 2017, and U.S. Application No. 15/497,149, filed April 25, 2017, the disclosures of each ation are incorporated herein by reference in their entireties.

Field ofthe Disclosure The subject matter described herein relates to bioinformatics, and more particularly to systems, apparatuses, and methods for implementing bioinformatic protocols, such as performing one or more functions for analyzing genomic data on an ated t, such as on a hardware processing platform.

Background to the Disclosure As described in detail , some major computational challenges for highthroughput DNA cing analysis is to address the explosive growth in available genomic data, the need for increased accuracy and sensitivity when gathering that data, and the need for fast, efficient, and accurate computational tools when performing is on a wide range ofsequencing data sets derived from such genomic data.

Keeping pace with such sed sequencing throughput generated by Next Gen Sequencers has typically been manifested as multithreaded re tools that have been executed on ever greater numbers of faster processors in computer clusters with expensive high availability storage that requires substantial power and significant IT support costs. antly, future increases in sequencing throughput rates will translate into rating real dollar costs for these secondary processing solutions.

The devices, systems, and methods of their use described herein are provided, at least in part, so as to address these and other such challenges.

Summary ofthe Disclosure The present disclosure is ed to devices, systems, and methods for ing the same in the performance of one or more genomics and/or bioinformatics protocols on data generated through a primary processing procedure, such as on genetic sequence data. For instance, in various aspects, the s, systems, and methods herein provided are configured for performing secondary and/or tertiary analysis protocols on genetic data, such as data generated by the sequencing of RNA and/or DNA, e.g., by a Next Gen Sequencer ("NGS"). In ular embodiments, one or more secondary processing pipelines for processing genetic sequence data is provided. In other ments, one or more tertiary processing pipelines for processing genetic sequence data is provided, such as where the pipelines, and/or individual elements thereof, deliver superior sensitivity and improved accuracy on a wider range of sequence derived data than is currently available in the art.

For example, provided herein is a system, such as for executing one or more of a sequence and/or genomic analysis pipeline on genetic sequence data and/or other data derived therefrom. In various embodiments, the system may include one or more of an electronic data source that provides digital signals enting a plurality of reads of genetic and/or genomic data, such as where each of the plurality of reads of genomic data e a sequence of nucleotides. The system may further e a memory, e.g., a DRAM, or a cache, such as for storing one or more of the sequenced reads, one or a plurality of genetic reference sequences, and one or more indices ofthe one or more genetic reference ces.

The system may additionally include one or more integrated circuits, such as a FPGA, ASIC, or sASIC, and/or a CPU and/or a GPU, which ated circuit, e.g., with respect to the FPGA, ASIC, or sASIC may be formed of a set of hardwired digital logic circuits that are interconnected by a plurality of physical electrical interconnects. The system may onally include a quantum computing processing unit, for use in implementing one or more ofthe methods sed .

In various embodiments, one or more of the plurality of electrical onnects may include an input to the one or more integrated circuits that may be connected or connectable, e.g., directly, via a suitable wired connection, or ctly such as via a wireless network connection (for instance, a cloud or hybrid cloud), with the electronic data source. Regardless of a connection with the sequencer, an integrated circuit of the disclosure may be configured for ing the plurality of reads of genomic data, e.g., directly from the cer or from an associated memory. The reads may be digitally encoded in a standard FASTQ or BCL file format. Accordingly, the system may include an integrated circuit having one or more electrical onnects that may be a physical interconnect that includes a memory interface so as to allow the integrated circuit to access the memory.

Particularly, the red digital logic t of the integrated circuit may be arranged as a set ofprocessing engines, such as where each processing engine may be formed of a subset of the hardwired digital logic ts so as to perform one or more steps in the sequence, genomic, and/or tertiary analysis pipeline, as described herein below, on the plurality of reads of genetic data as well as on other data derived rom. For instance, each subset of the hardwired digital logic circuits may be in a wired configuration to perform the one or more steps in the analysis pipeline. Additionally, where the integrated circuit is an FPGA, such steps in the sequence and/or further analysis process may e the partial reconfiguration ofthe FPGA during the analysis s.

Particularly, the set of processing engines may include a mapping module, e.g., in a wired configuration, to access, according to at least some of the sequence of nucleotides in a read of the plurality of reads, the index of the one or more genetic reference sequences, from the memory via the memory interface, so as to map the read to one or more segments of the one or more genetic reference sequences based on the index. Additionally, the set ofprocessing engines may include an alignment module in the wired configuration to access the one or more genetic reference sequences from the memory via the memory interface to align the read, e.g., the mapped read, to one or more positions in the one or more segments of the one or more genetic reference sequences, e.g., as received from the mapping module and/or stored in the memory.

Further, the set of processing s may include a sorting module so as to sort each aligned read according to the one or more positions in the one or more c reference sequences. Furthermore, the set of processing engines may include a variant call module, such as for processing the mapped, aligned, and/or sorted reads, such as with respect to a reference genome, to thereby produce an HMM readout and/or variant call file for use with and/or ing the variations between the sequenced c data and the reference genomic reference data. In various instances, one or more of the plurality of physical electrical interconnects may include an output from the integrated circuit for communicating result data from the mapping module and/or the alignment and/or sorting and/or variant call modules.

Particularly, with respect to the mapping module, in s embodiments, a system for executing a mapping analysis pipeline on a plurality ofreads of genetic data using an index of genetic reference data is provided. In various ces, the c sequence, e.g., read, and/or the genetic reference data may be ented by a sequence eotides, which may be stored in a memory of the system. The mapping module may be included within the integrated circuit and may be formed of a set of pre-configured and/or hardwired digital logic circuits that are interconnected by a plurality ofphysical electrical interconnects, which physical electrical onnects may e a memory interface for allowing the integrated circuit to access the memory. In more ular embodiments, the hardwired digital logic circuits may be arranged as a set of processing engines, such as where each sing engine is formed of a subset of the hardwired digital logic circuits to m one or more steps in the sequence analysis pipeline on the plurality ofreads ofgenomic data.

For instance, in one embodiment, the set of processing engines may e a mapping module in a hardwired uration, where the mapping module, and/or one or more processing engines thereof is configured for receiving a read of genomic data, such as via one or more of a plurality ofphysical electrical interconnects, and for ting a portion of the read in such a manner as to generate a seed therefrom. In such an instance, the read may be represented by a sequence of nucleotides, and the seed may represent a subset of the sequence of nucleotides represented by the read. The mapping module may include or be connectable to a memory that includes one or more of the reads, one or more of the seeds of the reads, at least a portion of one or more of the reference genomes, and/or one or more s, such an index built from the one or more reference genomes. In certain instances, a processing engine of the mapping module employ the seed and the index to calculate an address within the index based on the seed.

Once an address has been calculated or otherwise derived and/or stored, such as in an d or offboard memory, the address may be accessed in the index in the memory so as to receive a record from the address, such as a record representing position information in the genetic reference sequence. This position information may then be used to determine one or more matching positions from the read to the genetic reference sequence based on the record. Then at least one ofthe matching positions may be output to the memory via the memory interface.

In another embodiment, a set of the processmg engines may include an alignment module, such as in a pre-configured and/or hardwired configuration. In this instance, one or more ofthe processing engines may be configured to receive one or more of the mapped positions for the read data via one or more of the plurality of physical electrical interconnects. Then the memory (internal or external) may be accessed for each mapped position to retrieve a segment ofthe reference sequence/genome corresponding to the mapped position. An alignment of the read to each ved reference segment may be calculated along with a score for the alignment. Once calculated, at least one coring alignment of the read may be selected and output. In various instances, the ent module may also implement a dynamic programming algorithm when calculating the alignment, such as one or more of a Smith-Waterman algorithm, e.g., with linear or affine gap scoring, a gapped alignment algorithm, and/or a gapless alignment thm. In particular instances, the calculating of the alignment may include first performing a gapless alignment to each nce segment, and based on the gapless alignment results, selecting reference segments with which to further perform gapped alignments.

In various embodiments, a variant call module may be provided for performing improved t call functions that when implemented in one or both of re and/or hardware configurations generate superior processing speed, better processed result accuracy, and enhanced overall efficiency than the methods, devices, and systems currently known in the art. Specifically, in one , improved methods for performing variant call operations in software and/or in hardware, such as for performing one or more HMM operations on genetic sequence data, are ed. In another aspect, novel devices including an integrated circuit for performing such improved variant call operations, where at least a portion ofthe variant call operation is ented in hardware, are provided.

Accordingly, in s instances, the methods disclosed herein may include mapping, by a first subset of hardwired and/or quantum digital logic circuits, a plurality of reads to one or more segments of one or more genetic reference sequences. Additionally, the methods may e accessing, by the integrated and/or quantum circuits, e.g., by one or more of the ity of physical ical interconnects, from the memory or a cache associated therewith, one or more of the mapped reads and/or one or more of the c reference sequences; and aligning, by a second subset of the hardwired and/or quantum digital logic ts, the plurality of mapped reads to the one or more segments of the one or more genetic reference sequences.

In various embodiments, the method may additionally include accessing, by the integrated and/or quantum circuit, e.g., by one or more of the plurality of al electrical interconnects from a memory or a cache associated therewith, the aligned plurality of reads. In such an instance the method may include sorting, by a third subset of the hardwired and/or quantum digital logic circuits, the d plurality of reads according to their positions in the one or more genetic reference sequences. In certain instances, the method may further include outputting, such as by one or more of the plurality of physical ical interconnects ofthe integrated and/or quantum circuit, result data from the mapping and/or the ng and/or the sorting, such as where the result data includes positions of the mapped and/or aligned and/or sorted plurality s.

In some instances, the method may additionally include using the obtained result data, such as by a further subset ofthe hardwired and/or quantum digital logic circuits, for the purpose of ining how the mapped, aligned, and/or sorted data, derived from the subject's sequenced genetic sample, differs from a reference sequence, so as to produce a variant call file delineating the genetic differences between the two s. Accordingly, in various embodiments, the method may further include accessing, by the integrated and/or quantum circuit, e.g., by one or more ofthe plurality ofphysical electrical interconnects from a memory or a cache associated therewith, the mapped and/or aligned and/or sorted plurality of reads. In such an instance the method may include performing a variant call on, e.g., an HMM or paired HMM ion, on the accessed reads, by a third or fourth subset of the hardwired and/or m digital logic circuits, so as to produce a variant call file detailing how the mapped, aligned, and/or sorted reads vary from that of one or more reference, e.g., haplotype, sequences. ingly, in accordance with particular aspects ofthe disclosure, presented herein is a compact hardware, e.g., chip based, or m accelerated platform for performing secondary and/or tertiary es on genetic and/or genomic sequencing data.

Particularly, a platform or ne of hardwired and/or quantum digital logic circuits that have specifically been designed for performing secondary and/or tertiary genetic analysis, such as on sequenced genetic data, or genomic data derived therefrom, is provided.

Particularly, a set of hardwired digital and/or quantum logic circuits, which may be arranged as a set ofprocessing engines, may be provided, such as where the processing engines may be present in a preconfigured and/or hardwired and/or quantum configuration on a processing platform of the disclosure, and may be specifically designed for performing secondary mapping and/or aligning and/or variant call operations related to genetic analysis on DNA and/or RNA data, and/or may be specifically designed for performing other tertiary processing on the results data.

In particular instances, the present devices, systems, and methods of employing the same in the mance of one or more genomics and/or bioinformatics secondary and/or tertiary sing protocols, have been optimized so as to deliver an improvement in processing speed that is orders of magnitude faster than standard secondary processing pipelines that are implemented in software. onally, the pipelines and/or components thereof as set forth herein provide better sensitivity and accuracy on a wide range of sequence d data sets for the purposes of genomics and ormatics processing. In various instances, one or more of these operations may be performed on by an ated circuit that is part of or ured as a general purpose central processing unit and/or a graphics processing unit and/or a quantum processing unit.

For example, genomics and bioinformatics are fields concerned with the ation of information technology and computer science to the field of genetics and/or molecular biology. In particular, bioinformatics techniques can be applied to s and e various genetic and/or genomic data, such as from an individual, so as to determine qualitative and quantitative information about that data that can then be used by various practitioners in the development of prophylactic, therapeutic, and/or diagnostic methods for preventing, treating, ameliorating, and/or at least identifying diseased states and/or their potential, and thus, improving the safety, quality, and effectiveness of health care on an individualized level. Hence, because of their focus on advancing personalized healthcare, genomics and bioinformatics fields promote individualized healthcare that is proactive, instead of reactive, and this gives the subject in need of treatment the opportunity to become more involved in their own wellness. An advantage of employing the genetics, genomics, and/or bioinformatics technologies disclosed herein is that the qualitative and/or quantitative analyses of molecular biological, e.g., c, data can be performed on a broader range of sample sets at a much higher rate of speed and often times more accurately, thus expediting the emergence of a personalized healthcare system. Particularly, in s ments, the genomics and/or ormatics related tasks may form a genomics pipeline that includes one or more of a micro-array analysis pipeline, a genome, e.g., whole genome analysis ne, genotyping analysis pipeline, exome is ne, epigenome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analysis pipeline, WO 14320 PCT/0S2017/036424 including joint ping, variants is nes, including structural variants, somatic variants, and GATK, as well as RNA sequencing and other genetic analyses pipelines.

Accordingly, to make use of these advantages there exists enhanced and more accurate software implementations for performing one or a series of such bioinformatics based analytical techniques, such as for deployment by a general purpose CPU and/or GPU and/or may be implemented in one or more quantum circuits of a quantum processing platform. However, common teristics of traditionally configured software based bioinformatics methods and systems is that they are labor ive, take a long time to execute on such l purpose processors, and are prone to errors. Therefore, bioinformatics systems as implemented herein that could perform these algorithms, such as ented in software by a CPU and/or GPU of m processing unit in a less labor and/or processing intensive manner with a greater percentage accuracy would be useful.

Such implementations have been developed and are presented herein, such as where the cs and/or bioinformatics analyses are performed by optimized software run on a CPU and/or GPU and/or quantum computer in a system that makes use of the genetic sequence data derived by the processing units and/or integrated circuits of the disclosure.

Further, it is to be noted that the cost of analyzing, storing, and sharing this raw digital data has far outpaced the cost of producing it. Accordingly, also ted herein are "just in time" storage and/or retrieval methods that ze the storage of such data in a manner that substitutes the speed of regenerating the data in ge for the cost of storing such data collectively. Hence, the data generation, analysis, and "just in time" or "JIT" storage methods ted herein solve a key bottleneck that is a long felt but unmet obstacle standing between the ever-growing raw data generation and storage and the real l insight being sought from it.

Presented herein, ore, are systems, apparatuses, and methods for implementing genomics and/or bioinformatic protocols or portions thereof, such as for performing one or more functions for analyzing genomic data, for instance, on one or both of an integrated circuit, such as on a hardware processing platform, and a general e processor, such as for performing one or more bioanalytic operations in software and/or on firmware. For example, as set forth herein below, in various implementations, an integrated circuit and/or quantum circuit is provided so as to accelerate one or more processes in a primary, secondary, and/or tertiary processing platform. In various instances, the integrated circuit may be employed in performing genetic analytic related tasks, such as mapping, aligning, variant calling, compressing, decompressing, and the like, in an accelerated manner, and as such the ated circuit may include a hardware accelerated configuration.

Additionally, in various instances, an integrated and/or m circuit may be provided such as where the t is part of a processing unit that is configured for performing one or more genomics and/or bioinformatics protocols on the ted mapped and/or aligned and/or t called data.

Particularly, in a first embodiment, a first integrated circuit may be formed of an FPGA, ASIC, and/or sASIC that is coupled to or otherwise attached to the motherboard and configured, or in the case of an FPGA may be programmable by firmware to be configured, as a set of red digital logic circuits that are adapted to perform at least a first set of sequence analysis functions in a genomics analysis pipeline, such as where the integrated circuit is configured as bed herein above to include one or more digital logic circuits that are arranged as a set ofprocessing engines, which are adapted to perform one or more steps in a mapping, aligning, and/or variant calling operation on the genetic data so as to produce sequence analysis s data. The first integrated circuit may further include an , e.g., formed of a plurality of physical electrical interconnects, such as for communicating the result data from the mapping and/or the alignment and/or other procedures to the memory.

Additionally, a second integrated and/or quantum circuit may be included, coupled to or otherwise attached to the motherboard, and in communication with the memory via a communications interface. The second ated and/or quantum circuit may be formed as a central processing unit (CPU) or graphics processing unit (GPU) or quantum processing unit (QPU) that is configured for receiving the mapped and/or aligned and/or variant called sequence analysis result data and may be adapted to be responsive to one or more software algorithms that are configured to instruct the CPU or GPU to perform one or more genomics and/or bioinformatics functions of the genomic analysis pipeline on the , aligned, and/or variant called ce analysis result data. Specifically, the genomics and/or bioinformatics related tasks may form a genomics analysis pipeline that includes one or more of a micro-array analysis, a genome ne, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome is pipeline, epigenome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analyses pipelines, including joint genotyping, ts analyses pipelines, including structural variants, somatic WO 14320 PCT/0S2017/036424 ts, and GATK, as well as RNA sequencing analysis pipeline and other genetic es pipelines.

For instance, in one embodiment, the CPU and/or GPU and/or QPU of the second integrated circuit may include software that is configured for arranging the genome analysis pipeline for executing a whole genome analysis pipeline, such as a whole genome analysis pipeline that includes one or more of genome-wide variation analysis, whole-exome DNA analysis, whole transcriptome RNA analysis, gene function analysis, protein function analysis, protein binding is, quantitative gene analysis, and/or a gene assembly analysis. In certain instances, the whole genome analysis ne may be performed for the purposes of one or more of ancestry is, personal medical history analysis, disease diagnostics, drug discovery, and/or protein profiling. In a ular instance, the whole genome analysis pipeline is performed for the purposes of oncology analysis. In s instances, the results ofthis data may be made available, e.g. globally, throughout the system.

In various instances, the CPU and/or GPU and/or a quantum processing unit (QPU) of the second integrated and/or quantum circuit may include re that is configured for arranging the genome analysis pipeline for executing a genotyping analysis, such as a genotyping analysis including joint genotyping. For instance, the joint genotyping analysis may be performed using a Bayesian probability calculation, such as a Bayesian probability calculation that results in an absolute probability that a given determined genotype is a true genotype. In other instances, the software may be configured for ming a metagenome analysis so as to e metagenome result data that may in tum be ed in the mance of a microbiome analysis.

In certain instances, the first and/or second integrated circuit and/or the memory may be housed on an expansion card, such as a peripheral component interconnect (PCI) card. For instance, in various embodiments, one or more of the integrated circuits may be one or more chips coupled to a PCie card or otherwise associated with the motherboard. In various instances, the integrated and/or quantum circuit(s) and/or chip(s) may be a component within a sequencer or computer, or server, such as part of a server farm. In ular embodiments, the integrated and/or quantum circuit(s) and/or expansion card(s) and/or computer(s) and/or server(s) maybe accessible via the internet, e.g., cloud.

Further, in some ces, the memory may be a volatile random access memory (RAM), e.g., a direct access memory (DRAM). Particularly, in various embodiments, the memory may include at least two memories, such as a first memory that is an HMEM, e.g., for storing the reference haplotype sequence data, and a second memory that is an RMEM, e.g., for storing the read of c sequence data. In particular ces, each ofthe two memories may include a write port and/or a read port, such as where the write port and the read port each accessing a separate clock. Additionally, each of the two memories may include a flip-flop configuration for storing a multiplicity of genetic sequence and/or processing result data. ingly, in another aspect, the system may be configured for sharing memory resources amongst its component parts, such as in relation to performing some computational tasks via software, such as run by the CPU and/or GPU and/or quantum processing platform, and/or performing other computational tasks via firmware, such as via the hardware of an associated integrated circuit, e.g., FPGA, ASIC, and/or sASIC. This may be achieved in a number ofdifferent ways, such as by a direct loose or tight coupling between the CPU/GPU/QPU and the FPGA, e.g., chip or PCie card. Such configurations may be particularly useful when distributing operations related to the processing of the large data structures associated with genomics and/or bioinformatics es to be used and accessed by both the CPU/GPU/QPU and the associated ated circuit. Particularly, in various embodiments, when processing data through a genomics pipeline, as herein described, such as to accelerate overall processing function, timing, and efficiency, a number of different operations may be run on the data, which ions may e both software and hardware processing components.

Consequently, data may need to be shared and/or otherwise icated, between the software component(s) running on the CPU and/or GPU and/or QPU and/or the hardware component embodied in the chip, e.g., an FPGA. Accordingly, one or more of the various steps in the genomics and/or bioinformatics processing ne, or a portion thereof, may be performed by one device, e.g., the CPU/GPU/QPU, and one or more of the s steps may be med by a hardwired device, e.g., the FPGA. In such an instance, the CPU/GPU/QPU and/or the FPGA may be communicably coupled in such a manner to allow the efficient transmission of such data, which coupling may involve the shared use of memory resources. To e such distribution of tasks and the sharing of information for the performance of such tasks, the various PUs/QPUs may be loosely or tightly coupled to one another and/or the hardware devices, e.g., FPGA, or other chip set, such as by a quick path interconnect.

WO 14320 PCT/0S2017/036424 Particularly, m vanous embodiments, a genom1cs analysis platform is provided. For instance, the platform may include a motherboard, a memory, and ity of integrated and/or m circuits, such as forming one or more of a U/QPU, a mapping module, an alignment , a g module, and/or a variant call module. ically, in particular embodiments, the platform may include a first integrated and/or quantum circuit, such as an integrated circuit forming a central processing unit (CPU) or graphics processing unit (GPU), or a quantum circuit forming a m processor, that is responsive to one or more software or other algorithms that are configured to ct the CPU/GPU/QPU to perform one or more sets of genomics analysis functions, as described herein, such as where the CPU/GPU/QPU includes a first set of physical electronic onnects to connect with the motherboard. In s instances, the memory may also be attached to the motherboard and may further be electronically connected with the CPU/GPU/QPU, such as via at least a n of the first set of physical electronic interconnects. In such instances, the memory may be configured for storing a plurality of reads of genomic data, and/or at least one or more genetic reference sequences, and/or an index ofthe one or more c reference sequences.

Additionally, the platform may include one or more of another integrated circuit(s), such as where each of the other ated circuit forms a field programmable gate array (FPGA) having a second set of physical electronic interconnects to connect with the CPU/GPU/QPU and the memory, such as via a point-to-point interconnect protocol. In such an instance, such as where the integrated circuit is an FPGA, the FPGA may be programmable by firmware to configure a set of hardwired digital logic circuits that are interconnected by a plurality of physical interconnects to perform a second set of genomics analysis functions, e.g., mapping, aligning, variant calling, etc. Particularly, the hardwired digital logic ts of the FPGA may be arranged as a set ofprocessing engines to perform one or more pre-configured steps in a sequence analysis pipeline of the cs analysis, such as where the set(s) of processing engines include one or more of a mapping and/or aligning and/or variant call module, which modules may be formed of the separate or the same subsets ofprocessing engines.

As indicated, the system may be configured to include one or more processing engines, and in various embodiments, an included sing engine may itself be configured for determining one or more transition probabilities for the sequence of nucleotides of the read of genomic sequence going from one state to another, such as from a match state to an indel state, or match state to a delete state, and/or back again such as from an insert or delete state back to a match state. Additionally, in various instances, the integrated circuit may have a pipelined configuration and/or may include a second and/or third and/or fourth subset of hardwired digital logic circuits, such as ing a second set of processing engines, where the second set of processing s includes a mapping module configured to map the read of genomic sequence to the reference haplotype sequence to produce a mapped read. A third subset wired digital logic ts may also be included such as where the third set of processing engines includes an ng module configured to align the mapped read to one or more positions in the reference haplotype sequence. A fourth subset of hardwired digital logic circuits may additionally be included such as where the fourth set ofprocessing engines es a sorting module configured to sort the mapped and/or aligned read to its relative positions in the chromosome. Like above, in various of these instances, the mapping module and/or the aligning module and/or the sorting module, e.g., along with the variant call module, may be physically integrated on the expansion card. And in certain embodiments, the expansion card may be physically integrated with a genetic sequencer, such as a next gen sequencer and the like.

Accordingly, in one aspect, an tus for executing one or more steps of a ce analysis pipeline, such as on genetic data, is ed wherein the genetic data includes one or more of a genetic reference sequence(s), such as a ype or hypothetical haplotype sequence, an index of the one or more genetic reference sequence(s), and/or a plurality of reads, such as of genetic and/or genomic data, which data may be stored in one or more shared memory s, and/or processed by a distributed processing resource, such as a CPU/GPU/QPU and/or FPGA, which are coupled, e.g., y or loosely together. Hence, in various instances, the apparatus may include an integrated circuit, which integrated circuit may include one or more, e.g., a set, of hardwired digital logic circuits, n the set of hardwired digital logic circuits may be interconnected, such as by one or a plurality of physical ical interconnects.

Accordingly, the system may be configured to include an integrated circuit formed of one or more digital logic circuits that are interconnected by a plurality of physical electrical interconnects, one or more of the plurality of physical electrical interconnects having one or more of a memory interface and/or cache, for the integrated circuit to access the memory and/or data stored thereon and to retrieve the same, such as in a cache coherent manner between the CPU/GPU/QPU and associated chip, e.g., FPGA. In various instances, the digital logic circuits may include at least a first subset of digital logic circuits, such as where the first subset of digital logic circuits may be arranged as a first set of processing engines, which sing engine may be configured for accessing the data stored in the cache and/or direct or indirectly coupled memory. For instance, the first set of processing engines may be configured to perform one or more steps in a mapping and/or aligning and/or sorting analysis, as described above, and/or an HMM analysis on the read of genomic sequence data and the haplotype sequence data.

More particularly, a first set of processing engmes may include an HMM module, such as in a first configuration ofthe subset tal logic circuits, which is adapted to access in the memory, e.g., via the memory interface, at least some of the sequence of tides in the read of genomic sequence data and the haplotype sequence data, and may also be configured to perform the HMM analysis on the at least some of the sequence of nucleotides in the read of genomic sequence data and the at least some of the sequence of nucleotides in the ype sequence data so as to produce HMM result data. Additionally, the one or more of the plurality of physical ical interconnects may include an output from the integrated circuit such as for communicating the HMM result data from the HMM module, such as to a CPU/GPU/QPU ofa server or server cluster.

Accordingly, in one aspect, a method for ing a ce analysis pipeline such as on genetic sequence data is provided. The c data may e one or more genetic reference or haplotype sequences, one or more indexes of the one or more genetic reference and/or haplotype sequences, and/or a plurality of reads of genomic data.

The method may include one or more of receiving, accessing, mapping, aligning, sorting various iterations of the genetic sequence data and/or employing the results f in a method for producing one or more variant call files. For instance, in certain embodiments, the method may include receiving, on an input to an integrated t from an electronic data source, one or more of a plurality of reads of genomic data, wherein each read of genomic data may include a sequence ofnucleotides.

In various instances, the integrated circuit may be formed ofa set ofhardwired digital logic circuits that may be ed as one or more processing engines. In such an instance, a processing engine may be formed ofa subset ofthe hardwired digital logic circuits that may be in a wired configuration. In such an instance, the processing engine may be configured to perform one or more nfigured steps such as for implementing one or more of receiving, accessing, mapping, aligning, sorting various iterations of the genetic sequence data and/or employing the results thereof in a method for producing one or more variant call files. In some embodiments, the provided l logic circuits may be interconnected such as by a plurality ofphysical electrical interconnects, which may include an input.

The method may further include accessing, by the integrated circuit on one or more of the plurality ofphysical electrical interconnects from a memory, data for performing one or more ofthe operations detailed herein. In various ces, the integrated circuit may be part of a chipset such as embedded or otherwise contained as part of an FPGA, ASIC, or structured ASIC, and the memory may be directly or indirectly coupled to one or both of the chip and/or a CPU/GPU/QPU associated therewith. For instance, the memory may be a plurality of memories one of each coupled to the chip and a CPU/GPU/QPU that is itself d to the chip, e.g., y.

In other ces, the memory may be a single memory that may be coupled to a U/QPU that is itself tightly coupled to the FPGA, e.g., via a tight processing interconnect or quick path interconnect, e.g., QPI, and thereby accessible to the FPGA, such as in a cache coherent manner. Accordingly, the integrated circuit may be directly or indirectly coupled to the memory so as to access data relevant to performing the functions herein presented, such as for accessing one or more of a plurality of reads, one or more genetic reference or theoretical reference sequences, and/or an index of the one or more c reference ces, e.g., in the mance ofa mapping operation.

Hence, in various instances, implementations of s aspects of the disclosure may include, but are not limited to: apparatuses, systems, and methods including one or more es as described in detail herein, as well as articles that comprise a tangibly embodied machine-readable medium le to cause one or more machines (e.g., computers, etc.) to result in operations described herein. Similarly, computer systems are also described that may include one or more processors and/or one or more memories coupled to the one or more processors. Accordingly, computer implemented methods consistent with one or more implementations of the current subject matter can be implemented by one or more data processors ng in a single computing system or le computing systems containing multiple computers, such as in a ing or super-computing bank.

Such multiple computing systems can be connected and can exchange data and/or commands or other instructions or the like via one or more connections, including but not limited to a connection over a network (e.g. the Internet, a wireless wide area network, a local area network, a wide area network, a wired network, a physical electrical onnect, or the like), via a direct connection between one or more of the multiple computing systems, etc. A memory, which can include a computer-readable storage , may include, encode, store, or the like one or more programs that cause one or more processors to m one or more ofthe operations associated with one or more ofthe algorithms bed herein.

The details of one or more variations ofthe subject matter described herein are set forth in the accompanying drawings and the description below. Other features and ages of the subject matter described herein will be apparent from the description and drawings, and from the claims. While certain features of the tly disclosed subject matter are described for illustrative purposes in relation to an enterprise ce software system or other business software solution or architecture, it should be readily tood that such features are not ed to be limiting. The claims that follow this disclosure are intended to define the scope ofthe protected subject matter.

Brief Description ofthe Figures The accompanying drawings, which are incorporated in and constitute a part of this specification, show certain aspects of the subject matter disclosed herein and, together with the description, help n some of the principles associated with the disclosed implementations.

FIG. IA s a sequencing platform with a plurality of genetic samples thereon, a plurality of exemplary tiles are also depicted, as well as a three-dimensional representation ofthe sequenced reads.

FIG. IB depicts a representation of a flow cell with the vanous lanes represented.

FIG. IC depicts a lower comer ofthe flow cell platform ofFIG. IB, showing a constellation of sequenced reads.

FIG. ID depicts a virtual array of the results of the sequencing performed on the reads of FIGS. 1 and 2, where the reads are set forth in an output column by column order.

FIG. IE depicts the method by which the transposition of the outcome reads from column by column order to row by row read order may be implemented.

FIG. IF depicts the osition of the outcome reads from column by column order, to row by row read order.

FIG. IG depicts the system components for performing the transposition.

FIG IH depicts the transposition order.

FIG. II depicts the ecture for electronically transposing the sequenced data. s an HMM 3-state based model illustrating the transition probabilities ofgoing from one state to another. depicts a high-level view of an integrated circuit of the disclosure including a HMM interface structure. depicts the integrated t of , showing an HMM cluster features in greater detail. depicts an overview of HMM related data flow throughout the system including both re and hardware interactions. depicts exemplary HMM cluster collar connections. depicts a high-level view of the major functional blocks within an exemplary HMM hardware accelerator. depicts an exemplary HMM matrix ure and hardware processing flow. depicts an enlarged view of a n of showing the data flow and dependencies between nearby cells in the HMM M, I, and D state computations within the matrix. depicts exemplary computations useful for M, I, D state updates. depicts M, I, and D state update circuits, including the effects of fying assumptions of d to transition probabilities and the effect of sharing some M, I, D adder resources with the final sum operations. depicts Log domain M, I, D state calculation details. depicts an HMM state transition m showing the on between GOP, GCP and transition probabilities. depicts an HMM Transprobs and Priors generation circuit to t the general state transition diagram of. depicts a simplified HMM state transition diagram showing the relation n GOP, GCP and transition probabilities. depicts a HMM Transprobs and Priors tion circuit to support the simplified state transition. depicts an exemplary theoretical HMM matrix and illustrates how such an HMM matrix may be traversed.

A presents a method for performing a multi-region joint detection preprocessing procedure.

B presents an exemplary method for computing a connection matrix such as in the ocessing procedure ofA.

A depicts an exemplary event between two homologous sequenced regions in a pileup ofreads.

B depicts the constructed reads of A, demarcating nucleotide difference between the two sequences.

C depicts various bubbles of a De Brujin graph that may be used in performing an accelerated variant call operation.

D depicts a representation of a pruning the tree function as described herein.

E depicts one ofthe bubbles ofC. is a graphical representation of the exemplary pileup pursuant to the connection matrix of. is a processing matrix for performing the pre-processing ure of FIGS. 17A and B. is an example of a bubble formation m a De Brujin graph m ance with the methods of. is an example of a variant y through an exemplary De Brujin graph. is a graphical representation ofan exemplary g function. is another example of a processing matrix for a pruned multi-region joint detection ure. illustrates a joint pileup ofpaired reads for two regions. sets forth a probability table in accordance with the disclosed herein. is a further example of a processing matrix for a multi-region joint detection procedure. ents a selection of candidate solutions for the joint pile up of . ents a further selection of candidate solutions for the pile up of , after a pruning function has been performed. represents the final candidates of , and their associated probabilities, after the performance ofa MRJD on. illustrates the ROC curves for MRJD and a conventional detector. illustrates the same results of displayed as a function of the sequence similarity ofthe references.

A depicts an exemplary architecture illustrating a loose coupling between a CPU and an FPGA ofthe disclosure.

B depicts an ary architecture illustrating a tight coupling between a CPU and an FPGA ofthe disclosure.

A depicts a direct coupling ofa CPU and a FPGA ofthe disclosure.

B depicts an alternative embodiment of the direct coupling of a CPU and a FPGA ofA. depicts an embodiment of a package of a combined CPU and FPGA, where the two devices share a common memory and/or cache. illustrates a core of CPUs g one or more memories and/or caches, wherein the CPUs are configured for communicating with one or more FPGAs that may also include a shared or common memory or caches. illustrates an exemplary method of data er throughout the system. depicts the embodiment of in greater . depicts an exemplary method for the processing of one or more jobs of a system ofthe disclosure.

A depicts a block diagram for a genomic infrastructure for onsite and/or cloud based genomics processing and analysis.

B depicts a block diagram of a cloud-based genom1cs processmg platform for performing the BioIT analysis disclosed herein.

C depicts a block diagram for an exemplary genomic processing and analysis pipeline.

D depicts a block diagram for an exemplary genomic sing and analysis pipeline.

A depicts a block diagram of a local and/or cloud based computing function of A for a genomic infrastructure for onsite and/or cloud based genomics processing and analysis.

B depicts the block m of A illustrating greater detail regarding the computing function for a c infrastructure for onsite and/or cloud based genomics processing and analysis.

C depicts the block diagram of rating greater detail regarding the 3rd-Party analytics function for a genomic infrastructure for onsite and/or cloud based cs processing and analysis.

A depicts a block diagram illustrating a hybrid cloud configuration.

B depicts the block diagram ofA in r detail, rating a hybrid cloud configuration.

C depicts the block diagram ofA in greater detail, illustrating a hybrid cloud configuration.

A depicts a block diagram illustrating a primary, secondary, and/or tertiary analysis pipeline as presented herein.

B provides an exemplary tertiary processing epigenetics analysis for execution by the s and devices ofthe system herein.

C provides an exemplary tertiary processing methylation is for execution by the methods and devices ofthe system herein.

D provides an exemplary tertiary smg structural variants analysis for execution by the methods and devices ofthe system herein.

E provides an exemplary tertiary cohort processing analysis for execution by the methods and devices ofthe system herein.

F es an exemplary joint genotyping tertiary processing analysis for execution by the methods and devices ofthe system herein. depicts a flow m for an analysis pipeline ofthe sure. is a block diagram of a hardware processor architecture in accordance with an implementation ofthe disclosure. is a block diagram of a hardware processor architecture in accordance with another implementation. is a block diagram of a hardware processor architecture in accordance with yet another implementation. rates a genetic sequence analysis pipeline. illustrates processing steps using a genetic ce analysis hardware platform.

A rates an apparatus in accordance with an implementation ofthe sure.

B illustrates another tus m accordance with an alternative implementation ofthe disclosure. illustrates a genom1cs processmg system in accordance with an implementation.

Detailed Description ofthe Disclosure As summarized above, the present sure is directed to devices, systems, and methods for employing the same in the performance of one or more genomics and/or bioinformatics protocols, such as a mapping, aligning, sorting, and/or t call protocol on data generated through a primary processing procedure, such as on genetic sequence data. For instance, in various aspects, the s, systems, and methods herein provided are configured for performing secondary analysis protocols on c data, such as data generated by the sequencing of RNA and/or DNA, e.g., by a Next Gen Sequencer (''NGS").

In particular embodiments, one or more secondary processing nes for processing genetic sequence data is provided, such as where the pipelines, and/or individual elements thereof, may be implemented in software, hardware, or a combination thereof in a distributed and/or an optimized fashion so as to r superior sensitivity and improved cy on a wider range of sequence derived data than is currently available in the art. Additionally, as summarized above, the present disclosure is ed to devices, systems, and methods for employing the same in the performance of one or more genomics and/or bioinformatics tertiary protocols, such as a micro-array analysis protocol, a genome, e.g., whole genome analysis protocol, genotyping is protocol, exome analysis ol, epigenome analysis protocol, metagenome analysis protocol, microbiome is protocol, genotyping is protocol, including joint genotyping, variants analysis protocols, including structural variants, somatic variants, and GATK, as well as RNA sequencing protocols and other genetic analyses protocols such as on mapped, aligned, and/or other genetic sequence data, such as employing one or more variant call files.

Accordingly, provided herein are software and/or hardware e.g., chip based, accelerated platform analysis technologies for performing secondary and/or tertiary analysis of DNA/RNA sequencing data. More particularly, a platform, or pipeline, of processing engines, such as in a software implemented and/or red uration, which have specifically been designed for performing secondary c analysis, e.g., mapping, ng, sorting, and/or variant calling; and/or may be specifically designed for performing tertiary genetic is, such as a micro-array analysis, a genome, e.g., whole genome analysis, genotyping analysis, exome is, epigenome analysis, metagenome analysis, microbiome analysis, genotyping analysis, including joint ping analysis, variants analysis, including ural variants analysis, somatic variants analysis, and GATK analysis, as well as RNA sequencing analysis and other genetic analysis, such as with respect to genetic based sequencing data, which may have been generated in an optimized format that delivers an ement in sing speed that is magnitudes faster than standard pipelines that are implemented in known software alone. Additionally, the pipelines ted herein e better ivity and accuracy on a wide range of sequence derived data sets, such as on nucleic acid or protein derived sequences.

As ted above, in s instances, it 1s a goal of bioinformatics processing to determine individual genomes and/or protein sequences of people, which determinations may be used in gene discovery protocols as well as for prophylaxis and/or therapeutic regimes to better enhance the livelihood ofeach particular person and human kind as a whole. Further, knowledge of an individual's genome and/or protein compellation may be used such as in drug discovery and/or FDA trials to better predict with particularity which, if any, drugs will be likely to work on an individual and/or which would be likely to have deleterious side effects, such as by analyzing the individual's genome and/or a protein profile derived therefrom and comparing the same with predicted ical response from such drug administration.

Such bioinformatics processmg usually involves three well defined, but typically separate phases of ation processing. The first phase, termed primary processing, involves DNA/RNA sequencing, where a subject's DNA and/or RNA is obtained and subjected to various processes y the subject's genetic code is converted to a machine-readable l code, e.g., a FASTQ file. The second phase, termed secondary processing, involves using the subject's generated digital genetic code for the determination of the individual's genetic makeup, e.g., determining the individual's genomic nucleotide sequence. And the third phase, termed tertiary processing, es performing one or more analyses on the subject's genetic makeup so as to determine therapeutically useful information therefrom.

Accordingly, once a subject's genetic code is sequenced, such as by a NextGen sequencer, so as to produce a machine readable digital representation of the subject's genetic code, e.g., in a FASTQ and/or BCL file format, it may be useful to further process the lly encoded genetic sequence data obtained from the sequencer and/or WO 14320 PCT/0S2017/036424 sequencing protocol, such as by ting digitally represented data to secondary processing.

This secondary processing, for instance, can be used to map and/or align and/or otherwise assemble an entire genomic and/or protein e of an individual, such as where the individual's entire genetic makeup is ined, for instance, where each and every nucleotide of each and every chromosome is determined in sequential order such that the composition of the individual's entire genome has been identified. In such processing, the genome of the individual may be assembled such as by comparison to a reference genome, such as a reference standard, e.g., one or more genomes obtained from the human genome project or the like, so as to determine how the individual's genetic makeup differs from that of the nt(s). This process is ly known as variant calling. As the difference between the DNA of any one person to another is 1 in 1,000 base pairs, such a variant calling s can be very labor and time intensive, requiring many steps that may need to be performed one after the other and/or simultaneously, such as in a pipeline, so to analyze the subject's genomic data and determine how that genetic sequence differs from a given reference.

In performing a secondary analysis pipeline, such as for ting a variant call file for a given query sequence of an individual subject; a genetic sample, e.g., DNA, RNA, protein sample, or the like may be obtained, form the subject. The subject'sDNA/RNA may then be sequenced, e.g., by a NextGen Sequencer (NGS) and/or a sequencer-on-a-chip technology, e.g., in a primary processing step, so as to produce a multiplicity of read sequence segments ("reads") ng all or a portion of the individual's genome, such as in an mpled manner. The end product generated by the sequencing device may be a collection of short sequences, e.g., reads, that represent small segments of the t's genome, e.g., short genetic sequences representing the individual's entire genome. As indicated, typically, the information ented by these reads may be an image file or in a digital format, such as in FASTQ, BCL, or other similar file format.

Particularly, in a typical secondary processing protocol, a subject's genetic makeup is assembled by ison to a reference genome. This comparison involves the reconstruction of the individual's genome from millions upon millions of short read sequences and/or the comparison ofthe whole ofthe individual'sDNA to an exemplary DNA sequence model. In a typical secondary processing protocol an image, FASTQ, and/or BCL file is received from the cer ning the raw sequenced read data. In order to compare the subject's genome to that of the standard reference genome, it needs to be determined where each of these reads map to the reference genome, such as how each is aligned with t to one another, and/or how each read can also be sorted by chromosome order so as to determine at what position and in which chromosome each read belongs. One or more of these functions may take place prior to performing a variant call function on the entire full-length sequence, e.g., once assembled. Specifically, once it is determined where in the genome each read belongs, the full length genetic sequence may be determined, and then the differences between the t's genetic code and that ofthe referent can be assessed.

For instance, reference based assembly in a l secondary processing assembly protocol involves the comparison of sequenced genomic DNA/RNA of a subject to that of one or more standards, e.g., known reference sequences. Various g, aligning, g, and/or variant calling algorithms have been developed to help expedite these processes. These algorithms, therefore, may include some variation of one or more of: mapping, aligning, and/or sorting the millions of reads received from the image, FASTQ, and/or BCL file communicated by the sequencer, to ine where on each chromosome each particular read is located. It is noted that these processes may be ented in software or hardware, such as by the methods and/or s described in U.S. Patent Nos. 9,014,989 and 9,235,680 both assigned to Edico Genome Corporation and incorporated by reference herein in their entireties. Often a common feature behind the functioning of these s algorithms and/or hardware implementations is their use of an index and/or an array to expedite their processing on.

For example, with respect to mappmg, a large quantity, e.g., all, of the ced reads may be processed to determine the possible locations in the nce genome to which those reads could possibly align. One methodology that can be used for this purpose is to do a direct comparison of the read to the reference genome so as to find all the positions ofmatching. Another methodology is to employ a prefix or suffix array, or to build out a prefix or suffix tree, for the purpose of mapping the reads to various positions in the reference genome. A typical algorithm useful in ming such a function is a Burrows- Wheeler transform, which is used to map a selection of reads to a reference using a compression formula that compresses repeating sequences ofdata.

A further methodology is to employ a hash table, such as where a selected subset ofthe reads, a k-mer of a selected length "k", e.g., a seed, are placed in a hash table as keys and the reference sequence is broken into equivalent k-mer length portions and those portions and their location are ed by an thm into the hash table at those locations in the table to which they map according to a hashing function. A typical algorithm for performing this function is "BLAST", a Basic Local Alignment Search Tool. Such hash table based programs compare query nucleotide or n sequences to one or more standard reference sequence databases and calculates the statistical significance of matches. In such s as these, it may be determined where any given read is possibly located with respect to a reference genome. These algorithms are useful because they require less memory, fewer look ups, LUTs, and therefore require fewer processing resources and time in the performance of their functions, than would otherwise be the case, such as if the subject's genome were being assembled by direct comparison, such as without the use of these thms.

Additionally, an ng function may be performed to determine out of all the possible locations a given read may map to on a genome, such as in those instances where a read may map to multiple positions in the genome, which is in fact the location from which it actually was derived, such as by being sequenced therefrom by the al sequencing protocol. This function may be performed on a number of the reads, e.g., mapped reads, of the genome and a string of ordered nucleotide bases representing a portion or the entire genetic sequence of the subject's DNA/RNA may be obtained. Along with the ordered genetic sequence a score may be given for each nucleotide in a given position, enting the likelihood that for any given nucleotide position, the nucleotide, e.g., "A", "C", "G", "T" (or "U"), predicted to be in that position is in fact the nucleotide that belongs in that ed position. Typical algorithms for performing alignment functions include man-Wunsch and Smith-Waterman algorithms. In either case, these algorithms perform sequence alignments between a string of the subject's query genomic sequence and a string of the reference genomic sequence whereby instead of ing the entire genomic sequences, one with the other, segments ofa selection ofpossible lengths are compared.

Once the reads have been assigned a position, such as relative to the reference genome, which may include identifying to which chromosome the read belongs and/or its offset from the ing ofthat chromosome, the reads may be sorted by position. This may enable downstream analyses to take advantage of the oversampling procedures described . All of the reads that overlap a given on in the genome will be adjacent to each other after sorting and they can be organized into a pileup and readily ed to determine if the majority of them agree with the reference value or not. If they do not, a t can be flagged.

] For instance, in various embodiments, the methods of the disclosure may include generating a variant call file (VCF) identifying one or more, e.g., all, of the genetic variants in the individual who's DNA/RNA were sequenced, e.g., relevant to one or more reference genomes. For instance, once the actual sample genome is known and compared to the reference genome, the variations between the two can be determined, and a list of all the variations/deviations between the reference genome(s) and the sample genome may be called out, e.g., a t call file may be produced. Particularly, in one aspect, a variant call file containing all the variations of the subject's genetic sequence to the reference sequence(s) may be generated.

As indicated above, such variations between the two genetic sequences may be due to a number of reasons. Hence, in order to generate such a file, the genome ofthe subject must be sequenced and rebuilt prior to determining its ts. There are, however, l problems that may occur when attempting to generate such an assembly. For example, there may be problems with the chemistry, the sequencing machine, and/or human error that occur in the cing s. Furthermore, there may be c artifacts that make such reconstructions problematic. For instance, a typical problem with performing such assemblies is that there are sometimes huge ns of the genome that repeat themselves, such as long ns of the genome that include the same strings of tides. Hence, because any genetic ce is not unique everywhere, it may be difficult to determine where in the genome an identified read actually maps and aligns. Additionally, there may be a single nucleotide polymorphism (SNP), such as wherein one base in the subject's genetic sequence has been substituted for another; there may be more extensive substitutions of a plurality of tides; there may be an ion or a deletion, such as where one or a multiplicity of bases have been added to or deleted from the subject's c sequence, and/or there may be a structural variant, e.g., such as caused by the crossing of legs of two chromosomes, and/or there may simply be an offset causing a shift in the sequence.

Accordingly, there are two main possibilities for ion. For one, there is an actual variation at the particular location in question, for instance, where the person's genome is in fact different at a particular location than that of the reference, e.g., there is a natural variation due to an SNP (one base substitution), an Insertion or Deletion (of one or more nucleotides in length), and/or there is a structural variant, such as where the DNA material from one chromosome gets crossed onto a different chromosome or leg, or where a certain region gets copied twice in the DNA. Alternatively, a variation may be caused by there being a problem in the read data, either through chemistry or the machine, sequencer or aligner, or other human error. The methods disclosed herein may be ed in a manner so as to compensate for these types of errors, and more particularly so as to guish errors in variation due to chemistry, machine or human, and real variations in the ced genome.

More specifically, the methods, apparatuses, and systems for employing the same, as here in described, have been developed so as to clearly distinguish between these two different types of variations and therefore to better ensure the accuracy of any call files generated so as to correctly fy true variants.

Hence, in particular embodiments, a platform of technologies for performing genetic analyses are provided where the platform may include the performance of one or more of: mapping, aligning, sorting, local realignment, duplicate marking, base quality score recalibration, variant calling, compression, and/or decompression functions. For instance, in s aspects a pipeline may be provided wherein the pipeline es performing one or more analytic functions, as described , on a genomic sequence of one or more individuals, such as data ed in an image file and/or a digital, e.g., FASTQ or BCL, file format from an automated sequencer. A l pipeline to be executed may include one or more of sequencing genetic material, such as a portion or an entire genome, of one or more individual subjects, which genetic al may include DNA, ssDNA, RNA, rRNA, tRNA, and the like, and/or in some instances the genetic material may ent coding or noncoding regions, such as exomes and/or es of the DNA. The ne may include one or more of performing an image processing procedure, a base calling and/or error correction operation, such as on the digitized genetic data, and/or may include one or more of performing a mapping, an alignment, and/or a sorting function on the genetic data. In certain instances, the pipeline may include performing one or more of a realignment, a deduplication, a base quality or score recalibration, a reduction and/or ssion, and/or a decompression on the digitized genetic data. In certain instances the pipeline may include performing a variant g operation, such as a Hidden Markov Model, on the genetic data.

Accordingly, in certain instances, the implementation of one or more of these platform functions is for the purpose of performing one or more of determining and/or reconstructing a subject's consensus genomic sequence, comparing a subject's genomic sequence to a referent sequence, e.g., a reference or model c sequence, determining the manner in which the subject's genomic DNA or RNA s from a referent, e.g., variant calling, and/or for performing a tertiary analysis on the subject's genomic sequence, such as for genome-wide variation analysis, gene function analysis, protein function analysis, e.g., protein g analysis, tative and/or assembly analysis of genomes and/or transcriptomes, as well as for various diagnostic, and/or a prophylactic and/or therapeutic evaluation es.

As indicated above, in one aspect one or more of these platform functions, e.g., mapping, aligning, sorting, realignment, duplicate marking, base quality score recalibration, variant g, compression, and/or ression functions is configured for implementation in re. In some aspects, one or more of these platform functions, e.g., mapping, ng, sorting, local realignment, duplicate marking, base y score recalibration, decompression, variant calling, compression, and/or decompression functions is configured for implementation in hardware, e.g., firmware. In certain aspects, these genetic analysis technologies may employ improved algorithms that may be implemented by software that is run in a less processing intensive and/or less time consuming manner and/or with greater percentage accuracy, e.g., the hardware implemented functionality is faster, less processing intensive, and more accurate.

] For instance, in certain embodiments, improved algorithms for performing such primary, secondary, and/or tertiary processing, as disclosed , are provided. The improved algorithms are directed to more efficiently and/or more accurately performing one or more of mapping, aligning, sorting and/or variant calling functions, such as on an image file and/or a digital representation of DNA/RNA sequence data obtained from a sequencing platform, such as in a FASTQ or BCL file format obtained from an automated sequencer such as one of those set forth above. In particular embodiments, the improved thms may be ed to more efficiently and/or more accurately performing one or more of local realignment, duplicate marking, base quality score recalibration, variant calling, compression, and/or decompression functions. Further, as described in greater detail herein below, in certain ments, these genetic analysis technologies may employ one or more algorithms, such as improved algorithms, that may be ented by one or more of software and/or hardware that is run in a less processing intensive and/or less time consuming manner and/or with r tage accuracy than various traditional software implementations for doing the same. In various instances, improved algorithms for implementation on a quantum processing platform are provided.

Hence, in various aspects, presented herein are systems, apparatuses, and methods for implementing bioinformatics protocols, such as for performing one or more functions for analyzing genetic data, such as genomic data, for instance, via one or more optimized algorithms and/or on one or more optimized integrated and/or quantum circuits, such as on one or more hardware processing platforms. In one instance, s and methods are provided for implementing one or more algorithms, e.g., in software and/or in firmware and/or by a quantum processing circuit, for the performance of one or more steps for analyzing genomic data in a bioinformatics protocol, such as where the steps may e the performance of one or more of: mapping, aligning, sorting, local realignment, duplicate marking, base quality score recalibration, variant calling, compression, and/or decompression; and may further include one or more steps in a ry processing platform.

Accordingly, in certain ces, methods, including software, re, hardware, and/or quantum processing algorithms for performing the s, are presented herein where the methods involve the performance ofan algorithm, such as an algorithm for implementing one or more genetic analysis functions such as g, aligning, sorting, realignment, ate marking, base quality score recalibration, variant calling, compression, decompression, and/or one or more tertiary processing protocols where the algorithm, e.g., including firmware, has been optimized in accordance with the manner in which it is to be implemented.

In particular, where the algorithm is to be implemented in a re solution, the algorithm and/or its ant processes, has been optimized so as to be med faster and/or with better accuracy for execution by that media. Likewise, where the ons ofthe algorithm are to be ented in a hardware solution, e.g., as firmware, the hardware has been ed to perform these functions and/or their attendant processes in an optimized manner so as to be performed faster and/or with better accuracy for execution by that media.

Further, where the algorithm is to be implemented in a quantum processing solution, the algorithm and/or its attendant processes, has been optimized so as to be performed faster and/or with better accuracy for execution by that media. These methods, for instance, can be ed such as in an iterative mapping, ng, sorting, variant calling, and/or tertiary processing procedure. In another instance, systems and methods are ed for implementing the functions of one or more algorithms for the performance of one or more steps for analyzing genomic data in a bioinformatics protocol, as set forth herein, wherein the functions are implemented on a hardware and/or quantum accelerator, which may or may not be coupled with one or more general purpose processors and/or super computers and/or quantum computers.

More specifically, in some instances, s and/or machinery for implementing those s, for performing secondary analytics on data ning to the c composition of a subject are provided. In one instance, the analytics to be performed may involve reference based reconstruction of the subject genome. For instance, referenced based mapping es the use of a reference genome, which may be generated from sequencing the genome of a single or multiple individuals, or it may be an amalgamation of various people's DNA/RNA that have been combined in such a manner so as to e a ypical, standard reference genome to which any individual's genetic material, e.g., DNA/RNA, may be ed, for example, so as to determine and reconstruct the individual's genetic sequence and/or for determining the difference n their genetic makeup and that ofthe standard reference, e.g., variant calling.

Particularly, a reason for ming a secondary analysis on a subject's sequenced DNA/RNA is to determine how the t's DNA/RNA varies from that of the reference, such as to determine one, a multiplicity, or all, of the differences in the nucleotide sequence of the subject from that of the reference. For instance, the differences between the genetic sequences of any two random persons is 1 about in 1,000 base pairs, which when taken in view ofthe entire genome of over 3 billion base pairs amounts to a variation ofup to 3,000,000 divergent base pairs per person. Determining these differences may be useful such as in a tertiary analysis protocol, for instance, so as to t the potential for the occurrence of a diseased state, such as because of a c abnormality, and/or the likelihood of success of a prophylactic or therapeutic modality, such as based on how a prophylactic or therapeutic is expected to interact with the t's DNA or the proteins generated therefrom. In various instances, it may be useful to perform both a de novo and a reference based reconstruction of the subject's genome so as to confirm the results of one against the other, and to, where ble, enhance the accuracy ofa t calling protocol.

Accordingly, in one aspect, in various embodiments, once the subject's genome has been reconstructed and/or a VCF has been generated, such data may then be subjected to tertiary processing so as to interpret it, such as for determining what the data means with respect to identifying what es this person may or may have the potential for suffer from and/or for determining what treatments or lifestyle changes this subject may want to employ so as to ameliorate and/or prevent a diseased state. For example, the subject's genetic sequence and/or their variant call file may be analyzed to determine clinically relevant genetic markers that indicate the existence or potential for a diseased state and/or the efficacy ofa proposed therapeutic or prophylactic regimen may have on the subject. This data may then be used to e the t with one or more eutic or lactic regimens so as to better the subject's quality of life, such as treating and/or preventing a diseased state.

Particularly, once one or more of an individual's genetic variations are determined, such variant call file information can be used to develop medically useful information, which in tum can be used to determine, e.g., using various known statistical analysis , health related data and/or medical useful ation, e.g., for diagnostic purposes, e.g., diagnosing a disease or potential therefore, clinical interpretation (e.g., looking for markers that represent a disease variant), whether the subject should be included or excluded in various al , and other such purposes. More ularly, in various instances, the generated genomics and/or bioinformatics processed s data may be employed in the performance of one or more genomics and/or bioinformatics tertiary protocols, such as a micro-array analysis protocol, a genome, e.g., whole genome analysis protocol, a genotyping analysis protocol, an exome analysis ol, an epigenome analysis protocol, a metagenome analysis protocol, a microbiome analysis protocol, a genotyping analysis protocol, including joint genotyping, variants analyses ols, including structural variants, somatic variants, and GATK, as well as RNA sequencing protocols and other genetic analyses protocols.

As there are a finite number of diseased states that are caused by genetic malformations, in tertiary processing ts of a certain type, e.g., those known to be related to the onset of diseased states, can be queried for, such as by determining if one or more genetic based diseased markers are included in the variant call file of the subject. Consequently, in various instances, the methods herein disclosed may involve analyzing, e.g., scanning, the VCF and/or the generated sequence, against a known disease ce variant, such as in a data base of genomic markers therefore, so as to fy the presence of the genetic marker in the VCF and/or the generated sequence, and if present to make a call as to the presence or potential for a genetically induced diseased state. Since there are a large number of known genetic variations and a large number of individual's suffering from diseases caused by such variations, in some embodiments, the methods disclosed herein may entail the generation of one or more databases linking sequenced data for an entire genome and/or a t call file ning thereto, e.g., such as from an individual or a plurality of individuals, and a diseased state and/or searching the generated databases to determine if a particular subject has a genetic composition that would pose them to having such diseased state. Such searching may e a comparison of one entire genome with one or more others, or a fragment of a , such as a fragment containing only the variations, to one or more fragments of one or more other genomes such as in a database of reference genomes or fragments thereof.

Therefore, in various instances, a pipeline ofthe disclosure may include one or more modules, wherein the modules are configured for performing one or more functions, such as an image processing or a base calling and/or error correction ion and/or a g and/or an alignment, e.g., a gapped or gapless alignment, and/or a sorting function on genetic data, e.g., sequenced genetic data. And in various instances, the pipeline may include one or more modules, wherein the modules are configured for performing one more of a local realignment, a deduplication, a base quality score recalibration, a variant calling, e.g., HMM, a reduction and/or compression, and/or a decompression on the c data.

Additionally, the pipeline may e one or more modules, wherein the modules are configured for performing a tertiary analysis protocol, such as micro-array protocols, , e.g., whole genome protocols, ping protocols, exome protocols, epigenome protocols, metagenome protocols, microbiome ols, genotyping protocols, including joint ping ols, variants analysis ols, including structural variants protocols, c variants protocols, and GATK protocols, as well as RNA sequencing protocols and other genetic analyses protocols.

Many of these modules may either be performed by software or on hardware, locally or remotely, e.g., via software or hardware, such as on the cloud, e.g., on a remote server and/or server bank, such as a quantum computing cluster. Additionally, many of these modules and/or steps of the ne are optional and/or can be arranged in any logical order and/or omitted entirely. For instance, the software and/or hardware disclosed herein may or may not include an image processing and/or a base calling or sequence tion algorithm, such as where there may be a concern that such functions may result in a statistical bias.

Consequently, the system may include or may not include the base calling and/or sequence correction on, respectively, dependent on the level of accuracy and/or efficiency desired. And as indicated above, one or more of the pipeline functions may be employed in the generation of a genomic sequence of a subject such as through a reference based genomic reconstruction. Also, as indicated above, in certain instances, the output from the secondary WO 14320 2017/036424 processing pipeline may be a t call file (VCF, gVCF) ting a portion or all the variants in a genome or a portion thereof.

Particularly, once the reads are assigned a position relative to the reference , which may include identifying to which chromosome the read belongs and its offset from the beginning of that chromosome, they may be de-duplicated and/or sorted, such as by position. This enables downstream analyses to take advantage of the various oversampling protocols described herein. All of the reads that overlap a given position in the genome may be positioned adjacent to each other after sorting and they can be piled up, e.g., to form a pileup, and readily examined to determine if the majority of them agree with the reference value or not. Ifthey do not, as indicated above, a variant can be flagged.

Accordingly, as indicated above with respect to mapping, the image file, BCL file, and/or FASTQ file, obtained from the sequencer is comprised of a plurality, e.g., millions to a billion or more, of reads consisting of short strings of nucleotide sequence data representing a n or the entire genome of an individual. For instance, a first step in the secondary analysis pipelines, disclosed herein, is the receipt of c and/or bioinformatics data, such as from a genomics data ting apparatus, such as a sequencer.

Typically, the data produced by a sequencer, e.g., a NextGen Sequencer, may be in a BCL file format, which in some instances, may be converted into a FASTQ file format, either prior or subsequent to transmission, such as into a secondary processing platform herein described.

Particularly, when sequencing a human genome, a subject's DNA and/or RNA must be identified, on a base per base basis, where the results of such sequencing is a BCL file. A BCL file is a binary file that es the base calls and quality scores made for each base of each ce of the collection of sequences that compose at least a part of or the whole genome ofa subject.

Traditionally, the sequencer generated BCL file is converted to a FASTQ file, which then may be transmitted to a secondary processing platform, such as disclosed herein, for further processing, such as to determine the cs variance thereof. A FASTQ file is a text-based file format for transmitting and storing both a biological sequence (e.g., nucleotide sequence) and its corresponding quality scores, where both the sequence letter, e.g., A, C, G, T, and/or U, and the quality score may each be d with a single ASCII character for y. Accordingly, within this and other systems, it is the FASTQ file that is used for the es of further processing. Although the employment of a FASTQ file for genomics processing is useful, the conversion of the generated BCL file into a FASTQ file, as implemented in the sequencer tus, is time consuming and inefficient. Hence, in one aspect, devices and methods for directly converting a BCL file into a FASTQ file and/or for directly inputting such data into the present rm pipelines, as herein described, are provided.

For instance, in various embodiments, a Next Generation sequencer, or a sequencer on a chip technology, may be configured to perform a sequencing operation on received genetic data. For instance, as can be seen with respect to FIG. IA, the genetic data 6a may coupled to a sequencing platform 6 for ion into a Next Gen sequencer to be sequenced in an ive fashion, such that each sequence will be grown by the stepwise addition of one nucleotide after another. Specifically, the sequencing platform 6 may include a number oftemplate nucleotide sequences 6a from the subject that are arranged in a grid like fashion to form tiles 6b on the platform 6, which template sequences 6a are to be sequenced.

The platform 6 may be added to a flow cell 6c ofthe cer that is adapted for ming the sequencing reactions.

As the sequencing reactions take place, at each step a tide having a fluorescent tag 6d is added to the platform 6 of the flow cell 6c. If a izing reaction occurs, fluorescence is observed, an image is taken, the image is then processed, and an riate base call is made. This is ed base by base until all of the template sequences, e.g., the entire genome, has been sequenced and converted into reads, thereby producing the read data ofthe . Hence, once ced, the generated data, e.g., reads, need to be transferred from the sequencing platform into the secondary processing system.

For instance, typically, this image data is converted into a BCL and/or FASTQ file that can then be transported into the system.

However, in various instances, this conversion and/or transfer process may be made more efficient. Specifically, presented herein are methods and architectures for expedited BCL conversion into files that can be rapidly processed within the secondary processing system. More specifically, in particular ces, instead of transmitting the raw BCL or FASTQ files, the images produced representing each tile ofthe sequencing operation may be transferred directly into the system and prepared for mapping and aligning et al. For instance, the tiles may be streamed across a suitably configured PCie and into the ASIC, FPGA, or QPU, wherein the read data may be extracted therefrom directly, and the reads ed into the mapping and aligning and/or other processing engines. ularly, with respect to the transfer of the data from the tiles obtained by the sequencer to the FPGA/CPU/GPU/QPU, as can be seen with respect to FIG. IA, the sequencing platform 6 may be imaged as a 3-D cube 6c, within which the growing sequences 6a are generated. Essentially, as can be seen with respect to FIG. IB, the sequencing platform 6 may be composed of 16 lanes, 8 in the front and 8 in the back, which may be configured to form about 96 tiles 6b. Within each tile 6b are a number of template sequences 6a to be sequenced thereby forming reads, where each read represents the nucleotide sequence for a given region of the genome of a t, each column represents one file, and as digitally encoded represents 1 byte for every file, with 8 bits per file, such as where 2 bits represents the called base, and the remaining 6 bits represents the quality score.

More ularly, with respect to Next Gen cing, the sequencing is typically performed on glass plates 6 that form flow cells 6c that are entered into the automated sequencer for sequencing. As can be seen with t to FIG. IB, a flow cell 6c is a platform 6 composed of 8 vertical columns and 8 horizontal rows (front and back), er which form 16 lanes, where each lane is sufficient for the sequencing of an entire genome.

The DNA and/or RNA 6a of a subject to be sequenced is associated within designated positions in between fluidly isolated intersections of the columns and rows of the platform 6 so as to form the tiles 6b, where each tile es template genetic material 6a to be sequenced. The sequencing platform 6, therefore, includes a number of template nucleotide sequences from the subject, which sequences are ed in a grid like fashion oftiles on the rm. (See FIG. IB.) The genetic data 6 is then sequenced in an iterative fashion where each sequence is grown by the stepwise introduction of one nucleotide after another into the flow cell, where each iterative growth step represents a sequencing cycle.

As indicated, an image is captured after each step, and the growing sequence, e.g., of images, form the basis by which the BCL file is generated. As can be seen with respect to FIG. IC, the reads from the sequencing ure may form clusters, and it is these clusters that form the theoretical 3-D cube 6c. Accordingly, within this theoretical 3-D cube, each base of each g nucleotide strand being sequenced will have an x ion and a y dimension. The image data, or tiles 6b, from this 3-D cube 6c may be extracted and compiled into a two-dimensional map, from which a matrix, as seen in FIG. IAD may be formed. The matrix is formed of the sequencing cycles, which represent the horizontal axis, and the read identities, which ent the vertical axis. Accordingly, as can be seen with reference to FIG. IC, the sequenced reads form clusters in the flow cell 6c, which clusters WO 14320 PCT/0S2017/036424 may be defined by a vertical and horizontal axis, cycle by cycle, and the base by base data from each cycle for each read may be ed into the matrix of FIG. ID, such as in a streaming and/or pipelined fashion.

Specifically, each cycle represents the potential growth ofeach read within the flow cell by the addition of one nucleotide, which when sequencing one or several human genomes, may represent the growth of about 1 billion or more reads per lane. The growth of each read, e.g., by the addition of a nucleotide base, is identified by the iterative capturing of images, of the tiles 6b, of the flow cell 6c in between the growth steps. From these images base calls are made, and quality scores determined, and the virtual matrix of FIG ID is formed. Accordingly, there will be both a base call and a quality score entered into the , where each tile from each cycle represents a separate file. It is to be noted that where the sequencing is performed on an integrated circuit, sensed electronic data may be substituted for the image data.

For instance, as can be seen with respect to FIG. ID, the matrix itself will grow iteratively as the images are captured and processed, bases are called, and quality scores are ined for each read, cycle by cycle. This is repeated for each base in the read, for each tile ofthe flow cell. For example, the cluster s. IC may be numbered and entered into the matrix as the vertical axis. Likewise, the cycle number may be entered as the horizontal axis, and the base call and y score may then be entered so as to fill out the matrix column by column, row by row. Accordingly, each read will be represented by a number of bases, e.g., about 100 or 150 up to 1000 or more bases per read depending on the sequencer, and there may be up to 10 million or more reads per tile. So, if there are about 100 tiles each having 10 million reads, the matrix would contain about 1 billion reads, which need to be organized and streamed into the secondary processing apparatus.

Accordingly, such organization is fundamental to rapidly and efficiently processing the data. Hence, in one , presented herein are methods for transposing the data represented by the virtual sequencing matrix in a manner so that the data may be more directly and efficiently streamed into the pipelines of the system herein sed. For instance, the generation of the sequencing data, as represented by the star cluster of , is largely unorganized, which is problematic from a data processing standpoint. Particularly, as the data is generated by the sequencing operation, it is organized as one file per cycle, which means that by the end of the sequencing operation there are millions and ns of files ted, which files are represented in FIG. IE, by the data in the columns, demarcated by the solid lines. However, for the purposes of secondary and/or tertiary processmg, as disclosed herein, the file data needs to be re-organized into read data, demarcated by the dashed lines ofFIG. IE.

More particularly, in order to more efficiently stream the data generated by the sequencer into the secondary processing data, the data represented by the virtual matrix should be osed, such as by reorganizing the file data from a column by column basis of tiles per cycle, to a row by row basis identifying the bases of each of the reads. Specifically, the data structure ofthe ted files forming the matrix, as it is produced by the sequencer, is organized on a cycle by cycle, column by column, basis. By the processes disclosed herein, this data may be transposed, e.g., substantially simultaneously, so as to be ented, as seen within the virtual matrix, on a read by read, row by row basis, where each row represents an dual read, and each read is represented by a tial number of base calls and quality scores, thereby identifying both the ce for each read and its confidence. Thus, in a transpose operation as herein described, the data within the memory may be re-organized, e.g., within the virtual matrix, from a column by column basis, representing the input data order, to a row by row basis, representing the output data order, thereby transposing the data order from a vertical to a horizontal organization. r, although the process may be implemented efficiently in re, it may be made even more efficiently and faster, by being implemented in hardware and/or by a quantum processor.

] For instance, in various instances, this transposition process may be accelerated by being ented in hardware. For example, in one implementation, in a first step, the host software, e.g., of the sequencer, may write input data into the memory, associated with the FPGA, on a column by column basis, e.g., in the input order. Specifically, as the data is generated and stored into an ated memory, the data may be organized into files, cycle by cycle, where the data is saved as separate individual files. This data may be represented by the 3-D cube of FIG. IA. This generated column organized data may then be queued and/or streamed, e.g., in flight, into the hardware where dedicated processing engines will queue up the column zed data and transpose that data from a column by column, cycle order configuration, to a row by row, read order configuration, in a manner as described herein above, such as by converting the 3-D tile data into a 2-D matrix, whereby the column data may be reorganized into row data, e.g., on a read to read basis. This transposed data may then be stored in the memory in a more strategic order.

For example, the host software may be configured to write input data into the memory associated with the chip, e.g., FPGA, such as in a column-wise input order, and likewise the hardware may be configured to queue the data in a manner so that it is red into the memory in a strategic manner, such as set forth in FIG. IF. Specifically, the re may e an array of registers 8a into which the cycle files may be sed and reorganized into individual read data, such as by writing one base from a column into registers that are organized into rows. More specifically, as can be seen with respect to FIG. IG, the hardware device 1, including the transposition sing engine 8, may include a DRAM port 8a that may queue up the data to be transposed, where the port is operably coupled to a memory interface 8b that is associated with a plurality of registers and/or an external memory 8c, and is configured for ng an increased amount of ctions per cycle, where the queued data is itted in bursts.

Particularly, this transposition may take place one data segment at a time, such as where the memory accesses are queued up in such a manner as to take maximal advantage of the DDR transmission rate. For instance, with respect to DRAM, the minimal burst length of the DDR may be, for example, 64 bytes. Accordingly, the column arranged data stored in the host memory may be accessed in a manner such that with each memory access a column worth of corresponding, e.g., 64, bytes of data is obtained. Hence, with one access of the memory a portion of a tile, e.g., representing a corresponding "64" cycles or files, may be accessed, on a column by column basis.

However, as can be seen with respect to FIG. IF, although the data in the host memory is accessed as column data, when transmitted to the hardware, it may be uploaded into associated r memories, e.g., registers, in a different order y the data may be converted into bytes, e.g., 64 bytes, of row by row read data, such as in accordance with the minimal burst rate of the DDR, so as to generate a corresponding "64" memory units or blocks per access. This is exemplified by the virtual matrix of FIG. ID where a number of reads, e.g., 64 reads, are accessed in blocks, and read into memory in segments, as represented by FIG. IE, such as where each register, or flip-flop, accounts for a particular read, e.g., 64 cycles x 64 reads x 8 bits per read = 32K flip-flops. Specifically, this may be accomplished in various different ways in hardware, such as where the input wiring is organized to match the column ordering, and the output wiring is organized to match the row order. Hence in this configuration, the hardware may be adapted so as to both read and/or write to "64" different addresses per cycle.

More particularly, the hardware may be associated with an array of registers such that each base of a read is directed and written into a single register (or multiple registers in a row) such that when each block is complete, the newly ordered row data may be transmitted to memory as an output, e.g., FASTQ data, in a row by row organization. The FASTQ data may then be ed by one or more further sing s of the secondary processing system for further sing, such as by a mapping, aligning, and/or variant g engine, as described herein. It is to be noted, as described herein, the transpose is performed in small blocks, however, the system may be adapted for the processing of larger blocks as well, as the case may be.

As indicated, once a BCL file has been converted into a FASTQ file, as described above, and/or a BCL or FASTQ file has otherwise been received by the secondary processing platform, a mapping operation may be performed on the received data. Mapping, in general, involves plotting the reads to all the locations in the reference genome to where there is a match. For example, dependent on the size of the read there may be one or a plurality of ons where the read substantially matches a corresponding sequence in the reference genome. Hence, the mapping and/or other functions disclosed herein may be configured for determining where out of all the possible locations one or more reads may match to in the reference genome is actually the true location to where they map.

For ce, in various instances, an index of a reference genome may be generated or otherwise provided, so that the reads or portions of the reads may be looked up, e.g., within a Look-Up Table (LUT), in reference to the index, thereby ving indications of ons in the reference, so as to map the reads to the reference. Such an index of the reference can be constructed in various forms and queried in various manners. In some methods, the index may include a prefix and/or a suffix tree. In particular methods, the index may be derived from a Burrows/Wheeler transform of the reference. Hence, alternatively, or in addition to employing a prefix or a suffix tree, a s/Wheeler transform can be performed on the data. For instance, a Burrows/Wheeler transform may be used to store a tree-like data ure abstractly equivalent to a prefix and/or suffix tree, in a compact format, such as in the space allocated for storing the reference . In s instances, the data stored is not in a tree-like structure, but rather the reference ce data is in a linear list that may have been scrambled into a different order so as to transform it in a very particular way such that the accompanying algorithm allows the reference to be searched with reference to the sample reads so as to effectively walk the "tree".

Additionally, in various instances, the index may include one or more hash , and the methods disclosed herein may include a hash function that may be performed on one or more portions ofthe reads in an effort to map the reads to the reference, e.g., to the index of the nce. For instance, alternatively, or in addition to utilizing one or both a prefix/suffix tree and/or a Burrows/Wheeler transform on the reference genome and subject sequence data, so as to find where the one maps against the other, another such method involves the production of a hash table index and/or the performance of a hash function. The hash table index may be a large reference structure that is built up from sequences of the reference genome that may then be compared to one or more ns of the read to determine where the one may match to the other. Likewise, the hash table index may be built up from portions of the read that may then be compared to one or more sequences of the reference genome and thereby used to determine where the one may match to the other.

Implementation of a hash table is a fast method for performing a pattern match. Each lookup takes a nearly constant amount of time to perform. Such method may be contrasted with the Burrows-Wheeler method which may require many probes (the number may vary depending on how many bits are ed to find a unique pattern) per query to find a match, or a binary search method that takes log2(N) probes where N is the number of seed patterns in the table. Further, even though the hash function can break the reference genome down into ts ofseeds of any given length, e.g., 28 base pairs, and can then convert the seeds into a digital, e.g., binary, representation of 56 bits, not all 56 bits need be accessed entirely at the same time or in the same way. For instance, the hash function can be implemented in such a manner that the address for each seed is designated by a number less than 56 bits, such as about 18 to about 44 or 46 bits, such as about 20 to about 40 bits, such as about 24 to about 36 bits, including about 28 to about 32 or about 30 bits may be used as an l key or address so as to access the hash table. For e, in certain instances, about 26 to about 29 bits may be used as a primary access key for the hash table, leaving about 27 to about 30 bits left over, which may be employed as a means for double ng the first key, e.g., if both the first and second keys arrive at the same cell in the hash table, then it is vely clear that said location is where they belong.

For instance, a first portion of the digitally represented seed, e.g., about 26 to about 32, such as about 29 bits, can form a primary access key and be hashed and may be looked up in a first step. And, in a second step, the remaining about 27 to about 30 bits, e.g., a secondary access key, can be inserted into the hash table, such as in a hash chain, as a means WO 14320 PCT/0S2017/036424 for confirming the first pass. Accordingly, for any seed, its original address bits may be hashed in a first step, and the secondary address bits may be used in a second, confirmation step. In such an instance, the first portion of the seeds can be inserted into a primary record location, and the second portion may be fit into the table in a secondary record chain location.

And, as indicated above, in various instances, these two ent record locations may be positionally separated, such as by a chain format record.

In particular instances, a brute force linear scan can be employed to compare the reference to the read, or ns thereof. r, using a brute force linear search to scan the reference genome for locations where a seed matches, over 3 n locations may have to be checked. Which searching can be performed, in accordance with the methods disclosed herein, in software or hardware. Nevertheless, by using a hashing approach, as set forth herein, each seed lookup can occur in approximately a constant amount of time. Often, the location can be ascertained in a few, e.g., a single access. However, in cases where multiple seeds map to the same location in the table, e.g., they are not unique enough, a few additional accesses may be made to find the seed being currently looked up. Hence, even though there can be 30M or more possible locations for a given 100 nucleotide length read to match up to, with respect to a reference genome, the hash table and hash on can quickly determine where that read is going to show up in the reference genome. By using a hash table index, therefore, it is not necessary to search the whole reference genome, e.g., by brute force, to determine where the read maps and aligns.

In view of the above, any suitable hash function may be employed for these purposes, however, in various instances, the hash function used to determine the table address for each seed may be a cyclic redundancy check (CRC) that may be based on a 2k-bit primitive polynomial, as indicated above. Alternatively, a trivial hash on mapper may be employed such as by simply dropping some of the 2k bits. However, in various ces, the CRC may be a stronger hash function that may better separate r seeds while at the same time avoiding table congestion. This may ally be cial where there is no speed penalty when calculating CRCs such as with the dedicated hardware bed herein.

In such instances, the hash record populated for each seed may e the reference position where the seed occurred, and the flag indicating whether it was reverse complemented before hashing.

The output returned from the performance ofa mapping function may be a list of possibilities as to where one or more, e.g., each, read maps to one or more reference genomes. For instance, the output for each mapped read may be a list of possible locations the read may be mapped to a matching sequence in the reference genome. In s embodiments, an exact match to the reference for at least a piece, e.g., a seed of the read, if not all of the read may be sought. Accordingly, in various instances, it is not necessary for all portions ofall the reads to match exactly to all the portions of the reference genome.

As described herein, all of these operations may be performed via software or may be hardwired, such as into an integrated circuit, such as on a chip, for instance as part of a circuit board. For instance, the functioning of one or more of these algorithms may be embedded onto a chip, such as into a FPGA (field programmable gate array) or ASIC (application specific integrated circuit) chip, and may be optimized so as to m more efficiently because of their implementation in such hardware. Additionally, one or more, e.g., two or all three, of these g functions may form a , such as a mapping module, that may form part of a , e.g., a pipeline, that is used in a process for ining an actual entire genomic sequence, or a portion thereof, ofan dual.

An advantage of implementing the hash module in hardware is that the ses may be accelerated and therefore performed in a much faster manner. For instance, where software may include various instructions for performing one or more of these various functions, the implementation of such instructions often requires data and ctions to be stored and/or fetched and/or read and/or interpreted, such as prior to ion. As indicated above, however, and described in r detail herein, a chip can be hardwired to perform these functions without having to fetch, interpret, and/or m one or more of a sequence of instructions. Rather, the chip may be wired to perform such functions directly.

Accordingly, in various aspects, the disclosure is directed to a custom hardwired machine that may be configured such that ns or all of the above described mapping, e.g., hashing, module may be implemented by one or more network circuits, such as integrated circuits hardwired on a chip, such as an FPGA or ASIC.

For example, in s instances, the hash table index may be constructed and the hash function may be performed on a chip, and in other instances, the hash table index may be generated off of the chip, such as via software run by a host CPU, but once generated it is loaded onto or otherwise made accessible to the hardware and employed by the chip, such as in running the hash module. Particularly, in various instances, the chip, such as an FPGA, may be configured so as to be tightly d to the host CPU, such as by a low latency interconnect, such as a QPI interconnect. More particularly, the chip and CPU may be configured so as to be tightly coupled together in such a manner so as to share one or more memory resources, e.g., a DRAM, in a cache coherent configuration, as described in more detail below. In such an instance, the host memory may build and/or include the reference index, e.g., the hash table, which may be stored in the host memory but be made readily accessible to the FPGA such as for its use in the performance of a hash or other mapping function. In particular embodiments, one or both of the CPU and the FPGA may include one or more caches or registers that may be coupled er so as to be in a coherent configuration such that stored data in one cache may be substantially mirrored by the other.

Accordingly, in view of the above, at run-time, one or more previously constructed hash tables, e.g., containing an index of a reference genome, or a constructed or to be ucted hash table, may be loaded into onboard memory or may at least be made accessible by its host application, as described in greater detail herein below. In such an instance, reads, e.g., stored in FASTQ file , may be sent by the host application to the onboard processing engines, e.g., a memory or cache or other register associated ith, such as for use by a mapping and/or alignment and/or sorting engine, such as where the results thereof may be sent to and used for performing a variant call function. With respect thereto, as indicated above, in various instances, a pile up of overlapping seeds may be generated, e.g., via a seed generation function, and extracted from the sequenced reads, or read-pairs, and once generated the seeds may be hashed, such as against an index, and looked up in the hash table so as to ine candidate read mapping positions in the reference.

More particularly, in various ces, a mapping module may be provided, such as where the mapping module is ured to perform one or more g functions, such as in a hardwired configuration. Specifically, the hardwired mapping module may be configured to perform one or more functions typically performed by one or more algorithms run on a CPU, such as the functions that would typically be implemented in a software based algorithm that produces a prefix and/or suffix tree, a Burrows-Wheeler Transform, and/or runs a hash function, for instance, a hash function that makes use of, or otherwise relies on, a able indexing, such as of a reference, e.g., a reference genome sequence. In such ces, the hash function may be ured so as to implement a strategy, such as an optimized mapping strategy that may be configured to minimize the number of memory accesses, e.g., large-memory random accesses, being performed so as to thereby ze the utility of the on-board or otherwise ated memory bandwidth, which may fundamentally be constrained such as by space within the chip architecture.

Further, in certain instances, in order to make the system more efficient, the host CPU/GPU/QPU may be tightly coupled to the associated hardware, e.g., FPGA, such as by a low latency interface, e.g., Quick Path Interconnect ("QPI"), so as to allow the processing engines ofthe integrated circuit to have ready access to host memory. In particular instances, the interaction between the host CPU and the coupled chip and their respective associated memories, e.g., one or more DRAMs, may be configured so as to be cache coherent. Hence, in various embodiments, an integrated circuit may be provided n the integrated circuit has been pre-configured, e.g., ed, in such a manner as to include one or more digital logic circuits that may be in a wired configuration, which may be interconnected, e.g., by one or a plurality of physical electrical interconnects, and in various embodiments, the hardwired digital logic circuits may be arranged into one or more sing engines so as to form one or more modules, such as a mapping . ingly, in s instances, a mapping module may be provided, such as in a first nfigured wired, e.g., hardwired, configuration, where the mapping module is configured to perform various mapping functions. For instance, the mapping module may be configured so as to access, at least some of a sequence of nucleotides in a read of a plurality of reads, derived from a t's sequenced c , and/or a genetic nce sequence, and/or an index of one or more genetic reference sequences, from a memory or a cache associated ith, e.g., via a memory interface, such as a process interconnect, for instance, a Quick Path Interconnect, and the like. The mapping module may further be configured for mapping the read to one or more segments of the one or more genetic reference sequences, such as based on the index. For example, in various particular ments, the mapping algorithm and/or module presented herein, may be employed to build, or otherwise construct a hash table whereby the read, or a portion thereof, of the sequenced genetic material from the t may be ed with one or more segments of a reference genome, so as to produce mapped reads. In such an instance, once mapping has been performed, an alignment may be performed.

For example, after it has been determined where all the possible matches are for the seeds against the reference genome, it must be ined which out of all the possible locations a given read may match to is in fact the correct position to which it aligns.

Hence, after mapping there may be a multiplicity of positions that one or more reads appear to match in the reference genome. Consequently, there may be a plurality ofseeds that appear to be indicating the exact same thing, e.g., they may match to the exact same position on the WO 14320 PCT/0S2017/036424 reference, if you take into account the position of the seed in the read. The actual alignment, therefore, must be determined for each given read. This determination may be made in several different ways.

In one instance, all the reads may be evaluated so as to determine their correct alignment with respect to the reference genome based on the positions indicated by every seed from the read that returned position information during the g, e.g., hash lookup, process. However, in various ces, prior to performing an alignment, a seed chain filtering function may be performed on one or more of the seeds. For ce, in certain instances, the seeds associated with a given read that appear to map to the same l place as against the reference genome may be ated into a single chain that references the same general . All of the seeds associated with one read may be grouped into one or more seed chains such that each seed is a member of only one chain. It is such s) that then cause the read to be aligned to each indicated position in the reference genome.

Specifically, in various instances, all the seeds that have the same supporting ce indicating that they all belong to the same general location(s) in the reference may be gathered together to form one or more chains. The seeds that group together, therefore, or at least appear as they are going to be near one another in the reference genome, e.g., within a certain band, will be grouped into a chain ofseeds, and those that are outside ofthis band will be made into a different chain of seeds. Once these various seeds have been aggregated into one or more various seed chains, it may be determined which ofthe chains actually represents the correct chain to be aligned. This may be done, at least in part, by use of a filtering algorithm that is a heuristic designed to eliminate weak seed chains which are highly unlikely to be the correct one.

] The outcome from performing one or more of these mapping, ing, and/or editing functions is a list of reads which includes for each read a list of all the possible locations to where the read may matchup with the reference genome. Hence, a g function may be performed so as to quickly determine where the reads ofthe image file, BCL file, and/or FASTQ file obtained from the sequencer map to the reference genome, e.g., to where in the whole genome the various reads map. However, if there is an error in any ofthe reads or a genetic variation, you may not get an exact match to the reference and/or there may be several places one or more reads appear to match. It, therefore, must be determined where the various reads actually align with respect to the genome as a whole. ingly, after mappmg and/or filtering and/or editing, the location positions for a large number ofreads have been ined, where for some ofthe individual reads a multiplicity of on positions have been determined, and it now needs to be determined which out of all the possible locations is in fact the true or most likely location to which the various reads align. Such aligning may be performed by one or more thms, such as a dynamic programming algorithm that matches the mapped reads to the reference genome and runs an alignment function thereon. An ary aligning function compares one or more, e.g., all of the reads, to the reference, such as by placing them in a graphical relation to one another, e.g., such as in a table, e.g., a virtual array or matrix, where the sequence of one of the reference genome or the mapped reads is placed on one dimension or axis, e.g., the horizontal axis, and the other is placed on the opposed dimensions or axis, such as the vertical axis. A conceptual scoring wave front is then passed over the array so as to ine the alignment of the reads with the reference genome, such as by computing alignment scores for each cell in the matrix.

The scoring wave front represents one or more, e.g., all, the cells of a matrix, or a portion of those cells, which may be scored independently and/or simultaneously according to the rules of dynamic programming applicable in the alignment thm, such as Smith-Waterman, and/or Needleman-Wunsch, and/or related algorithms. Alignment scores may be computed sequentially or in other orders, such as by computing all the scores in the top row from left to right, followed by all the scores in the next row from left to right, etc. In this manner the diagonally sweeping diagonal wave front represents an optimal sequence of batches ofscores computed simultaneously or in parallel in a series ofwave front steps.

] For instance, in one embodiment, a window of the reference genome containing the segment to which a read was mapped may be placed on the horizontal axis, and the read may be positioned on the vertical axis. In a manner such as this an array or matrix is generated, e.g., a virtual matrix, whereby the nucleotide at each position in the read may be compared with the tide at each position in the nce window. As the wave front passes over the array, all ial ways of aligning the read to the reference window are considered, ing if changes to one sequence would be required to make the read match the reference sequence, such as by changing one or more nucleotides of the read to other nucleotides, or inserting one or more new nucleotides into one sequence, or deleting one or more nucleotides from one sequence.

An alignment score, enting the extent of the changes that would be required to be made to achieve an exact alignment, is generated, wherein this score and/or other associated data may be stored in the given cells of the array. Each cell of the array corresponds to the possibility that the nucleotide at its position on the read axis aligns to the nucleotide at its position on the reference axis, and the score generated for each cell represents the l alignment terminating with the cell's positions in the read and the reference window. The highest score ted in any cell represents the best overall alignment of the read to the reference window. In various instances, the alignment may be global, where the entire read must be aligned to some portion of the reference window, such as using a Needleman-Wunsch or similar algorithm; or in other instances, the alignment may be local, where only a portion of the read may be aligned to a portion of the reference window, such as by using a Smith-Waterman or similar algorithm.

Accordingly, in various instances, an alignment function may be performed, such as on the data obtained from the mapping module. Hence, in various instances, an alignment function may form a module, such as an alignment module, that may form part ofa system, e.g., a ne, that is used, such as in addition with a mapping module, in a s for ining the actual entire genomic sequence, or a portion thereof, of an individual. For instance, the output returned from the performance of the mapping function, such as from a mapping module, e.g., the list ofpossibilities as to where one or more or all ofthe reads maps to one or more positions in one or more reference genomes, may be employed by the alignment function so as to determine the actual sequence alignment of the subject's sequenced DNA.

] Such an ent function may at times be useful because, as described above, often times, for a variety of different reasons, the ced reads do not always match exactly to the reference genome. For instance, there may be an SNP (single nucleotide polymorphism) in one or more of the reads, e.g., a substitution of one nucleotide for another at a single position; there may be an "indel," insertion or on of one or more bases along one or more of the read sequences, which insertion or deletion is not t in the reference genome; and/or there may be a sequencing error (e.g., errors in sample prep and/or sequencer read and/or sequencer output, etc.) causing one or more of these apparent variations.

Accordingly, when a read varies from the reference, such as by an SNP or Indel, this may be e the nce s from the true DNA sequence sampled, or because the read differs from the true DNA sequence sampled. The problem is to figure out how to correctly align the reads to the reference genome given the fact that in all likelihood the two sequences are going to vary from one another in a licity ofdifferent ways.

] In various instances, the input into an alignment function, such as from a mapping on, such as a /suffix tree, or a Burrows/Wheeler transform, or a hash table and/or hash on, may be a list of possibilities as to where one or more reads may match to one or more positions of one or more reference sequences. For instance, for any given read, it may match any number of positions in the reference genome, such as at 1 location or 16, or 32, or 64, or 100, or 500, or 1,000 or more locations where a given read maps to in the genome. However, any individual read was derived, e.g., sequenced, from only one specific portion of the genome. Hence, in order to find the true location from where a given particular read was d, an alignment function may be performed, e.g., a Smith- an gapped or gapless alignment, a Needleman-Wunsch alignment, etc., so as to determine where in the genome one or more of the reads was actually derived, such as by comparing all of the possible locations where a match occurs and determining which of all the possibilities is the most likely location in the genome from which the read was sequenced, on the basis ofwhich location's alignment score is greatest.

As indicated, typically, an algorithm is used to perform such an alignment function. For example, a Smith-Waterman and/or a Needleman-Wunsch alignment thm may be employed to align two or more sequences against one another. In this instance, they may be employed in a manner so as to determine the probabilities that for any given position where the read maps to the reference genome that the mapping is in fact the position from where the read originated. Typically these algorithms are configured so as to be performed by software, however, in various instances, such as herein presented, one or more of these algorithms can be configured so as to be executed in hardware, as bed in greater detail herein below.

In particular, the alignment function operates, at least in part, to align one or more, e.g., all, of the reads to the reference genome despite the presence of one or more portions of mismatches, e.g., SNPs, ions, deletions, structural cts, etc. so as to determine where the reads are likely to fit in the genome correctly. For instance, the one or more reads are compared against the reference , and the best possible fit for the read against the genome is determined, while accounting for substitutions and/or Indels and/or structural variants. However, to better determine which of the modified versions of the read best fits against the reference genome, the proposed changes must be accounted for, and as such a scoring on may also be performed.

For example, a scoring function may be med, e.g., as part of an overall alignment function, whereby as the alignment module performs its function and introduces one or more changes into a sequence being compared to another, e.g., so as to achieve a better or best fit between the two, for each change that is made so as to achieve the better alignment, a number is ted from a starting score, e.g., either a perfect score, or a zero starting score, in a manner such that as the alignment is performed the score for the alignment is also determined, such as where matches are detected the score is increased, and for each change introduced a penalty is incurred, and thus, the best fit for the possible alignments can be determined, for example, by ng out which of all the possible modified reads fits to the genome with the highest score. Accordingly, in various instances, the alignment function may be configured to determine the best combination of changes that need to be made to the read(s) to achieve the t scoring alignment, which alignment may then be determined to be the correct or most likely alignment.

In view of the above, there are, therefore, at least two goals that may be achieved from performing an alignment function. One is a report of the best ent, ing position in the reference genome and a ption ofwhat changes are necessary to make the read match the reference segment at that position, and the other is the alignment quality score. For instance, in various instances, the output from the alignment module may be a Compact Idiosyncratic Gapped Alignment Report, e.g., a CIGAR string, wherein the CIGAR string output is a report detailing all the s that were made to the reads so as to e their best fit alignment, e.g., detailed alignment instructions indicating how the query actually aligns with the reference. Such a CIGAR string readout may be useful in further stages essing so as to better determine that for the given subject's genomic nucleotide sequence, the predicted variations as compared against a reference genome are in fact true variations, and not just due to e, re, or human error.

As set forth above, in s embodiments, alignment is typically performed in a sequential manner, wherein the algorithm and/or firmware receives read sequence data, such as from a mapping module, pertaining to a read and one or more possible locations where the read may potentially map to the one or more nce genomes, and further receives genomic sequence data, such as from one or more memories, such as associated DRAMs, pertaining to the one or more positions in the one or more reference genomes to which the read may map. In particular, in vanous embodiments, the mapping module processes the reads, such as from a FASTQ file, and maps each of them to one or more positions in the reference genome to where they may possibly align. The aligner then takes these predicted positions and uses them to align the reads to the reference genome, such as by building a virtual array by which the reads can be ed with the reference genome.

In performing this function the aligner evaluates each mapped position for each individual read and particularly evaluates those reads that map to le possible locations in the reference genome and scores the possibility that each position is the correct position. It then compares the best scores, e.g., the two best scores, and makes a decision as to where the particular read actually aligns. For instance, in comparing the first and second best alignment scores, the aligner looks at the difference between the scores, and if the difference between them is great, then the confidence score that the one with the bigger score is correct will be high. However, where the ence between them is small, e.g., zero, then the confidence score in being able to tell from which of the two positions the read actually is derived is low, and more processing may be useful in being able to clearly determine the true location in the reference genome from where the read is derived.

Hence, the aligner in part is looking for the biggest difference n the first and second best confidence scores in making its call that a given read maps to a given location in the reference . y, the score ofthe best possible choice nment is icantly greater than the score for the second best alignment for that sequence. There are many different ways an ent scoring methodology may be implemented, for instance, each cell of the array may be scored or a rtion of cells may be scored, such as in accordance with the methods disclosed herein. In various instances, scoring parameters for nucleotide matches, nucleotide mismatches, insertions, and deletions may have any various positive or negative or zero values. In various instances, these scoring parameters may be modified based on available information. For instance, accurate alignments may be achieved by making scoring ters, including any or all of nucleotide match scores, nucleotide mismatch , gap (insertion and/or deletion) penalties, gap open penalties, and/or gap extend penalties, vary according to a base quality score associated with the current read nucleotide or position. For e, score bonuses and/or penalties could be made smaller when a base y score indicates a high probability a cing or other error being present. Base quality sensitive scoring may be implemented, for example, using a fixed or configurable lookup-table, accessed using a base quality score, which returns corresponding scoring parameters.

In a hardware implementation in an integrated circuit, such as an FPGA or ASIC, a scoring wave front may be implemented as a linear array of scoring cells, such as 16 cells, or 32 cells, or 64 cells, or 128 cells or the like. Each ofthe scoring cells may be built of digital logic ts in a wired configuration to compute alignment scores. Hence, for each step of the wave front, for instance, each clock cycle, or some other fixed or variable unit of time, each ofthe scoring cells, or a portion ofthe cells, computes the score or scores required for a new cell in the virtual alignment matrix. Notionally, the various scoring cells are considered to be in various positions in the alignment matrix, corresponding to a scoring wave front as discussed herein, e.g., along a straight line extending from bottom-left to topright in the matrix. As is well understood in the field of digital logic design, the physical scoring cells and their comprised digital logic need not be physically arranged in like manner on the integrated circuit.

Accordingly, as the wave front takes steps to sweep through the virtual alignment matrix, the notional ons ofthe g cells correspondingly update each cell, for example, notionally "moving" a step to the right, or for e, a step downward in the ent matrix. All scoring cells make the same relative notional movement, g the diagonal wave front arrangement intact. Each time the wave front moves to a new position, e.g., with a vertical downward step, or a horizontal rightward step in the matrix, the scoring cells arrive in new notional positions, and compute alignment scores for the virtual alignment matrix cells they have d. In such an entation, oring scoring cells in the linear array are coupled to communicate query (read) nucleotides, reference nucleotides, and usly calculated alignment scores. The nucleotides of the reference window may be fed sequentially into one end of the wave front, e.g., the top-right scoring cell in the linear array, and may shift from there sequentially down the length of the wave front, so that at any given time, a segment of reference nucleotides equal in length to the number of scoring cells is present within the cells, one successive nucleotide in each successive scoring cell.

For ce, each time the wave front steps horizontally, another reference nucleotide is fed into the top-right cell, and other reference nucleotides shift down-left h the wave front. This shifting of reference tides may be the underlying reality of the notional movement of the wave front of scoring cells rightward through the ent matrix. Hence, the nucleotides of the read may be fed sequentially into the opposite end of the wave front, e.g. the bottom-left scoring cell in the linear array, and shift from there sequentially up the length of the wave front, so that at any given time, a segment of query nucleotides equal in length to the number of scoring cells is present within the cells, one successive nucleotide in each successive scoring cell. Likewise, each time the wave front steps vertically, another query nucleotide is fed into the bottom-left cell, and other query nucleotides shift up-right through the wave front. This shifting of query nucleotides is the underlying reality of the notional movement of the wave front of scoring cells downward through the alignment matrix. Accordingly, by commanding a shift of reference nucleotides, the wave front may be moved a step horizontally, and by commanding a shift of query nucleotides, the wave front may be moved a step vertically. Hence, to produce generally diagonal wave front movement, such as to follow a l alignment of query and reference ces without insertions or deletions, wave front steps may be commanded in alternating vertical and horizontal directions. ingly, oring scoring cells in the linear array may be coupled to communicate previously calculated alignment scores. In various alignment scoring algorithms, such as a Smith-Waterman or Needleman-Wunsch, or such variant, the alignment score(s) in each cell of the l alignment matrix may be calculated using previously calculated scores in other cells ofthe matrix, such as the three cells positioned immediately to the left of the current cell, above the current cell, and diagonally up-left of the current cell.

When a scoring cell calculates new score(s) for another matrix position it has entered, it must retrieve such previously calculated scores corresponding to such other matrix positions.

These previously calculated scores may be obtained from e of previously calculated scores within the same cell, and/or from storage ofpreviously calculated scores in the one or two neighboring scoring cells in the linear array. This is because the three contributing score positions in the l alignment matrix (immediately left, above, and ally up-left) would have been scored either by the current scoring cell, or by one of its neighboring scoring cells in the linear array.

For instance, the cell ately to the left in the matrix would have been scored by the t scoring cell, if the most recent wave front step was horizontal (rightward), or would have been scored by the neighboring cell down-left in the linear array, if the most recent wave front step was vertical (downward). Similarly, the cell ately above in the matrix would have been scored by the current scoring cell, if the most recent wave front step was vertical ard), or would have been scored by the neighboring cell up-right in the linear array, if the most recent wave front step was horizontal (rightward).

Particularly, the cell diagonally up-left in the matrix would have been scored by the current scoring cell, if the most recent two wave front steps were in ent directions, e.g., down then right, or right then down, or would have been scored by the neighboring cell up-right in the linear array, if the most recent two wave front steps were both horizontal (rightward), or would have been scored by the neighboring cell down-left in the linear array, if the most recent two wave front steps were both vertical (downward). ingly, by ering information on the last one or two wave front step ions, a scoring cell may select the appropriate previously calculated scores, accessing them within itself, and/or within neighboring scoring cells, utilizing the ng between neighboring cells. In a variation, scoring cells at the two ends ofthe wave front may have their outward score inputs hard-wired to invalid, or zero, or minimum-value scores, so that they will not affect new score calculations in these e cells. A wave front being thus implemented in a linear array of scoring cells, with such coupling for shifting reference and query nucleotides through the array in opposing ions, in order to notionally move the wave front in vertical and horizontal, e.g., diagonal, steps, and coupling for accessing scores previously computed by neighboring cells in order to compute alignment score(s) in new l matrix cell positions entered by the wave front, it is accordingly possible to score a band of cells in the virtual matrix, the width of the wave front, such as by commanding successive steps ofthe wave front to sweep it through the matrix.

For a new read and reference window to be aligned, therefore, the wave front may begin positioned inside the scoring matrix, or, advantageously, may gradually enter the scoring matrix from outside, beginning e.g., to the left, or above, or diagonally left and above the top-left comer of the matrix. For instance, the wave front may begin with its top-left scoring cell positioned just left of the top-left cell of the virtual matrix, and the wave front may then sweep rightward into the matrix by a series zontal steps, scoring a horizontal band of cells in the top-left region of the . When the wave front reaches a predicted alignment relationship between the reference and query, or when matching is detected from increasing alignment scores, the wave front may begin to sweep diagonally down-right, by alternating vertical and horizontal steps, scoring a diagonal band of cells through the middle of the matrix. When the bottom-left wave front scoring cell reaches the bottom of the alignment matrix, the wave front may begin sweeping rightward again by successive horizontal steps, until some or all wave front cells sweep out of the boundaries of the alignment matrix, scoring a horizontal band s in the bottom-right region ofthe matrix.

One or more of such alignment procedures may be performed by any suitable alignment algorithm, such as a Needleman-Wunsch ent algorithm and/or a Smith- Waterman alignment algorithm that may have been modified to accommodate the functionality herein described. In general both of these algorithms and those like them basically perform, in some instances, in a r manner. For instance, as set forth above, these alignment algorithms typically build the virtual array in a similar manner such that, in various instances, the horizontal top boundary may be configured to ent the genomic reference sequence, which may be laid out across the top row of the array according to its base pair composition. se, the vertical boundary may be ured to represent the ced and mapped query sequences that have been positioned in order, downwards along the first column, such that their nucleotide sequence order is generally matched to the nucleotide sequence of the reference to which they mapped. The intervening cells may then be populated with scores as to the probability that the relevant base of the query at a given position, is positioned at that location relative to the reference. In performing this function, a swath may be moved diagonally across the matrix populating scores within the intervening cells and the probability for each base of the query being in the indicated position may be determined.

With respect to a Needleman-Wunsch alignment function, which generates optimal global (or semi-global) alignments, aligning the entire read sequence to some segment of the reference genome, the wave front steering may be configured such that it typically sweeps all the way from the top edge of the ent matrix to the bottom edge.

When the wave front sweep is complete, the maximum score on the bottom edge of the alignment matrix sponding to the end of the read) is selected, and the alignment is back-traced to a cell on the top edge of the matrix (corresponding to the beginning of the read). In various of the ces disclosed herein, the reads can be any length long, can be any size, and there need not be extensive read parameters as to how the alignment is med, e.g., in various instances, the read can be as long as a chromosome. In such an instance, however, the memory size and chromosome length may be limiting factor.

With respect to a Waterman algorithm, which generates l local alignments, aligning the entire read sequence or part ofthe read sequence to some segment of the reference genome, this algorithm may be configured for finding the best scoring possible based on a full or partial alignment of the read. Hence, in various instances, the wave frontscored band may not extend to the top and/or bottom edges ofthe alignment , such as if a very long read had only seeds in its middle mapping to the reference , but commonly the wave front may still score from top to bottom ofthe matrix. Local alignment is typically achieved by two adjustments. First, ent scores are never d to fall below zero (or some other floor), and if a cell score otherwise calculated would be negative, a zero score is substituted, representing the start of a new ent. Second, the maximum alignment score produced in any cell in the , not necessarily along the bottom edge, is used as the terminus ofthe alignment. The alignment is backtraced from this maximum score up and left through the matrix to a zero score, which is used as the start on of the local alignment, even if it is not on the top row ofthe matrix.

In view ofthe above, there are several different possible pathways through the virtual array. In various embodiments, the wave front starts from the upper left comer of the virtual array, and moves downwards towards identifiers of the m score. For instance, the results of all le aligns can be gathered, processed, correlated, and scored to determine the maximum score. When the end of a boundary or the end of the array has been reached and/or a computation leading to the highest score for all of the processed cells is ined (e.g., the overall highest score identified) then a backtrace may be performed so as to find the pathway that was taken to achieve that highest score. For example, a pathway that leads to a predicted maximum score may be identified, and once identified an audit may be performed so as to determine how that maximum score was derived, for instance, by moving backwards following the best score alignment arrows retracing the pathway that led to achieving the identified maximum score, such as calculated by the wave front scoring cells.

This backwards reconstruction or backtrace involves starting from a determined m score, and working backward through the previous cells navigating the path of cells having the scores that led to achieving the maximum score all the way up the table and back to an initial boundary, such as the beginning ofthe array, or a zero score in the case of local alignment. During a backtrace, having d a particular cell in the alignment matrix, the next backtrace step is to the neighboring cell, immediately leftward, or above, or diagonally up-left, which contributed the best score that was selected to uct the score in the current cell. In this manner, the evolution of the m score may be determined, thereby figuring out how the maximum score was achieved. The backtrace may end at a comer, or an edge, or a boundary, or may end at a zero score, such as in the upper left hand comer of the array. ingly, it is such a back trace that identifies the proper alignment and thereby produces the CIGAR strand readout that represents how the sample genomic sequence d from the individual, or a portion thereof, matches to, or otherwise aligns with, the genomic sequence ofthe reference DNA.

Once it has been determined where each read is mapped, and further determined where each read is aligned, e.g., each relevant read has been given a position and a quality score reflecting the probability that the position is the correct alignment, such that the nucleotide sequence for the subject's DNA is known, then the order of the s reads and/or genomic nucleic acid sequence ofthe subject may be verified, such as by performing a back trace function moving backwards up h the array so as to determine the identity of every nucleic acid in its proper order in the sample genomic sequence. Consequently, in some aspects, the present disclosure is directed to a back trace function, such as is part of an alignment module that performs both an alignment and a back trace function, such as a module that may be part of a pipeline of modules, such as a pipeline that is directed at taking raw sequence read data, such as form a genomic sample form an individual, and mapping and/or aligning that data, which data may then be sorted.

To facilitate the backtrace operation, it is useful to store a scoring vector for each scored cell in the alignment matrix, encoding the selection on. For classical Smith-Waterman and/or Needleman-Wunsch scoring implementations with linear gap penalties, the g vector can encode four possibilities, which may optionally be stored as a 2-bit integer from 0 to 3, for e: 0=new alignment (null score selected); 1=vertical alignment (score from the cell above selected, modified by gap y); 2=horizontal alignment (score from the cell to the left selected, modified by gap penalty); 3=diagonal alignment (score from the cell up and left selected, modified by nucleotide match or mismatch score). Optionally, the ed score(s) for each scored matrix cell may also be stored (in addition to the maximum ed alignment score which is standardly stored), but this is not generally necessary for backtrace, and can consume large amounts of memory.

Performing backtrace then s a matter of following the scoring vectors; when the backtrace has reached a given cell in the matrix, the next backtrace step is determined by the stored scoring vector for that cell, e.g.: 0=terminate backtrace; 1=backtrace upward; 2=backtrace leftward; trace ally up-left.

Such sconng vectors may be stored in a two-dimensional table arranged according to the dimensions of the alignment matrix, wherein only entries corresponding to cells scored by the wave front are populated. Alternatively, to conserve memory, more easily record g vectors as they are ted, and more easily accommodate alignment matrices of s sizes, scoring vectors may be stored in a table with each row sized to store scoring vectors from a single wave front of scoring cells, e.g. 128 bits to store 64 2-bit scoring vectors from a 64-cell wave front, and a number of rows equal to the maximum number of wave front steps in an alignment operation. Additionally, for this option, a record may be kept ofthe ions ofthe various wavefront steps, e.g., storing an extra, e.g., 129th, bit in each table row, encoding e.g., 0 for vertical wavefront step preceding this wavefront position, and 1 for horizontal wavefront step preceding this wavefront position. This extra bit can be used during backtrace to keep track of which virtual scoring matrix positions the scoring vectors in each table row correspond to, so that the proper scoring vector can be retrieved after each successive backtrace step. When a backtrace step is vertical or horizontal, the next scoring vector should be retrieved from the previous table row, but when a backtrace step is al, the next g vector should be retrieved from two rows previous, because the wavefront had to take two steps to move from scoring any one cell to scoring the cell diagonally down from it.

] In the case of affine gap scoring, scoring vector information may be extended, e.g. to 4 bits per scored cell. In addition to the e.g., 2-bit score-choice direction indicator, two I-bit flags may be added, a vertical extend flag, and a horizontal extend flag. According to the methods of affine gap scoring extensions to Smith-Waterman or Needleman-Wunsch or similar alignment algorithms, for each cell, in addition to the primary alignment score representing the best-scoring alignment terminating in that cell, a cal score' should be generated, corresponding to the maximum alignment score reaching that cell with a final al step, and a 'horizontal score' should be generated, corresponding to the maximum alignment score reaching that cell with a final horizontal step; and when computing any of the three scores, a al step into the cell may be computed either using the primary score from the cell above minus a gap-open y, or using the vertical score from the cell above minus a gap-extend y, whichever is greater; and a horizontal step into the cell may be computed either using the primary score from the cell to the left minus a gap-open y, or using the horizontal score from the cell to the left minus a gap-extend penalty, whichever is greater. In cases where the vertical score minus a gap extend penalty is selected, the vertical extend flag in the scoring vector should be set, e.g. '1', and otherwise it should be unset, e.g.

In cases when the horizontal score minus a gap extend penalty is selected, the horizontal extend flag in the scoring vector should be set, e.g. '1', and otherwise it should be unset, e.g. '0'.During backtrace for affine gap scoring, any time backtrace takes a vertical step upward from a given cell, if that cell's g vector's al extend flag is set, the following backtrace step must also be vertical, regardless of the scoring vector for the cell above. Likewise, any time backtrace takes a horizontal step leftward from a given cell, if that cell's scoring vector's horizontal extend flag is set, the following ace step must also be horizontal, regardless ofthe scoring vector for the cell to the left. Accordingly, such a table of scoring vectors, e.g. 129 bits per row for 64 cells using linear gap scoring, or 257 bits per row for 64 cells using affine gap scoring, with some number NR of rows, is adequate to t ace after concluding alignment scoring where the scoring ont took NR steps or fewer.

For example, when aligning 300-nucleotide reads, the number of wavefront steps required may always be less than 1024, so the table may be 257x1024 bits, or approximately 32 kilobytes, which in many cases may be a reasonable local memory inside the integrated circuit. But if very long reads are to be aligned, e.g. 100,000 nucleotides, the memory requirements for scoring vectors may be quite large, e.g. 8 megabytes, which may be very costly to include as local memory inside the integrated circuit. For such support, scoring vector information may be recorded to bulk memory outside the integrated circuit, e.g.

DRAM, but then the bandwidth requirements, e.g. 257 bits per clock cycle per aligner module, may be excessive, which may bottleneck and dramatically reduce r performance. Accordingly, it is desirable to have a method for disposing of scoring vectors before completing ent, so their e requirements can be kept bounded, e.g. to perform incremental backtraces, generating incremental partial CIGAR strings for example, from early portions of an alignment's g vector y, so that such early portions ofthe scoring vectors may then be discarded. The challenge is that the backtrace is supposed to begin in the alignment's terminal, maximum scoring cell, which n until the alignment scoring completes, so any backtrace begun before alignment completes may begin from the wrong cell, not along the eventual final optimal alignment path.

Hence, a method is given for performing incremental backtrace from partial alignment information, e.g., sing partial scoring vector information for alignment matrix cells scored so far. From a currently completed alignment boundary, e.g., a particular scored wave front position, backtrace is initiated from all cell positions on the boundary.

Such backtrace from all boundary cells may be performed sequentially, or advantageously, especially in a hardware implementation, all the backtraces may be performed together. It is not necessary to extract alignment notations, e.g., CIGAR strings, from these multiple backtraces; only to determine what alignment matrix positions they pass through during the backtrace. In an implementation of simultaneous backtrace from a scoring boundary, a number of I-bit registers may be utilized, corresponding to the number of alignment cells, initialized e.g., all to '1 's, representing whether any of the backtraces pass through a corresponding position. For each step of simultaneous backtrace, scoring vectors corresponding to all the current '1 'sin these registers, e.g. from one row ofthe g vector table, can be examined, to determine a next ace step corresponding to each '1'in the registers, leading to a following position for each '1' in the registers, for the next simultaneous backtrace step.

Importantly, it is easily possible for multiple '1 'sinthe registers to merge into common ons, ponding to multiple of the simultaneous backtraces merging together onto common backtrace paths. Once two or more of the simultaneous backtraces merge together, they remain merged indefinitely, because henceforth they will utilize scoring vector ation from the same cell. It has been observed, empirically and for theoretical reasons, that with high probability, all of the simultaneous aces merge into a singular backtrace path, in a relatively small number of ace steps, which e.g. may be a small le, e.g. 8, times the number of scoring cells in the wavefront. For example, with a 64- cell wavefront, with high probability, all backtraces from a given wavefront boundary merge into a single backtrace path within 512 backtrace steps. Alternatively, it is also possible, and not uncommon, for all backtraces to terminate within the number, e.g. 512, of backtrace steps.

Accordingly, the multiple simultaneous backtraces may be performed from a g boundary, e.g. a scored wavefront on, far enough back that they all either terminate or merge into a single ace path, e.g. in 512 backtrace steps or fewer. If they all merge together into a ar backtrace path, then from the location in the scoring matrix where they merge, or any distance further back along the singular backtrace path, an incremental backtrace from partial alignment information is possible. Further backtrace from the merge point, or any ce further back, is commenced, by normal singular backtrace methods, ing ing the corresponding ent notation, e.g., a partial CIGAR string. This incremental backtrace, and e.g., partial CIGAR string, must be part of any possible final backtrace, and e.g., full CIGAR string, that would result after alignment completes, unless such final backtrace would terminate before reaching the scoring boundary where simultaneous backtrace began, because if it reaches the scoring boundary, it must follow one of the simultaneous backtrace paths, and merge into the singular backtrace path, now incrementally extracted.

Therefore, all scoring vectors for the matrix regions corresponding to the incrementally extracted backtrace, e.g., in all table rows for wave front positions preceding the start of the extracted singular backtrace, may be safely discarded. When the final backtrace is med from a maximum scoring cell, if it terminates before reaching the scoring boundary (or alternatively, if it terminates before reaching the start of the extracted singular backtrace), the incremental alignment notation, e.g. partial CIGAR string, may be discarded. If the final backtrace continues to the start of the extracted singular backtrace, its alignment notation, e.g., CIGAR string, may then be grafted onto the incremental alignment notation, e.g., partial CIGAR string. Furthermore, in a very long alignment, the process of performing a simultaneous ace from a scoring boundary, e.g., scored wave front on, until all backtraces terminate or merge, followed by a singular backtrace with alignment notation extraction, may be repeated multiple times, from various successive scoring boundaries. The incremental alignment notation, e.g. partial CIGAR string, from each successive incremental backtrace may then be grafted onto the accumulated us alignment notations, unless the new simultaneous ace or singular backtrace terminates early, in which case accumulated previous ent notations may be ded. The eventual final backtrace likewise grafts its alignment notation onto the most recent accumulated alignment notations, for a complete backtrace description, e.g., CIGAR string.

Accordingly, in this , the memory to store scoring vectors may be kept bounded, ng aneous backtraces always merge er in a bounded number of steps, e.g. 512 steps. In rare cases where simultaneous backtraces fail to merge or terminate in the d number of steps, various exceptional actions may be taken, including failing the current alignment, or repeating it with a higher bound or with no bound, perhaps by a different or traditional method, such as storing all scoring vectors for the complete ent, such as in external DRAM. In a variation, it may be reasonable to fail such an alignment, because it is extremely rare, and even rarer that such a failed alignment would have been a best-scoring alignment to be used in alignment reporting.

In an optional variation, scoring vector storage may be divided, physically or logically, into a number ofdistinct blocks, e.g. 512 rows each, and the final row in each block may be used as a scoring boundary to ce a simultaneous backtrace. Optionally, a simultaneous ace may be required to ate or merge within the single block, e.g. 512 steps. Optionally, if simultaneous backtraces merge in fewer steps, the merged backtrace may nevertheless be continued through the whole block, before commencing an extraction of a singular backtrace in the previous block. Accordingly, after scoring vectors are fully written to block N, and begin writing to block N+1, a simultaneous backtrace may commence in block N, followed by a singular backtrace and ent on extraction in block N-1. If the speed of the simultaneous backtrace, the singular backtrace, and alignment scoring are all r or identical, and can be performed simultaneously, e.g., in parallel hardware in an integrated circuit, then the singular backtrace in block N-1 may be simultaneous with scoring vectors filling block N+2, and when block N+3 is to be filled, block N-1 may be released and recycled.

Thus, in such an implementation, a minimum of 4 scoring vector blocks may be employed, and may be utilized cyclically. Hence, the total scoring vector storage for an aligner module may be 4 blocks of 257 x 512 bits each, for example, or approximately 64 kilobytes. In a variation, if the current m alignment score ponds to an earlier block than the current wavefront position, this block and the previous block may be preserved rather than recycled, so that a final ace may commence from this position if it remains the maximum score; having an extra 2 blocks to keep preserved in this manner brings the minimum, e.g., to 6 blocks.

In r variation, to support overlapped alignments, the g wave front crossing gradually from one alignment matrix to the next as described above, additional blocks, e.g. 1 or 2 additional , may be utilized, e.g., 8 blocks total, e.g., approximately 128 kilobytes. Accordingly, if such a limited number of blocks, e.g., 4 blocks or 8 blocks, is used cyclically, alignment and backtrace of arbitrarily long reads is possible, e.g., 100,000 nucleotides, or an entire chromosome, without the use of external memory for scoring vectors. It is to be understood, such as with reference to the above, that although a mapping function may in some instances have been described, such as with reference to a mapper, and/or an ent function may have in some ces been described, such as with reference to an aligner, these ent functions may be performed sequentially by the same architecture, which has ly been referenced in the art as an aligner. Accordingly, in various instances, both the mapping function and the aligning on, as herein described may be performed by a common architecture that may be understood to be an aligner, especially in those instances wherein to perform an alignment function, a g function need first be performed.

In various ces, the devices, systems, and their methods of use of the t sure may be configured for performing one or more of a full-read gapless and/or gapped alignments that may then be scored so as to determine the appropriate alignment for the reads in the dataset. For instance, in various instances, a gapless alignment procedure may be performed on data to be processed, which gapless alignment procedure may then be ed by one or more of a gapped alignment, and/or by a selective Smith- an alignment procedure. For instance, in a first step, a gapless alignment chain may be generated. As described herein, such gapless alignment functions may be performed quickly, such as without the need for accounting for gaps, which after a first step of performing a gapless alignment, may then be followed by then performing a gapped alignment.

For example, an ent function may be performed in order to ine how any given nucleotide sequence, e.g., read, aligns to a reference sequence without the need for inserting gaps in one or more of the reads and/or refemce. An important part of performing such an alignment function is determining where and how there are mismatches in the sequence in on versus the sequence of the reference genome. However, because ofthe great homology within the human genome, in theory, any given nucleotide sequence is going to largely match a representative reference ce. Where there are mismatches, these will likely be due to a single nucleotide polymorphism, which is relatively easy to detect, or they will be due to an insertion or deletion in the sequences in question, which are much more difficult to detect.

] Consequently, in performing an alignment function, the majority of the time, the ce in question is going to match the reference sequence, and where there is a mismatch due to an SNP, this will easily be determined. Hence, a relatively large amount of processing power is not required to m such analysis. Difficulties arise, however, where there are insertions or deletions in the sequence in question with respect to the reference sequence, because such insertions and deletions amount to gaps in the alignment. Such gaps require a more extensive and complicated processing platform so as to determine the correct alignment. Nevertheless, because there will only be a small percentage of , only a relatively smaller percentage of gapped alignment ols need be performed as ed to the millions of gapless alignments performed. Hence, only a small percentage of all of the gapless alignment functions result in a need for further processing due to the presence of an indel in the sequence, and therefore will need a gapped ent.

When an indel is indicated in a gapless alignment procedure, only those sequences get passed on to an alignment engine for r processing, such as an alignment engine configured for performing an advanced alignment on, such as a Smith Waterman alignment (SWA). Thus, because either a s or a gapped alignment is to be performed, the devices and systems disclosed herein are a much more efficient use of resources. More particularly, in certain embodiments, both a gapless and a gapped alignment may be performed on a given selection of sequences, e.g., one right after the other, then the results are compared for each sequence, and the best result is chosen. Such an ement may be implemented, for instance, where an enhancement in accuracy is desired, and an increased amount oftime and resources for performing the required processing is acceptable.

Particularly, in various instances, a first alignment step may be performed without engaging a processing intensive Smith Waterman function. Hence, a plurality of gapless ents may be performed in a less resource intensive, less time-consuming manner, and because less resources are needed less space need be dedicated for such processing on the chip. Thus, more processing may be performed, using less processing elements, requiring less time, therefore, more alignments can be done, and better accuracy can be achieved. More particularly, less chip resource-implementations for performing Smith Waterman ents need be dedicated using less chip area, as it does not require as much chip area for the processing elements required to perform gapless alignments as it does for performing a gapped alignment. As the chip resource requirements go down, the more processing can be med in a shorter period of time, and with the more processing that can be med, the better the accuracy can be achieved.

Accordingly, in such instances, a gapless alignment protocol, e.g., to be performed by suitably configured gapless alignment resources, may be employed. For e, as disclosed herein, in s embodiments, an alignment processing engine is provided such as where the processing engine is configured for ing digital signals, e.g., representing one or more reads of genomic data, such as digital data denoting one or more nucleotide sequences, from an electronic data source, and mapping and/or aligning that data to a reference sequence, such as by first performing a gapless alignment function on that data, which gapless alignment function may then be followed, if necessary, by a gapped alignment function, such as by performing a Smith Waterman alignment protocol.

Consequently, in various instances, a s alignment function is performed on a contiguous portion of the read, e.g., employing a s aligner, and if the gapless alignment goes from end to end, e.g., the read is te, a gapped alignment is not performed. However, if the s of the gapless alignment are indicative of their being an indel present, e.g., the read is clipped or otherwise incomplete, then a gapped alignment may be performed. Thus, the ungapped alignment results may be used to determine if a gapped ent is needed, for instance, where the ed alignment is extended into a gap region but does not extend the entire length of the read, such as where the read may be clipped, e.g., soft clipped to some , and where clipped then a gapped alignment may be performed.

Hence, in s embodiments, based on the completeness and alignment scores, it is only if the s alignment ends up being clipped, e.g., does not go end to end, that a gapped alignment is performed. More particularly, in various embodiments, the best identifiable gapless and/or gapped alignment score may be estimated and used as a cutoff line for deciding if the score is good enough to warrant further analysis, such as by performing a gapped alignment. Thus, the completeness ofalignment, and its score, may be employed such that a high score is indicative of the alignment being complete, and therefore, ungapped, and a lower score is indicative of the alignment not being complete, and a gapped alignment needing to be performed. Hence, where a high score is ed a gapped alignment is not performed, but only when the score is low enough is the gapped alignment performed. Of course, in various instances a brute force alignment ch may be employed such that the number of gapped and/or gapless aligners are deployed in the chip architecture, so as to allow for a greater number of ents to be performed, and thus a larger amount of data may be looked at.

More particularly, in various embodiments, each mapping and/or aligning engine may include one or more, e.g., two Smith-Waterman, aligner modules. In certain instances, these modules may be configured so as to support global (end-to-end) gapless alignment and/or local ed) gapped alignment, perform affine gap scoring, and can be configured for generating unclipped score s at each end. uality sensitive match and mismatch scoring may also be supported. Where two alignment modules are included, e.g., as part of the integrated circuit, for example, each Smith-Waterman aligner may be constructed as an iagonal wavefront of scoring cells, which wavefront 'moves'through a virtual alignment rectangle, scoring cells that it sweeps through.

However, for longer reads, the Smith-Waterman ont may also be configured to support automatic steering, so as to track the best alignment h accumulated indels, such as to ensure that the ent wavefront and cells being scored do not escape the g band. In the background, logic engines may be configured to examine current wavefront scores, find the maximums, flag the s of cells over a old distance below the maximum, and target the midpoint between the two extreme flags. In such an instance, auto-steering may be configured to run diagonally when the target is at the wavefront center, but may be configured to run straight ntally or vertically as needed to re-center the target if it drifts, such as due to the presence ofindels.

The output from the alignment module is a SAM (Text) or BAM (e.g., binary version of a SAM) file along with a mapping quality score (MAPA), which quality score reflects the confidence that the predicted and aligned location of the read to the reference is actually where the read is derived. Accordingly, once it has been determined where each read is mapped, and further ined where each read is aligned, e.g., each relevant read has been given a position and a quality score reflecting the probability that the position is the correct alignment, such that the tide sequence for the subject's DNA is known as well as how the subject's DNA differs from that of the reference (e.g., the CIGAR string has been determined), then the various reads representing the genomic nucleic acid sequence of the subject may be sorted by chromosome location, so that the exact location of the read on the chromosomes may be determined. Consequently, in some aspects, the present disclosure is directed to a sorting function, such as may be performed by a sorting module, which sorting module may be part of a pipeline of modules, such as a pipeline that is directed at taking raw sequence read data, such as form a c sample form an dual, and mapping and/or aligning that data, which data may then be sorted.

More particularly, once the reads have been assigned a position, such as ve to the reference genome, which may include identifying to which chromosome the read belongs and/or its offset from the beginning of that chromosome, the reads may be sorted by position. Sorting may be useful, such as in ream analyses, whereby all ofthe reads that overlap a given position in the genome may be formed into a pile up so as to be adjacent to one another, such as after being processed through the sorting module, whereby it can be readily determined if the majority of the reads agree with the reference value or not.

Hence, where the majority ofreads do not agree with the reference value a variant call can be flagged. Sorting, therefore, may involve one or more of sorting the reads that align to the relatively same position, such as the same chromosome position, so as to produce a pileup, such that all the reads that cover the same location are physically grouped together; and may further involve analyzing the reads of the pileup to determine where the reads may indicate an actual variant in the genome, as compared to the nce genome, which variant may be distinguishable, such as by the consensus ofthe pileup, from an error, such as a machine read error or error an error in the cing methods which may be exhibited by a small minority ofthe reads.

Once the data has been obtained there are one or more other modules that may be run so as to clean up the data. For instance, one module that may be included, for example, in a sequence analysis pipeline, such as for determining the genomic ce of an individual, may be a local realignment module. For example, it is often difficult to determine insertions and deletions that occur at the end ofthe read. This is because the Smith-Waterman or equivalent alignment s lacks enough context beyond the indel to allow the scoring to detect its ce. Consequently, the actual indel may be reported as one or more SNPs. In such an instance, the accuracy of the ted location for any given read may be enhanced by performing a local realignment on the mapped and/or aligned and/or sorted read data.

In such instances, pileups may be used to help clarify the proper alignment, such as where a position in on is at the end of any given read, that same position is likely to be at the middle of some other read in the pileup. ingly, in performing a local realignment the various reads in a pileup may be analyzed so as to determine if some of the reads in the pile up indicate that there was an insertion or a deletion at a given position where an other read does not include the indel, or rather es a substitution, at that position, then the indel may be ed, such as into the reference, where it is not present, and the reads in the local pileup that overlap that region may be realigned to see if collectively a better score is achieved then when the insertion and/or on was not there. Ifthere is an improvement, the whole set of reads in the pileup may be reviewed and if the score of the l set has improved then it is clear to make the call that there really was an indel at that position. In a manner such as this, the fact that there is not enough context to more accurately align a read at the end of a chromosome, for any individual read, may be compensated for. Hence, when performing a local realignment, one or more pileups where one or more indels may be positioned are examined, and it is ined if by adding an indel at any given position the overall alignment score may be enhanced. r module that may be included, for example, in a ce analysis pipeline, such as for determining the genomic sequence of an individual, may be a duplicate marking module. For instance, a duplicate marking function may be performed so as to compensate for chemistry errors that may occur during the sequencing phase. For example, as described above, during some sequencing procedures nucleic acid sequences are attached to beads and built up from there using labeled nucleotide bases. Ideally there will be only one read per bead. However, sometimes multiple reads become attached to a single bead and this results in an ive number of copies of the attached read. This phenomenon is known as read duplication.

] After an alignment is performed and the results obtained, and/or a sorting function, local realignment, and/or a de-duplication is performed, a variant call function may be employed on the resultant data. For instance, a typical variant call function or parts thereof may be configured so as to be implemented in a software and/or hardwired uration, such as on an integrated circuit. Particularly, t calling is a process that involves positioning all the reads that align to a given location on the reference into groupings such that all overlapping regions from all the various d reads form a "pile up." Then the pileup of reads covering a given region of the reference genome are ed to determine what the most likely actual content of the sampled dual's DNA/RNA is within that region. This is then repeated, step wise, for every region of the genome. The determined content tes a list of ences termed "variations" or "variants" from the reference genome, each with an associated confidence level along with other metadata.

The most common variants are single nucleotide polymorphisms (SNPs), in which a single base differs from the reference. SNPs occur at about 1 in 1000 positions in a human . Next most common are insertions (into the reference) and deletions (from the reference), or "indels" collectively. These are more common at shorter lengths, but can be of any length. Additional complications arise, however, because the collection of sequenced segments ("reads") is random, some regions will have deeper coverage than others. There are also more complex variants that e multi-base tutions, and combinations of indels and substitutions that can be thought of as length-altering substitutions. Standard software based variant callers have difficulty identifying all of these, and with various limits on variant lengths. More specialized variant callers in both software and/or hardware are needed to identify longer variations, and many varieties of exotic "structural variants" involving large alterations ofthe chromosomes. r, variant calling is a difficult procedure to ent in software, and worlds of magnitude more difficult to deploy in hardware. In order to account for and/or detect these types of errors, typical variant callers may perform one or more of the following tasks. For instance, they may come up with a set ofhypothesis pes (content of the one or two chromosomes at a , use Bayesian calculations to estimate the posterior probability that each genotype is the truth given the observed evidence, and report the most likely genotype along with its confidence level. As such variant callers may be simple or x. Simpler variant s look only at the column of bases in the aligned read pileup at the precise position of a call being made. More advanced variant s are "haplotype based callers", which may be configured to take into account context, such as in a window, around the call being made.

A "haplotype" is particular DNA content (nucleotide ce, list ofvariants, etc.) in a single common "strand", e.g. one oftwo diploid strands in a region, and a haplotype based caller considers the Bayesian implications ofwhich differences are linked by appearing in the same read. Accordingly, a variant call ol, as ed herein, may implement one or more improved functions such as those performed in a Genome Analysis Tool Kit (GATK) haplotype caller and/or using a Hidden Markov Model (HMM) tool and/or a De Bruijn Graph function, such as where one or more these functions typically employed by a GATK haplotype caller, and/or a HMM tool, and/or a De Bruijn Graph function may be implemented in software and/or in hardware.

More particularly, as implemented herein, vanous different variant call ions may be configured so as to be performed in software or hardware, and may include one or more ofthe following steps. For instance, variant call function may include an active region identification, such as for identifying places where multiple reads disagree with the reference, and for ting a window around the identified active region, so that only these regions may be selected for further processing. Additionally, localized haplotype ly may take place, such as where, for each given active region, all the overlapping reads may be led into a "De Bruijn graph" (DBG) matrix. From this DBG, various paths through the matrix may be extracted, where each path constitutes a ate haplotype, e.g., hypotheses, for what the true DNA sequence may be on at least one strand.

Further, haplotype alignment may take place, such as where each extracted haplotype candidate may be aligned, e.g., Smith-Waterman aligned, back to the reference genome, so as to ine what variation(s) from the reference it implies. rmore, a read likelihood calculation may be performed, such as where each read may be tested against each haplotype, or hypothesis, to estimate a probability of observing the read assuming the haplotype was the true original DNA sampled.

] With respect to these processes, the read likelihood calculation will typically be the most resource intensive and time ing operation to be performed, often requiring a pair HMM evaluation. Additionally, the ucting ofDe Bruijn graphs for each pileup of reads, with associated operations of identifying locally and globally unique , as described below may also be resource intensive and/or time consuming. Accordingly, m various ments, one or more of the various ations involved in ming one or more ofthese steps may be configured so as to be implemented in optimized software fashion or hardware, such as for being performed in an accelerated manner by an integrated circuit, as herein described.

As ted above, in vanous embodiments, a Haplotype Caller of the disclosure, implemented in software and/or in hardware or a combination thereof may be configured to include one or more of the following operations: Active Region Identification, zed Haplotype Assembly, Haplotype Alignment, Read Likelihood Calculation, and/or Genotyping. For instance, the devices, systems, and/or methods of the disclosure may be configured to perform one or more of a mapping, aligning, and/or a sorting operation on data obtained from a subject's sequenced A to generate mapped, aligned, and/or sorted results data. This results data may then be cleaned up, such as by performing a de duplication operation on it and/or that data may be communicated to one or more dedicated haplotype caller processing engines for performing a t call operation, including one or more ofthe entioned steps, on that results data so as to generate a variant call file with respect thereto. Hence, all the reads that have been sequenced and/or been mapped and/or aligned to particular positions in the reference genome may be ted to further processing so as to determine how the determined sequence differs from a reference sequence at any given point in the reference genome.

Accordingly, in various embodiments, a device, system, and/or method of its use, as herein disclosed, may include a variant or haplotype caller system that is implemented in a software and/or hardwired uration to perform an active region identification operation on the ed results data. Active region identification involves identifying and ining places where multiple reads, e.g., in a pile up of reads, disagree with a reference, and further es generating one or more windows around the disagreements ("active s") such that the region within the window may be selected for further processing. For example, during a mapping and/or aligning step, identified reads are mapped and/or aligned to the regions in the reference genome where they are expected to have ated in the t'sgenetic sequence.

However, as the sequencing is performed in such a manner so as to create an oversampling of sequenced reads for any given region ofthe genome, at any given position in the reference sequence may be seen a pile up of any and/ all of the sequenced reads that line up and align with that region. All ofthese reads that align and/or overlap in a given region or pile up position may be input into the t caller system. Hence, for any given read being analyzed, the read may be ed to the reference at its suspected region of overlap, and that read may be compared to the reference to determine if it shows any ence in its sequence from the known sequence of the reference. If the read lines up to the reference, without any insertions or deletions and all the bases are the same, then the alignment is determined to be good.

] Hence, for any given mapped and/or aligned read, the read may have bases that are different from the reference, e.g., the read may include one or more SNPs, creating a position where a base is mismatched; and/or the read may have one or more of an insertion and/or deletion, e.g., creating a gap in the alignment. Accordingly, in any of these instances, there will be one or more mismatches that need to be accounted for by further processing.

Nevertheless, to save time and increase efficiency, such further processing should be limited to those instances where a perceived mismatch is non-trivial, e.g., a non-noise difference. In determining the significance of a mismatch, places where multiple reads in a pile up ee from the reference may be identified as an active region, a window around the active region may then be used to select a locus of disagreement that may then be subjected to further processing. The disagreement, however, should be non-trivial. This may be determined in many ways, for instance, the non-reference probability may be calculated for each locus in question, such as by analyzing base match vs ch quality scores, such as above a given threshold deemed to be a sufficiently significant amount of indication from those reads that disagree with the reference in a significant way.

] For instance, if 30 of the mapped and/or aligned reads all line up and/or overlap so as to form a pile up at a given position in the reference, e.g., an active region, and only 1 or 2 out of the 30 reads disagrees with the reference, then the minimal threshold for r processing may be deemed to not have been met, and the non-agreeing read(s) can be disregarded in view of the 28 or 29 reads that do agree. However, if 3 or 4, or 5, or 10, or more of the reads in the pile up disagree, then the eement may be tically significant enough to warrant further processing, and an active region around the fied region(s) of difference might be determined. In such an instance, an active region window ascertaining the bases surrounding that difference may be taken to give enhanced context to the region surrounding the difference, and additional processing steps, such as performing a Gaussian distribution and sum of non-reference probabilities distributed across neighboring positions, may be taken to further investigate and process that region to figure out if and active region should be declared and if so what variances from the reference actually are present within that region if any. Therefore, the determining of an active region identifies those regions where extra processing may be needed to clearly ine if a true variance or a read error has occurred.

Particularly, because in many instances it is not desirable to subject every region in a pile up of sequences to further processing, an active region can be identified whereby it is only those regions where extra processing may be needed to clearly determine if a true variance or a read error has occurred that may be determined as needing of further processing. And, as ted above, it may be the size of the supposed variance that determines the size ofthe window of the active . For instance, in s ces, the bounds of the active window may vary from 1 or 2 or about 10 or 20 or even about 25 or about 50 to about 200 or about 300, or about 500 or about 1000 bases long or more, where it is only within the bounds of the active window that further processing is taking place. Of course, the size of the active window can be any suitable length so long as it provides the context to determine the tical importance ofa difference.

Hence, if there are only one or two isolated differences, then the active window may only need to cover one or more to a few dozen bases in the active region so as to have enough context to make a statistical call that an actual variant is present. However, if there is a r or a bunch of differences, or if there are indels present for which more context is desired, then the window may be configured so as to be larger. In either instance, it may be desirable to analyze any and all the differences that might occur in clusters, so as to analyze them all in one or more active regions, because to do so can provide supporting ation about each individual difference and will save processing time by decreasing the number of active s d. In various instances, the active region boundaries may be determined by active ilities that pass a given threshold, such as about 0.00001 or about 0.00001 or about 0.0001 or less to about 0.002 or about 0.02 or about 0.2 or more. And if the active region is longer than a given threshold, e.g., about 300 - 500 bases or 1000 bases or more, then the region can be broken up into sub-regions, such as by sub-regions defined by the locus with the lowest active probability score.

In various instances, after an active region is identified, a localized haplotype assembly ure may be performed. For instance, in each active region, all the piled up and/or overlapping reads may be assembled into a "De Bruijn Graph" (DBG). A DBG may be a directed graph based on all the reads that pped the selected active region, which active region may be about 200 or about 300 to about 400 or about 500 bases long or more, within which active region the presence and/or identity of variants are to be determined. In various instances, as indicated above, the active region can be extended, e.g., by including another about 100 or about 200 or more bases in each direction ofthe locus in question so as to generate an extended active region, such as where additional context surrounding a difference may be desired. ingly, it is from the active region window, extended or not, that all of the reads that have portions that overlap the active region are piled up, e.g., to produce a , the overlapping portions are identified, and the read sequences are threaded into the haplotype caller system and are thereby assembled together in the form ofa De Bruin graph, much like the pieces ofa puzzle.

Accordingly, for any given active window there will be reads that form a pile up such that en masse the pile up will include a sequence pathway through which the overlapping regions of the various overlapping reads in the pile up covers the entire sequence within the active window. Hence, at any given locus in the active region, there will be a plurality of reads overlapping that locus, albeit any given read may not extend the entire active region. The result of this is that various regions of various reads within a pileup are employed by the DBG in determining whether a variant ly is present or not for any given locus in the sequence within the active . As it is within the active window that this ination is being made, it is those portions of any given read within the borders of the active window that are considered, and those portions that are outside of the active window may be discarded.

As indicated, it is those sections of the reads that overlap the reference within the active region that are fed into the DBG system. The DBG system then assembles the reads like a puzzle into a graph, and then for each position in the sequence, it is determined based on the collection of overlapping reads for that position, r there is a match or a ch for any given, and if there is a mismatch, what the probability of that mismatch is.

For instance, where there are discrete places where segments of the reads in the pile up overlap each other, they may be aligned to one another based on their areas of matching, and from stringing or stitching the matching reads together, as determined by their points of matching, it can be ished for each position within that segment, whether and to what extent the reads at any given position match or mismatch each other. Hence, if two or more reads being compiled line up and match each other identically for a while, a graph having a single string will result; however, when the two or more reads come to a point ofdifference, a branch in the graph will form, and two or more divergent strings will result, until matching between the two or more reads resumes.

Hence, the ys through the graph are often not a straight line. For instance, where the k-mers of a read varies from the k-mers of the reference and/or the kmers from one or more overlapping reads, e.g., in the pileup, a "bubble" will be formed in the graph at the point of ence resulting in two divergent strings that will continue along two different path lines until matching n the two sequences resumes. Each vertex may be given a weighted score fying how many times the respective k-mers overlap in all ofthe reads in the pileup. Particularly, each pathway extending through the generated graph from one side to the other may be given a count. And where the same k-mers are generated from a licity of reads, e.g., where each k-mer has the same sequence pattern, they may be accounted for in the graph by increasing the count for that pathway where the k-mer ps an already existing k-mer pathway. Hence, where the same k-mer is generated from a multiplicity of overlapping reads having the same ce, the pattern of the pathway between the graph will be repeated over and over again and the count for traversing this pathway through the graph will be increased incrementally in correspondence therewith. In such an instance, the n is only recorded for the first instance ofthe k-mer, and the count is incrementally increased for each k-mer that s that pattern. In this mode the various reads in the pile up can be harvested to determine what variations occur and where.

In a manner such as this, a graph matrix may be formed by taking all possible N base k-mers, e.g., 10 base k-mers, which can be generated from each given read by sequentially g the length ofthe read in ten base segments, where the beginning of each new ten base segment is off set by one base from the last generated 10 base segment. This procedure may then be repeated by doing the same for every read in the pile up within the active window. The ted k-mers may then be aligned with one another such that areas of identical matching between the generated k-mers are matched to the areas where they overlap, so as to build up a data structure, e.g., graph, that may then be scanned and the percentage of matching and ching may be determined. Particularly, the reference and any usly processed k-mers aligned therewith may be scanned with respect to the next generated k-mer to determine if the instant generated k-mer matches and/or ps any portion of a previously generated k-mer, and where it is found to match the instant generated k-mer can then be inserted into the graph at the appropriate position.

Once built, the graph can be scanned and it may be determined based on this matching whether any given SNPs and/or indels in the reads with respect to the reference are likely to be an actual variation in the subject's genetic code or the result of a processing or other error. For instance, if all or a significant portion of the k-mers, of all or a significant portion of all of the reads, in a given region include the same SNP and/or indel mismatch, but differ from the reference in the same manner, then it may be determined that there is an actually SNP and/or indel variation in the subject's genome as ed to the nce genome. However, if only a limited number of k-mers from a limited number of reads evidence the ct, it is likely to be caused by machine and/or processing and/or other error and not indicative ofa true variation at the on in question.

As indicated, where there is a suspected variance, a bubble will be formed within the graph. Specifically, where all ofthe k-mers within all of a given region ofreads all match the reference, they will line up in such a manner as to form a linear graph. However, where there is a difference between the bases at a given locus, at that locus of difference that graph will branch. This ing may be at any position within the k-mer, and consequently at that point of difference the 10 base k-mer, including that difference, will diverge from the rest of the k-mers in the graph. In such an instance, a new node, forming a different pathway through the graph will be formed.

Hence, where everything may have been agreeing, e.g., the sequence in the given new k-mer being graphed is matching the ce to which it aligns in the graph, up to the point of difference the pathway for that k-mer will match the pathway for the graph lly and will be linear, but post the point of difference, a new pathway through the graph will emerge to accommodate the difference ented in the sequence of the newly graphed k-mer. This divergence being represented by a new node within the graph. In such an instance, any new k-mers to be added to the graph that match the newly divergent pathway will increase the count at that node. Hence, for every read that supports the arc, the count will be increased incrementally.

In various of such instances, the k-mer and/or the read it represents will once again start matching, e.g., after the point of divergence, such that there is now a point of convergence where the k-mer begins matching the main y through the graph represented by the k-mers of the reference sequence. For instance, naturally after a while the read(s) that support the ed node should rejoin the graph over time. Thus, over time, the k-mers for that read will rejoin the main pathway again. More particularly, for an SNP at a given locus within a read, the k-mer starting at that SNP will diverge from the main graph and will stay separate for about 10 nodes, because there are 10 bases per k-mer that overlap that locus of mismatching between the read and the nce. Hence, for an SNP, at the 11 th position, the k-mers covering that locus within the read will rejoin the main pathway as exact matching is resumed. Consequently, it will take ten shifts for the k-mers of a read having an SNP at a given locus to rejoin the main graph represented by the reference sequence.

As indicated above, there is typically one main path or line or backbone that is the reference path, and where there is a divergence a bubble is formed at a node where there is a difference between a read and the backbone graph. Thus there are some reads that diverge from the backbone and form a bubble, which divergence may be indicative of the presence of a t. As the graph is sed, bubbles within bubbles within bubbles may be formed along the reference backbone, so that they are stacked up and a plurality of pathways h the graph may be created. In such an instance, there may be a main path represented by the reference backbone, one path of a first divergence, and a further path of a second divergence within the first ence, all within a given window, each pathway through the graph may represent an actual variation or may be an artifact such as caused by sequencing error, and/or PCR error, and/or a processing error, and the like.

Once such a graph has been produced, it must be determined which pathways h the graph ent actual variations present within the sample genome and which are mere artifacts. Albeit, it is expected that reads containing handling or machine errors will not be supported by the majority of reads in the sample pileup, however, this is not always the case. For instance, errors in PCR processing may typically be the result of a cloning WO 14320 PCT/0S2017/036424 mistake that occurs when preparing the DNA , such mistakes tend to result in an insertion and/or a deletion being added to the cloned sequence. Such indel errors may be more consistent among reads, and can wind up with generating multiple reads that have the same error from this mistake in PCR cloning. Consequently, a higher count line for such a point ofdivergence may result because ofsuch errors.

Hence, once a graph matrix has been formed, with many paths through the graph, the next stage is to traverse and thereby extract all ofthe paths through the graph, e.g., left to right. One path will be the reference backbone, but there will be other paths that follow various bubbles along the way. All paths must be traversed and their count tabulated. For instance, if the graph includes a pathway with a two level bubble in one spot and a three level bubble in another spot, there will be (2 x 3)6 paths through that graph. So each of the paths will dually need to be extracted, which ted paths are termed as candidate haplotypes. Such candidate haplotypes represent es for what could really be representative of the subject's actual DNA that was sequenced, and the following processing steps, including one or more of haplotype alignment, read likelihood calculation, and/or genotyping may be employed to test these theories so as to find out the probabilities that anyone and/or each of these theories is correct. The implementation of a De Bruijn graph reconstruction therefore ents a way to reliably extract a good set ofhypotheses to test.

For instance, in ming a variant call function, as disclosed herein, an active region identification operation may be implemented, such as for identifying places where multiple reads in a pile up within a given region disagree with the reference, and for generating a window around the identified active region, so that only these regions may be selected for further processing. Additionally, localized haplotype assembly may take place, such as where, for each given active , all the pping reads in the pile up may be assembled into a "De Bruijn graph" (DBG) matrix. From this DBG, various paths h the matrix may be extracted, where each path constitutes a candidate haplotype, e.g., hypotheses, for what the true DNA sequence may be on at least one strand.

] Further, haplotype ent may take place, such as where each extracted haplotype candidate may be aligned, e.g., Smith-Waterman aligned, back to the reference genome, so as to determine what variation(s) from the reference it implies. Furthermore, a read likelihood calculation may be performed, such as where each read may be tested against each haplotype, to estimate a probability of observing the read assuming the haplotype was the true original DNA sampled. Finally, a ping operation may be implement, and a t call file ed. As indicated above, any or all of these operations may be configured so as to be implemented in an optimized manner in software and/or in re, and in various instances, because of the resource intensive and time consuming nature of building a DBG matrix and extracting candidate haplotypes therefrom, and/or because of the resource intensive and time consuming nature of performing a haplotype alignment and/or a read likelihood calculation, which may include the ment of an Hidden Markov Model (HMM) evaluation, these operations (e.g., localized haplotype assembly, and/or haplotype alignment, and/or read likelihood calculation) or a portion thereof may be configured so as to have one or more functions of their operation implemented in a hardwired form, such as for being performed in an accelerated manner by an integrated circuit as described . In various instances, these tasks may be configured to be implemented by one or more quantum circuits such as in a quantum computing device.

] Accordingly, in s instances, the devices, systems, and methods for performing the same may be ured so as to perform a haplotype alignment and/or a read likelihood calculation. For instance, as indicated, each extracted haplotype may be aligned, such as Smith-Waterman aligned, back to the reference genome, so as to determine what ion(s) from the nce it implies. In various exemplary ces, scoring may take place, such as in accordance with the following exemplary g parameters: a match = .0; a mismatch= -15.0; a gap open -26.0; and a gap extend= -1.1, other scoring parameters may be used. Accordingly, in this manner, a CIGAR strand may be generated and associated with the haplotype to produce an led haplotype, which assembled haplotype may eventually be used to identify variants. Accordingly, in a manner such as this, the likelihood of a given read being associated with a given haplotype may be calculated for all read/haplotype combinations. In such instances, the likelihood may be calculated using a Hidden Markov Model (HMM).

For instance, the various assembled haplotypes may be aligned in accordance with a dynamic programing model similar to a SW alignment. In such an instance, a virtual matrix may be generated such as where the candidate haplotype, e.g., generated by the DBG, may be positioned on one axis of a virtual array, and the read may be positioned on the other axis. The matrix may then be filled out with the scores ted by traversing the extracted paths through the graph and calculating the probabilities that any given path is the true path.

Hence, in such an ce, a difference in this alignment protocol from a typical SW ent protocol is that with respect to finding the most likely path through the array, a maximum likelihood calculation is used, such as a calculation performed by an HMM model that is configured to provide the total ility for alignment of the reads to the haplotype.

Hence, an actual CIGAR strand alignment, in this instance, need not be produced. Rather all possible alignments are considered and their possibilities are summed. The pair HMM evaluation is resource and time intensive, and thus, implementing its operations within a hardwired configuration within an integrated circuit or via quantum circuits on a quantum computing rm is very advantageous.

For example, each read may be tested against each candidate haplotype, so as to estimate a probability of observing the read assuming the ype is the true representative of the original DNA sampled. In various instances, this ation may be performed by ting a "pair hidden Markov model" (HMM), which may be configured to model the various possible ways the ype candidate might have been ed, such as by PCR or sequencing errors, and the like, and a variation introduced into the read observed.

In such instances, the HMM evaluation may employ a c programming method to calculate the total ility of any series of Markov state transitions arriving at the observed read in view of the possibility that any ence in the read may be the result of an error model. Accordingly, such HMM calculations may be configured to analyze all the possible SNPs and Indels that could have been introduced into one or more ofthe reads, such as by amplification and/or sequencing artifacts.

Particularly, paired HMM considers m a virtual matrix all the possible alignments of the read to the reference candidate haplotypes along with a probability associated with each of them, where all probabilities are added up. The sum of all of the probabilities of all the variants along a given path is added up to get one overarching probability for each read. This process is then performed for every pair, for every haplotype, read pair. For example, if there is a six pile up cluster overlapping a given region, e.g., a region of six haplotype candidates, and if the pile up includes about one hundred reads, 600 HMM operations will then need to be performed. More particularly, if there are 6 haplotypes then there are going to be 6 branches through the path and the probability that each one is the correct pathway that matches the subject's actual genetic code for that region must be calculated. Consequently, each y for all of the reads must be considered, and the probability for each read that you would arrive at this given haplotype is to be calculated.

] The pair Hidden Markov Model is an approximate model for how a true haplotype in the d DNA may transform into a possible different detected read. It has been ed that these types of transformations are a combination of SNPs and Indels that have been introduced into the genetic sample set by the PCR process, by one or more of the other sample preparation steps, and/or by an error caused by the sequencing process, and the like. As can be seen with respect to to account for these types of errors, an underlying e base model may be employed, such as where: (M = alignment match, I = insertion, D = deletion), further where any transition is possible except I <-> D.

As can be seen with respect to the 3-state base model transitions are not in a time sequence, but rather are in a sequence of progression through the candidate haplotype and read sequences, beginning at position Oin each sequence, where the first base is position 1. A transition to M implies position +1 in both sequences; a transition to I implies position + 1 in the read sequence only; and a transition to D implies position +1 in the haplotype sequence only. The same 3-state model may be configured to underlie the Smith- Waterman and/or Needleman-Wunsch ents, as herein described, as well. ingly, such a 3-state model, as set forth , may be employed in a SW and/or NW s thereby allowing for affine gap (indel) scoring, in which gap opening (entering the I or D state) is assumed to be less likely than gap extension (remaining in the I or D state). Hence, in this instance, the pair HMM can be seen as alignment, and a CIGAR string may be produced to encode a sequence ofthe various state transitions.

In various instances, the 3-state base model may be complicated by allowing the transition probabilities to vary by position. For instance, the probabilities of all M transitions may be multiplied by the prior probabilities of observing the next read base given its base quality score, and the corresponding next haplotype base. In such an instance, the base quality scores may ate to a probability of a cing SNP error. When the two bases match, the prior probability is taken as one minus this error ility, and when they mismatch, it is taken as the error probability divided by 3, since there are 3 possible SNP results.

The above discussion is regarding an abstract "Markovish" model. In various instances, the m-likelihood transition sequence may also be determined, which is termed herein as an alignment, and may be performed using a Needleman-Wunsch or other dynamic mming thm. But, in various instances, in performing a variant calling function, as disclosed , the maximum likelihood alignment, or any particular alignment, need not be a primary concern. Rather, the total probability may be computed, for instance, by computing the total probability of observing the read given the haplotype, which is the sum of the probabilities of all le transition paths through the graph, from read position zero at any haplotype position, to the read end position, at any haplotype position, each component path probability being simply the product of the various constituent tion probabilities.

Finding the sum of pathway probabilities may also be med by employing a virtual array and using a dynamic programming algorithm, as described above, such that in each cell of a (0 ... N) x (0 ... M) matrix, there are three probability values calculated, ponding to M, D, and I transition states. (Or equivalently, there are 3 matrices.) The top row (read position zero) ofthe matrix may be initialized to probability 1.0 in the D , and 0.0 in the I and M states; and the rest of the left column (haplotype on zero) may be initialized to all zeros. (In software, the initial D probabilities may be set near the double-precision max value, e.g. 2,-\ 1020, so as to avoid underflow, but this factor may be normalized out ) This 3-to-1 computation dependency cts the order that cells may be computed. They can be computed left to right in each row, progressing through rows from top to , or top to bottom in each column, progressing rightward. Additionally, they may be computed in anti-diagonal wavefronts, where the next step is to compute all cells (n,m) where n+m equals the incremented step number. This wavefront order has the advantage that all cells in the anti-diagonal may be computed independently of each other.

The bottom row ofthe matrix then, at the final read on, may be configured to represent the completed alignments. In such an instance, the Haplotype Caller will work by summing the I and M probabilities of all bottom row cells. In various embodiments, the system may be set up so that no D transitions are permitted within the bottom row, or a D transition ility of0.0 may be used there, so as to avoid double counting.

As described herein, in various instances, each HMM evaluation may operate on a sequence pair, such as on a candidate haplotype and a read pair. For instance, within a given active region, each of a set of ypes may be HMM-evaluated vs. each of a set of reads. In such an instance, the software and/or hardware input bandwidth may be reduced and/or minimized by transferring the set of reads and the set of haplotypes once, and letting the software and/or hardware generate the NxM pair operations. In certain instances, a Smith- Waterman evaluator may be ured to queue up individual HMM operations, each with its own copy ofread and haplotype data. A Smith-Waterman (SW) alignment module may be configured to run the pair HMM calculation in linear space or may operate in log probability space. This is useful to keep precision across the huge range of probability values with fixedpoint values. However, in other ces, floating point operations may be used.

There are three el multiplications (e.g., additions in log space), then two serial additions (6 stage approximation pipelines), then an onal multiplication. In such an instance, the full ne may be about L = 12-16 cycles long. The I & D calculations may be about half the length. The pipeline may be fed a multiplicity of input probabilities, such as 2 or 3 or 5 or 7 or more input probabilities each cycle, such as from one or more already ed neighboring cells (M and/or D from the left, M and/or I from above, and/or M and/or I and/or D from above-left). It may also include one or more haplotype bases, and/or one or more read bases such as with associated parameters, e.g., preprocessed parameters, each cycle. It outputs the M & I & D result set for one cell each cycle, after fall-through y.

As indicated above, in performing a variant call function, as sed herein, a De Bruijn Graph may be formulated, and when all ofthe reads in a pile up are identical, the DBG will be linear. r, where there are ences, the graph will form "bubbles" that are indicative of regions of differences resulting in multiple paths diverging from matching the reference alignment and then later re-joining in matching alignment. From this DBG, various paths may be extracted, which form candidate haplotypes, e.g., hypotheses for what the true DNA sequence may be on at least one strand, which hypotheses may be tested by performing an HMM, or modified HMM, operation on the data. Further still, a genotyping function may be employed such as where the possible diploid combinations of the candidate ypes may be formed, and for each of them, a conditional probability of observing the entire read pileup may be calculated. These results may then be fed into a Bayesian formula module to ate an absolute probability that each genotype is the truth, given the entire read pileup observed.

Hence, in accordance with the devices, systems, and methods of their use bed herein, in various instances, a genotyping operation may be performed, which genotyping operation may be configured so as to be implemented in an optimized manner in software and/or in hardware and/or by a quantum processing unit. For instance, the possible diploid combinations of the candidate haplotypes may be formed, and for each combination, a conditional ility of observing the entire read pileup may be calculated, such as by using the constituent probabilities of observing each read given each haplotype from the pair HMM evaluation. The results of these calculations feed into a Bayesian formula so as to calculate an absolute probability that each genotype is the truth, given the entire read pileup observed.

Accordingly, in various aspects, the present disclosure is directed to a system for performing a haplotype or variant call operation on generated and/or supplied data so as to produce a variant call file with respect thereto. Specifically, as bed herein above, in particular instances, a variant call file may be a digital or other such file that s the difference between one sequence and another, such as a the ence between a sample sequence and a reference ce. Specifically, in various instances, the variant call file may be a text file that sets forth or otherwise details the genetic and/or structural variations in a person's genetic makeup as ed to one or more reference genomes.

For instance, a haplotype is a set of genetic, e.g., DNA and/or RNA, variations, such as polymorphisms that reside in a person's chromosomes and as such may be passed on to offspring and thereby inherited together. Particularly, a haplotype can refer to a combination of alleles, e.g., one of a plurality ofalternative forms ofa gene such as may arise by mutation, which allelic variations are typically found at the same place on a chromosome.

Hence, in determining the identity of a person's genome it is important to know which form of various different possible s a specific person's genetic sequence codes for. In particular instances, a ype may refer to one or more, e.g., a set, of nucleotide polymorphisms (e.g., SNPs) that may be found at the same position on the same chromosome. lly, in various embodiments, in order to determine the genotype, e.g., allelic haplotypes, for a subject, as described herein and above, a software based algorithm may be engaged, such as an algorithm employing a haplotype call program, e.g., GATK, for simultaneously determining SNPs and/or ions and/or deletions, i.e., indels, in an dual's c sequence. In particular, the thm may involve one or more haplotype assembly protocols such as for local de-novo assembly of a ype in one or more active regions of the genetic sequence being processed. Such processing typically involves the deployment of a processing function called a Hidden Markov Model (HMM) that is a stochastic and/or statistical model used to exemplify randomly changing systems such as where it is d that future states within the system depend only on the present state and not on the sequence ofevents that precedes it.

In such instances, the system being modeled bears the characteristics or is otherwise d to be a Markov process with unobserved (hidden) . In particular ces, the model may involve a simple dynamic an network. Particularly, with respect to determining genetic variation, in its simplest form, there is one of four possibilities for the identity of any given base in a sequence being processed, such as when comparing a t of a reference sequence, e.g., a hypothetical haplotype, and that of a subject's DNA or RNA, e.g., a read derived from a sequencer. However, in order to determine such variation, in a first instance, a subject's DNA/RNA must be sequenced, e.g., via a Next Gen Sequencer ("NGS"), to produce a readout or "reads" that identify the subject's genetic code.

Next, once the subject's genome has been sequenced to produce one or more reads, the various reads, representative of the subject's DNA and/or RNA need to be mapped and/or aligned, as herein described above in great detail. The next step in the process then is to determine how the genes of the subject that have just been determined, e.g., having been mapped and/or aligned, vary from that of a prototypical reference sequence. In performing such analysis, ore, it is assumed that the read potentially representing a given gene of a subject is a representation of the prototypical haplotype albeit with various SNPs and/or indels that are to presently be determined.

Specifically, in particular aspects, devices, systems, and/or s for practicing the same, such as for performing a haplotype and/or variant call function, such as ing an HMM function, for instance, in an accelerated haplotype caller is provided. In various instances, in order to overcome these and other such various problems known in the art, the HMM accelerator herein presented may be configured to be operated in a manner so as to be implemented in software, implemented in hardware, or a combination of being implemented and/or otherwise controlled in part by software and/or in part by hardware and/or may include quantum computing implementations. For ce, in a ular aspect, the disclosure is directed to a method by which data pertaining to the DNA and/or RNA sequence identity of a subject and/or how the subject's genetic information may differ from that ofa reference genome may be determined.

In such an instance, the method may be performed by the implementation of a haplotype or t call function, such as employing an HMM protocol. Particularly, the HMM function may be med in hardware, software, or via one or more m circuits, such as on an accelerated device, in accordance with a method described . In such an instance, the HMM accelerator may be configured to receive and process the sequenced, mapped, and/or aligned data, to process the same, e.g., to produce a variant call file, as well as to transmit the processed data back throughout the system. Accordingly, the method may include deploying a system where data may be sent from a processor, such as a re-controlled CPU or GPU or even a QPU, to a haplotype caller implementing an accelerated HMM, which ype caller may be deployed on a microprocessor chip, such as an FPGA, ASIC, or structured ASIC or implemented by one or more m circuits. The method may further include the steps for processing the data to produce HMM result data, which results may then be fed back to the CPU and/or GPU and/or QPU.

] Particularly, in one embodiment, as can be seen with respect to , a bioinformatics pipeline system including an HMM accelerator is ed. For instance, in one instance, the bioinformatics pipeline system may be configured as a variant call system 1.

The system is illustrated as being implemented in hardware, but may also be implemented via one or more quantum circuits, such as of a quantum computing rm. Specifically, provides a high-level view of an HMM interface structure. In particular embodiments, the variant call system 1 is configured to accelerate at least a portion of a variant call operation, such as an HMM operation. Hence, in various instances, the t call system may be referenced herein as an HMM system 1. The system 1 includes a server having one or more central processing units (CPU/GPU/QPU) 1000 configured for performing one or more routines d to the cing and/or processing of genetic information, such as for comparing a sequenced genetic sequence to one or more reference sequences.

Additionally, the system 1 includes a peripheral device 2, such as an expansion card, that includes a microchip 7, such as an FPGA, ASIC, or sASIC. In some instances, one or more quantum circuits may be provided and configured for performing the various operations set forth herein. It is also to be noted that the term ASIC may refer equally to a structured ASIC (sASIC), where appropriate. The peripheral device 2 includes an interconnect 3 and a bus interface 4, such as a parallel or serial bus, which connects the U/QPU 1000 with the chip 7. For instance, the device 2 may comprise a peripheral component interconnect, such as a PCI, PCI-X, PCie, or QPI (quick path interconnect), and may include a bus interface 4, that is d to operably and/or communicably connect the CPU/GPU/QPU 1000 to the peripheral device 2, such as for low latency, high data transfer rates. Accordingly, in particular instances, the interface may be a peripheral component interconnect s (PCie) 4 that is associated with the hip 7, which hip es an HMM accelerator 8. For example, in particular instances, the HMM accelerator 8 1s configured for performing an accelerated HMM on, such as where the HMM function, in certain embodiments, may at least partially be implemented in the hardware of the FPGA, AISC, or sASIC or via one or more suitably configured quantum circuits.

] Specifically, presents a high-level figure of an HMM accelerator 8 having an ary organization of one or more engines 13, such as a plurality of processmg engines 13a - l3m+l, for performing one or more processes of a t call function, such as including an HMM task. Accordingly, the HMM accelerator 8 may be composed of a data distributor 9, e.g., CentCom, and one or a multiplicity of processing clusters 11 - lln+l that may be organized as or otherwise e one or more instances 13, such as where each instance may be configured as a processing engine, such as a small engine 13a - l3m+l• For instance, the distributor 9 may be configured for receiving data, such as from the CPU/GPU/QPU 1000, and distributing or ise transferring that data to one or more ofthe multiplicity ofHMM processing clusters 11.

Particularly, in certain embodiments, the distributor 9 may be positioned lly between the on-board PCie interface 4 and the HMM accelerator module 8, such as where the interface 4 icates with the distributor 9 such as over an interconnect or other suitably configured bus 5, e.g., PCie bus. The distributor module 9 may be d for communicating with one or more HMM accelerator clusters 11 such as over one or more cluster buses 10. For instance, the HMM accelerator module 8 may be configured as or otherwise include an array of clusters 1la-1 ln+l, such as where each HMM cluster 11 may be configured as or otherwise includes a cluster hub 11 and/or may include one or more instances 13, which ce may be configured as a processing engine 13 that is adapted for performing one or more operations on data ed thereby. Accordingly, in various embodiments, each cluster 11 may be formed as or otherwise include a cluster hub 1la-1 ln+l, where each of the hubs may be operably associated with multiple HMM accelerator engine instances 13a-13m+1, such as where each cluster hub 11 may be configured for ing data to a ity ofthe processing engines 13a - 13m+1 within the cluster 11.

In various instances, the HMM accelerator 8 is configured for comparing each base of a subject's sequenced genetic code, such as in read format, with the various known or generated candidate haplotypes of a reference sequence and determining the ility that any given base at a position being considered either matches or doesn't match the relevant haplotype, e.g., the read includes an SNP, an insertion, or a deletion, thereby ing in a variation of the base at the position being considered. Particularly, in various embodiments, the HMM accelerator 8 is configured to assign transition probabilities for the sequence ofthe bases of the read going between each of these states, Match ("M"), Insert ("I"), or Delete ("D") as described in greater detail herein below.

More particularly, dependent on the configuration, the HMM acceleration function may be implemented in either software, such as by the CPU/GPU/QPU 1000 and/or microchip 7, and/or may be implemented in hardware and may be present within the microchip 7, such as positioned on the eral expansion card or board 2. In various embodiments, this functionality may be implemented lly as software, e.g., run by the CPU/GPU/QPU 1000, and partially as hardware, implemented on the chip 7 or via one or more m processing circuits. Accordingly, in various embodiments, the chip 7 may be t on the motherboard of the U/QPU 1000, or it may be part of the peripheral device 2, or both. Consequently, the HMM accelerator module 8 may e or otherwise be ated with various interfaces, e.g., 3, 5, 10, and/or 12 so as to allow the efficient transfer of data to and from the processing engines 13.

Accordingly, as can be seen with respect to FIGS. 2 and 3, in vanous embodiments, a microchip 7 configured for performing a variant, e.g., haplotype, call function is provided. The microchip 7 may be associated with a CPU/GPU/QPU 1000 such as directly coupled therewith, e.g., ed on the motherboard of a computer, or indirectly coupled thereto, such as being included as part of a peripheral device 2 that is ly d to the CPU/GPU/QPU 1000, such as via one or more interconnects, e.g., 3, 4, 5, 10, and/or 12. In this instance, the microchip 7 is present on the peripheral device 2. It is to be understood that although configured as a hip, the accelerator could also be configured as one or more quantum circuits of a quantum sing unit, wherein the quantum circuits are configured as one or more processing engines for performing one or more ofthe functions disclosed .

Hence, the peripheral device 2 may include a parallel or serial expansion bus 4 such as for connecting the peripheral device 2 to the central processing unit (CPU/GPU/QPU) 1000 of a computer and/or server, such as via an interface 3, e.g., DMA. In particular instances, the peripheral device 2 and/or serial ion bus 4 may be a Peripheral ent Interconnect express (PCie) that is configured to communicate with or otherwise include the microchip 7, such as via connection 5. As described herein, the microchip 7 may at least partially be configured as or may otherwise include an HMM accelerator 8. The HMM accelerator 8 may be configured as part ofthe microchip 7, e.g., as hardwired and/or as code to be run in association therewith, and is ured for performing a variant call function, such as for performing one or more operations of a Hidden Markov Model, on data supplied to the microchip 7 by the CPU/GPU/QPU 1000, such as over the PCie interface 4.

Likewise, once one or more t call functions have been performed, e.g., one or more HMM operations run, the results thereof may be transferred from the HMM accelerator 8 of the chip 7 over the bus 4 to the CPU/GPU/QPU 1000, such as via tion 3.

For instance, in particular instances, a CPU/GPU/QPU 1000 for processing and/or transferring ation and/or executing instructions is provided along with a microchip 7 that is at least lly configured as an HMM accelerator 8. The CPU/GPU/QPU 1000 communicates with the microchip 7 over an interface 5 that is adapted to facilitate the communication between the CPU/GPU/QPU 1000 and the HMM accelerator 8 of the microchip 7 and ore may icably connect the CPU/GPU/QPU 1000 to the HMM accelerator 8 that is part of the microchip 7. To facilitate these functions, the microchip 7 es a distributor module 9, which may be a CentCom, that is configured for transferring data to a multiplicity of HMM engines 13, e.g., via one or more clusters 11, where each engine 13 is ured for receiving and processing the data, such as by running an HMM protocol thereon, computing final , outputting the results thereof, and repeating the same. In various instances, the performance of an HMM protocol may include determining one or more transition probabilities, as bed herein below. Particularly, each HMM engine 13 may be configured for ming a job such as including one or more of the generating and/or evaluating ofan HMM virtual matrix to produce and output a final sum value with respect thereto, which final sum expresses the probable likelihood that the called base matches or is different from a corresponding base in a hypothetical haplotype ce, as described herein below. presents a detailed depiction of the HMM cluster 11 of . In various embodiments, each HMM cluster 11 includes one or more HMM instances 13. One or a number of clusters may be provided, such as desired in accordance with the amount of resources provided, such as on the chip or quantum computing processor. Particularly, a HMM cluster may be provided, where the cluster is configured as a cluster hub 11. The r hub 11 takes the data pertaining to one or more jobs 20 from the distributor 9, and is further communicably connected to one or more, e.g., a plurality of, HMM instances 13, such as via one or more HMM instance busses 12, to which the cluster hub 11 transmits the job data 20.

The bandwidth for the er ofdata throughout the system may be relatively low bandwidth s, and once a job 20 is received, the system 1 may be configured for ting the job, such as without having to go off chip 7 for . In various embodiments, one job 20a is sent to one processing engine 13a at any given time, but several jobs 20a-n may be distributed by the cluster hub 11 to several different processing engines m+l, such as where each of the processing engines 13 will be g on a single job , e.g., a single comparison between one or more reads and one or more ype sequences, in parallel and at high speeds. As described below, the performance of such a job may typically involve the generation of a virtual matrix whereby the t's "read" sequences may be compared to one or more, e.g., two, hypothetical haplotype sequences, so as to determine the differences there between. In such instances, a single job 20 may involve the processing of one or more matrices having a multiplicity of cells therein that need to be processed for each comparison being made, such as on a base by base basis. As the human genome is about 3 billion base pairs, there may be on the order of 1 to 2 billion ent jobs to be performed when analyzing a 30X oversampling of a human genome (which is equitable to about 20 trillion cells in the matrices of all associated HMM jobs).

Accordingly, as described herein, each HMM instance 13 may be adapted so as to perform an HMM protocol, e.g., the generating and sing of an HMM , on sequence data, such as data received thereby from the CPU/GPU/QPU 1000. For e, as explained above, in sequencing a subject's genetic material, such as DNA or RNA, the DNA/RNA is broken down into segments, such as up to about 100 bases in length. The identity ofthese 100 base segments are then determined, such as by an automated sequencer, and "read" into a FASTQ text based file or other format that stores both each base identity of the read along with a Phred quality score (e.g., typically a number between 0 and 63 in log scale, where a score of 0 indicates the least amount of confidence that the called base is correct, with scores between 20 to 45 generally being acceptable as relatively accurate).

Particularly, as indicated above, a Phred quality score is a quality indicator that measures the quality of the identification of the nucleobase identities generated by the sequencing processor, e.g., by the automated DNA/RNA sequencer. Hence, each read base includes its own quality, e.g., Phred, score based on what the sequencer evaluated the quality of that ic identification to be. The Phred represents the confidence with which the sequencer estimates that it got the called base ty correct. This Phred score is then used by the implemented HMM module 8, as described in detail below, to further determine the accuracy of each called base in the read as compared to the haplotype to which it has been mapped and/or aligned, such as by determining its Match, Insertion, and/or Deletion transition probabilities, e.g., in and out of the Match state. It is to be noted that in various embodiments, the system 1 may modify or otherwise adjust the initial Phred score prior to the performance of an HMM protocol thereon, such as by taking into account neighboring bases/scores and/or fragments ofneighboring DNA and allowing such factors to influence the Phred score ofthe base, e.g., cell, under ation.

In such instances, as can be seen with respect to the system 1, e.g., computer/quantum software, may determine and identify various active regions 500n within the sequenced genome that may be explored and/or otherwise subjected to further processing as herein described, which may be broken down into jobs 20n that may be parallelized amongst the various cores and ble threads 1007 throughout the system 1. For instance, such active regions 500 may be fied as being s of variation between the sequenced and reference genomes. Particularly, the CPU/GPU/QPU 1000 may have multiple threads 1007 running, identifying active regions 500a, 500b, and 500c, compiling and ating various different jobs 20 n to be worked on, e.g., via a suitably configured aggregator 1008, based on the active region(s) 500a-c currently being examined. Any suitable number of threads 1007 may be employed so as to allow the system 1 to run at maximum efficiency, e.g., the more s present the less active time spent g.

Once identified, compiled, and/or ated, the threads 1007/1008 will then transfer the active jobs 20 to the data distributor 9, e.g., CentCom, of the HMM module 8, such as via PCie ace 4, e.g., in a fire and forget manner, and will then move on to a different process while g for the HMM 8 to send the output data back so as to be matched back up to the corresponding active region 500 to which it maps and/or aligns. The data butor 9 will then distribute the jobs 20 to the s different HMM clusters 11, such as on a job-by-job manner. If everything is running efficiently, this may be on a first in first out format, but such does not need to be the case. For instance, in various embodiments, raw jobs data and processed job results data may be sent h and across the system as they become available.

Particularly, as can be seen with respect to FIGS. 2, 3, and 4, the various job data 20 may be aggregated into 4K byte pages of data, which may be sent via the PCie 4 to and through the CentCom 9 and on to the processing engines 13, e.g., via the clusters 11. The amount ofdata being sent may be more or less than 4K bytes, but will typically include about 100 HMM jobs per 4K (e.g., 1024) page of data. Particularly, these data then get digested by the data distributor 9 and are fed to each r 11, such as where one 4K page is sent to one cluster 11. However, such need not be the case as any given job 20 may be sent to any given cluster 11, based on the clusters that become available and when.

Accordingly, the r 11 approach as presented here efficiently distributes incoming data to the processing engines 13 at high-speed. Specifically, as data arrives at the PCie interface 4 from the CPU/GPU/QPU 1000, e.g., over DMA connection 3, the ed data may then be sent over the PCie bus 5 to the CentCom distributor 9 of the variant caller microchip 7. The butor 9 then sends the data to one or more HMM processing clusters 11, such as over one or more cluster dedicated buses 10, which cluster 11 may then transmit the data to one or more processing instances 13, e.g., via one or more instance buses 12, such as for processing. In this instance, the PCie interface 4 is adapted to e data through the peripheral expansion bus 5, distributor 9, and/or cluster 10 and/or instance 12 busses at a rapid rate, such as at a rate that can keep one or more, e.g., all, of the HMM accelerator instances 13a-(m+l) within one or more, e.g., all, of the HMM clusters 1la-(n+l) busy, such as over a prolonged period of time, e.g., full time, during the period over which the system 1 is being run, the jobs 20 are being processed, and whilst also keeping up with the output of the sed HMM data that is to be sent back to one or more CPUs 1000, over the PCie interface 4.

For instance, any inefficiency in the interfaces 3, 5, 10, and/or 12 that leads to idle time for one or more of the HMM rator ces 13 may directly add to the overall processing time ofthe system 1. Particularly, when analyzing a human genome, there may be on the order of two or more n different jobs 20 that need to be distributed to the various HMM clusters 11 and processed over the course of a time period, such as under 1 hour, under 45 minutes, under 30 minutes, under 20 minutes including 15 minutes, 10 minutes, 5 minutes, or less. ingly, sets forth an overview of an exemplary data flow throughout the software and/or hardware of the system 1, as described generally above. As can be seen with t to the system 1 may be configured in part to transfer data, such as between the PCie interface 4 and the distributor 9, e.g., CentCom, such as over the PCie bus 5. Additionally, the system 1 may further be configured in part to transfer the received data, such as between the distributor 9 and the one or more HMM clusters 11, such as over the one or more cluster buses 10. Hence, in various embodiments, the HMM accelerator 8 may include one or more clusters 11, such as one or more clusters 11 configured for performing one or more processes of an HMM on. In such an instance, there is an interface, such as a cluster bus 10, that connects the CentCom 9 to the HMM r 11.

For instance, is a high-level diagram depicting the interface in to and out of the HMM module 8, such as into and out of a cluster . As can be seen with t to each HMM cluster 11 may be configured to communicate with, e.g., receive data from and/or send final result data, e.g., sum data, to the CentCom data distributor 9 through a dedicated cluster bus 10. Particularly, any suitable interface or bus 5 may be provided so long as it allows the PCie ace 4 to communicate with the data distributor 9.

More ularly, the bus 5 may be an interconnect that includes the interpretation logic useful in talking to the data distributor 9, which interpretation logic may be configured to accommodate any protocol ed to provide this functionality. Specifically, in various instances, the interconnect may be configured as a PCie bus 5.

Additionally, the cluster 11 may be configured such that single or multiple clock s may be employed therein, and hence, one or more clocks may be present within the cluster 11. In particular instances, multiple clock domains may be provided. For example, a slower clock may be provided, such as for communications, e.g., to and from the r 11. Additionally, a faster, e.g., a high speed, clock may be provided which may be employed by the HMM instances 13 for use in performing the various state calculations described herein.

Particularly, in various embodiments, as can be seen with respect to the system 1 may be set up such that, in a first ce, as the data distributor 9 ges the existing CentCom IP, a collar, such as a gasket, may be provided, where the gasket is configured for translating signals to and from the CentCom interface 5 from and to the HMM cluster interface or bus 10. For ce, an HMM cluster bus 10 may communicably and/or operably connect the CPU/GPU 1000 to the s clusters 11 of the HMM accelerator module 8. Hence, as can be seen with respect to structured write and/or read data for each haplotype and/or for each read may be sent throughout the system 1.

Following a job 20 being input into the HMM engine, an HMM engine 13 may typically start either: a) immediately, if it is IDLE, or b) after it has completed its currently assigned task. It is to be noted that each HMM accelerator engine 13 can handle ping and pong inputs (e.g., can be working on one data set while the other is being loaded), WO 14320 PCT/0S2017/036424 thus minimizing downtime between jobs. Additionally, the HMM r collar 11 may be configured to automatically take the input job 20 sent by the data distributor 9 and assign it to one ofthe HMM engine instances 13 in the cluster 11 that can receive a new job. There need not be a control on the software side that can select a specific HMM engine instance 13 for a specific job 20. However, in various instances, the software can be configured to control such instances.

Accordingly, in view of the above, the system 1 may be lined when transferring the results data back to the CPU/GPU/QPU, and because of this efficiency there is not much data that needs to go back to the CPU/GPU/QPU to achieve the usefulness ofthe results. This allows the system to achieve about a 30 minute or less, such as about a 25 or about a 20 minute or less, for instance, about a 18 or about a 15 minute or less, including about a 10 or about a 7 minute or less, even about a 5 or about a 3 minute or less variant call operation, dependent on the system configuration. presents a high-level view of various functional blocks within an exemplary HMM engine 13 within a hardware rator 8, on the FPGA or ASIC 7.

Specifically, within the hardware HMM rator 8 there are multiple clusters 11, and within each cluster 11 there are multiple engines 13. presents a single instance of an HMM engine 13. As can be seen with respect to the engine 13 may include an instance bus interface 12, a plurality of memories, e.g., an HMEM 16 and an RMEM 18, various other ents 17, HMM control logic 15, as well as a result output interface 19.

Particularly, on the engine side, the HMM ce bus 12 is operably connected to the memories, HMEM 16 and RMEM 18, and may include interface logic that communicates with the cluster hub 11, which hub is in communications with the butor 9, which in tum is communicating with the PCie interface 4 that communicates with the variant call software being run by the CPU/GPU and/or server 1000. The HMM instance bus 12, therefore, receives the data from the CPU 1000 and loads it into one or more of the memories, e.g., the HMEM and RMEM. This configuration may also be implemented in one or more quantum circuits and adapted ingly.

In these instances, enough memory space should be allocated such that at least one or two or more haplotypes, e.g., two haplotypes, may be , e.g., in the HMEM 16, per given read sequence that is loaded, e.g., into the RMEM 18, which when multiple haplotypes are loaded results in an easing of the burden on the PCie bus 5 bandwidth. In particular instances, two haplotypes and two read sequences may be loaded into their WO 14320 PCT/0S2017/036424 respective es, which would allow the four sequences to be processed together in all relevant combinations. In other instances four, or eight, or sixteen sequences, e.g., pairs of sequences, may be loaded, and in like manner be processed in combination, such as to further ease the bandwidth when desired. onally, enough memory may be reserved such that a ping-pong structure may be implemented therein such that once the es are loaded with a new job 20a, such as on the ping side ofthe memory, a new job signal is indicated, and the control logic 15 may begin processing the new job 20a, such as by generating the matrix and performing the requisite calculations, as bed herein and below. Accordingly, this leaves the pong side of the memory ble so as to be loaded up with another job 20b, which may be loaded therein while the first job 20a is being processed, such that as the first job 20a is finished, the second job 20b may immediately begin to be processed by the control logic 15.

In such an instance, the matrix for job 20b may be preprocessed so that there is virtually no down time, e.g., one or two clock cycles, from the ending of processing of the first job 20a, and the beginning of processing of the second job 20b. Hence, when utilizing both the ping and pong side of the memory structures, the HMEM 16 may typically store 4 haplotype ces, e.g., two a piece, and the RMEM 18 may typically store 2 read sequences. This ping-pong configuration is useful because it simply requires a little extra memory space, but allows for a doubling ofthe throughput ofthe engine 13.

During and/or after processing the memories 16, 18 feed into the transition probabilities calculator and lookup table (LUT) block 17a, which is configured for calculating various information related to "Priors" data, as explained below, which in tum feeds the Prior results data into the M, I, and D state ator block 17b, for use when calculating transition probabilities. One or more scratch RAMs 17c may also be included, such as for holding the M, I, and D states at the boundary of the swath, e.g., the values ofthe bottom row of the processing swath, which as indicated, in various ces, may be any suitable amount of cells, e.g., about 10 cells, in length so as to be commensurate with the length ofthe swath 35.

Additionally, a separate results output interface block 19 may be included so that when the sums are finished they, e.g., a 4 32-bit word, can ately be transmitted back to the variant call software of the CPU/GPU/QPU 1000. It is to be noted that this configuration may be d so that the system 1, specifically the M, I, and D calculator 17b is not held up waiting for the output interface 19 to clear, e.g., so long as it does not take as long to clear the results as it does to perform the job 20. Hence, in this configuration, there may be three pipeline steps functioning in concert to make an l systems pipeline, such as loading the memory, performing the MID calculations, and outputting the results. Further, it is noted that any given HMM engine 13 is one of many with their own output interface 19, however they may share a common interface 10 back to the data distributor 9. Hence, the cluster hub 11 will include management capabilities to manage the transfer ") of information through the HMM accelerator 8 so as to avoid collisions.

Accordingly, the following details the processes being performed within each module of the HMM engines 13 as it receives the haplotype and read sequence data, processes it, and outputs results data pertaining to the same, as generally outlined above.

Specifically, the high-bandwidth computations in the HMM engine 13, within the HMM cluster 11, are directed to computing and/or ng the match (M), insert (I), and delete (D) state values, which are ed in determining whether the particular read being examined matches the haplotype nce as well as the extent of the same, as described above.

Particularly, the read along with the Phred score anf GOP value for each base in the read is transmitted to the cluster 11 from the distributor 9 and is thereby assigned to a particular processing engine 13 for processing. These data are then used by the M, I, and D ator 17 of the processing engine 13 to determine whether the called base in the read is more or less likely to be correct and/or to be a match to its respective base in the haplotype, or to be the product of a variation, e.g., an insert or deletion; and/or if there is a variation, whether such variation is the likely result of a true variability in the haplotype or rather an artifact of an error in the sequence generating and/or mapping and/or aligning systems.

As indicated above, a part of such is includes the MID calculator 17 determining the transition probabilities from one base to r in the read going from one M, I, or D state to another in comparison to the reference, such as from a matching state to another matching state, or a matching state to either an insertion state or to a deletion state. In making such determinations each of the associated transition probabilities is determined and considered when evaluating whether any observed variation between the read and the reference is a true variation and not just some e or processing error. For these purposes, the Phred score for each base being considered is useful in determining the transition ilities in and out of the match state, such as going from a match state to an insert or on, e.g., a gapped, state m the comparison. Likewise, the transition probabilities of continuing a gapped state or going from a gapped state, e.g., an insert or deletion state, back to a match state are also determined. In particular instances, the probabilities in or out of the delete or insert state, e.g., exiting a gap continuation state, may be a fixed value, and may be referenced herein as the gap continuation probability or penalty.

Nevertheless, in various instances, such gap continuation penalties may be floating and therefore subject to change dependent on the accuracy demands ofthe system configuration.

] Accordingly, as depicted with respect to FIGS. 7 and 8 each ofthe M, I, and D state values are computed for each le read and haplotype base pairing. In such an instance, a virtual matrix 30 ofcells containing the read sequence being evaluated on one axis ofthe matrix and the associated haplotype sequence on the other axis may be formed, such as where each cell in the matrix represents a base on in the read and haplotype reference.

Hence, if the read and ype ces are each 100 bases in length, the matrix 30 will include 100 by 100 cells, a given portion of which may need to be processed in order to determine the likelihood and/or extent to which this particular read s up with this particular reference. Hence, once virtually formed, the matrix 30 may then be used to ine the s state transitions that take place when moving from one base in the read sequence to another and comparing the same to that of the haplotype sequence, such as depicted in FIGS. 7 and 8. Specifically, the processing engine 13 is configured such that a multiplicity of cells may be processed in el and/or sequential fashion when traversing the matrix with the control logic 15. For instance, as ed in a virtual processing swath 35 is propagated and moves across and down the matrix 30, such as from left to right, processing the individual cells ofthe matrix 30 down the right to left diagonal.

More specifically, as can be seen with respect to each individual virtual cell within the matrix 30 includes an M, I, and D state value that needs to be calculated so as to asses the nature ofthe identity ofthe called base, and as depicted in the data dependencies for each cell in this process may y be seen. Hence, for determining a given M state of a present cell being processed, the Match, Insert, and Delete states of the cell diagonally above the present cell need to be pushed into the present cell and used in the calculation of the M state of the cell tly being calculated (e.g., thus, the diagonal downwards, forwards progression through the matrix is indicative ofmatching).

However, for determining the I state, only the Match and Insert states for the cell directly above the present cell need be pushed into the present cell being sed (thus, the vertical downwards "gapped" progression when continuing in an insertion state).

Likewise, for determining the D state, only the Match and Delete states for the cell directly left of the present cell need be pushed into the present cell (thus, the horizontal cross-wards "gapped" progression when continuing in a deletion state). As can be seen with t to after computation of cell 1 (the shaded cell in the top most row) begins, the processing of cell 2 (the shaded cell in the second row) can also begin, without waiting for any results from cell 1, because there is no data dependencies between this cell in row 2 and the cell ofrow 1 where processing begins. This forms a reverse diagonal 35 where processing proceeds downwards and to the left, as shown by the red arrow. This reverse diagonal 35 processing approach increases the processing efficiency and throughput ofthe overall system.

Likewise, the data generated in cell 1, can immediately be pushed forward to the cell down and forward to the right ofthe top most cell 1, thereby advancing the swath 35 forward.

] For ce, s an exemplary HMM matrix structure 35 showing the re processing flow. The matrix 35 includes the haplotype base index, e.g., containing 36 bases, positioned to run along the top edge of the horizontal axis, and further includes the base read index, e.g., 10 bases, positioned to fall along the side edge of the vertical axis in such a manner to from a structure of cells where a selection of the cells may be populated with an M, I, and D probability state, and the tion probabilities of transitioning from the present state to a neighboring state. In such an ce, as described in greater detail above, a move from a match state to a match state results in a forwards diagonal progression through the matrix 30, while moving from a match state to an ion state results in a vertical downwards progressing gap, and a move from a match state to a deletion state results in a horizontal progressing gap. Hence, as depicted in for a given cell, when determining the match, insert, and delete states for each cell, the match, , and delete probabilities of its three adjoining cells are employed.

] The downwards arrow in represents the parallel and tial nature of the processing engine(s) that are configured so as to produce a processing swath or wave that moves progressively along the virtual matrix in accordance with the data dependencies, see FIGS. 7 and 8, for determining the M, I, and D states for each particular cell in the structure 30. Accordingly, in certain instances, it may be desirable to calculate the identities of each cell in a downwards and diagonal manner, as explained above, rather than simply calculating each cell along a vertical or ntal axis exclusively, although this can be done if desired. This is due to the increased wait time, e.g., latency, that would be required when sing the virtual cells of the matrix 35 individually and sequentially along the al or horizontal axis alone, such as via the hardware configuration.

For instance, in such an instance, when moving linearly and sequentially through the virtual matrix 30, such as in a row by row or column by column manner, in order to process each new cell the state computations of each preceding cell would have to be completed, thereby increasing latency time overall. However, when propagating the M, I, D probabilities of each new cell in a downwards and diagonal fashion, the system 1 does not have to wait for the processing of its preceding cell, e.g., of row one, to te before beginning the sing of an adjoining cell in row two of the matrix. This allows for parallel and sequential processing of cells in a diagonal arrangement to occur, and further allows the various computational delays of the pipeline associated with the M, I, and D state ations to be hidden. Accordingly, as the swath 35 moves across the matrix 30 from left to right, the computational processing moves diagonally downwards, e.g., towards the left (as shown by the arrow in . This configuration may be particularly useful for hardware and/or m circuit implementations, such as where the memory and/or clock-by-clock latency are a primary concern.

In these configurations, the actual value output from each call of an HMM engine 13, e.g., after having calculated the entire matrix 30, may be a bottom row (e.g., Row of) containing M, I, and D , where the Mand I states may be summed (the D states may be ignored at this point having already fulfilled their function in processing the calculations above), so as to produce a final sum value that may be a single probability that tes, for each read and haplotype index, the probability of observing the read, e.g., assuming the haplotype was the true original DNA sampled.

Particularly, the outcome of the processing of the matrix 30, e.g., of may be a single value representing the ility that the read is an actual representation of that haplotype. This ility is a value between Oand 1 and is formed by summing all of the M and I states from the bottom row of cells in the HMM matrix 30. Essentially, what is being assessed is the possibility that something could have gone wrong in the sequencer, or associated DNA preparation methods prior to cing, so as to incorrectly produce a mismatch, insertion, or deletion into the read that is not actually present within the subject's genetic sequence. In such an instance, the read is not a true reflection of the subject's actual Hence, accounting for such production errors, it can be determined what any given read actually represents with respect to the ype, and thereby allows the system to better determine how the subject's genetic sequence, e.g., en masse, may differ from that of a reference sequence. For instance, many ypes may be run against many read sequences, generating scores for all of them, and determining based on which matches have the best scores, what the actual genomic sequence identity of the individual is and/or how it truly varies from a reference genome.

] More particularly, depicts an enlarged view of a portion of the HMM state matrix 30 from As shown in given the internal composition of each cell in the matrix 30, as well as the structure of the matrix as a whole, the M, I, and D state probability for any given "new" cell being calculated is dependent on the M, I, and D states of several of its surrounding neighbors that have already been calculated. Particularly, as shown in greater detail with respect to FIGS. 1 and 16, in an exemplary configuration, there may be an approximately a .9998 probability of going from a match state to another match state, and there may be only a .0001 ility (gap open penalty) of going from a match state to either an insertion or a deletion, e.g., gapped, state. Further, when in either a gapped insertion or gapped on state there may be only a 0.1 probability (gap extension or continuation penalty) of staying in that gapped state, while there is a .9 probability of returning to a match state. It is to be noted that according to this model, all ofthe probabilities in to or out of a given state should sum to one. Particularly, the processing of the matrix 30 revolves around calculating the transition probabilities, accounting for the various gap open or gap continuation penalties and a final sum is calculated.

Hence, these calculated state transition probabilities are derived mainly from the directly adjoining cells in the matrix 30, such as from the cells that are immediately to the left of, the top of, and diagonally up and left of that given cell presently being calculated, as seen in . Additionally, the state transition probabilities may in part be d from the "Phred" quality score that accompanies each read base. These tion probabilities, therefore, are useful in computing the M, I, and D state values for that particular cell, and likewise for any associated new cell being calculated. It is to be noted that as described herein, the gap open and gap continuation penalties may be fixed values, however, in various instances, the gap open and gap continuation ies may be variable and therefore programmable within the , albeit by employing additional hardware ces dedicated to determining such variable transition probability calculations. Such instances may be useful where greater accuracy is desired. Nevertheless, when such values are assumed to be constant, smaller resource usage and/or chip size may be achieved, leading to greater processing speed, as explained below.

Accordingly, there is a multiplicity of calculations and/or other mathematical computations, such as multiplications and/or additions, which are involved in deriving each new M, I, and D state value. In such an instance, such as for calculating maximum throughput, the primitive mathematical ations involved in each M, I, and D transition state calculation may be pipelined. Such ning may be configured in a way that the corresponding clock frequencies are high, but where the pipeline depth may be ivial.

Further, such a pipeline may be configured to have a finite depth, and in such instances it may take more than one clock cycle to complete the operations.

For instance, these computations may be run at high speeds inside the sor 7, such as at about . This may be achieved such as by pipelining the FPGA or ASIC heavily with registers so little mathematical computation occurs between each op.

This pipeline structure results in multiple cycles of latency in going from the input of the match state to the output, but given the reverse diagonal computing structure, set forth in above, these latencies may be hidden over the entire HMM matrix 30, such as where each cell represents one clock cycle.

Hence, the number ofM, I, and D state calculations may be d. In such an instance, the processing engine 13 may be configured in such a manner that a ng, e.g., swath 35, of cells in a number ofrows ofthe matrix 30 may be sed as a group (such as in a down-and-left-diagonal fashion as illustrated by the arrow in before proceeding to the processing of a second swath below, e.g., where the second swath contains the same number of cells in rows to be processed as the first. In a manner such as this, a hardware implementation of an accelerator 8, as bed herein, may be adapted so as to make the overall system more efficient, as described above.

Particularly, sets forth an exemplary computational structure for performing the various state processing calculations herein described. More particularly, sets forth three dedicated logic blocks 17 of the processing engine 13 for computing the state computations involved in generating each M, I, and D state value for each particular cell, or grouping of cells, being processed in the HMM matrix 30. These logic blocks may be implemented in hardware, but in some instances, may be ented in software, such as for being performed by one or more quantum circuits. As can be seen with respect to the match state computation 15a is more involved than either ofthe insert 15b or deletion 15c computations, this is because in ating the match state 15a of the present cell being processed, all ofthe previous match, insert, and delete states ofthe adjoining cells along with various "Priors" data are included in the present match computation (see FIGS. 9 and 10), whereas only the match and either the insert and delete states are included in their respective calculations. Hence, as can be seen with respect to in calculating a match state, three state multipliers, as well as two adders, and a final multiplier, which accounts for the Prior, e.g. Phred, data are included. However, for calculating the I or D state, only two multipliers and one adder are included. It is noted that in hardware, multipliers are more resource intensive than adders.

Accordingly, to various extents, the M, I, and D state values for processing each new cell in the HMM matrix 30 uses the knowledge or pre-computation ofthe following , such as the ous" M, I, and D state values from left, above, and/or diagonally left and above of the currently-being-computed cell in the HMM matrix. Additionally, such values representing the prior information, or "Priors", may at least in part be based on the "Phred" quality score, and whether the read base and the reference base at a given cell in the matrix 30 match or are different. Such information is particularly useful when ining a match state. Specifically, as can be seen with respect to in such ces, there are basically seven ition probabilities" (M-to-M, , D-to-M, I-to-I, M-to-I, D-to-D, and M-to-D) that indicate and/or estimate the probability of seeing a gap open, e.g., ng a transition from a match state to an insert or delete state; seeing a gap close; e.g., going from an insert or delete state back to a match state; and seeing the next state continuing in the same state as the us state, e.g., Match-to-Match, Insert-to-Insert, Delete-to-Delete.

The state values (e.g., in any cell to be processed in the HMM matrix 30), Priors, and transition probabilities are all values in the range of [0,1]. Additionally, there are also known starting ions for cells that are on the left or top edge of the HMM matrix . As can be seen from the logic 15a of there are four multiplication and two addition computations that may be employed in the particular M state calculation being determined for any given cell being processed. Likewise, as can be seen from the logic of 15b and 15c there are two multiplications and one addition involved for each I state and each D state calculation, respectively. Collectively, along with the priors multiplier this sums to a total of eight multiplications and four addition operations for the M, I, and D state calculations associated with each single cell in the HMM matrix 8 to be processed.

] The final sum output, e.g., row 34 ofPIG. 16, ofthe computation ofthe matrix , e.g., for a single job 20 ofcomparing one read to one or two haplotypes, is the summation ofthe final M and I states across the entire bottom row 34 ofthe matrix 30, which is the final sum value that is output from the HMM accelerator 8 and delivered to the CPU/GPU/QPU 1000. This final summed value represents how well the read matches the haplotype(s). The value is a probability, e.g., ofless than one, for a single job 20a that may then be compared to the output resulting from another job 20b such as form the same active region 500. It is noted that there are on the order of 20 trillion HMM cells to evaluate in a "typical" human genome at 30X ge, where these 20 trillion HMM cells are spread across about 1 to 2 billion HMM matrices 30 ofall associated HMM jobs 20.

The results of such calculations may then be compared one against the other so as to determine, in a more precise manner, how the genetic sequence of a subject differs, e.g., on a base by base comparison, from that ofone or more nce genomes. For the final sum calculation, the adders already employed for calculating the M, I, and/or D states of the individual cells may be re-deployed so as to e the final sum value, such as by including a mux into a selection of the re-deployed adders thereby including one last additional row, e.g., with respect to calculation time, to the matrix so as to calculate this final sum, which if the read length is 100 bases amounts to about a 1% ad. In alternative embodiments, dedicated hardware resources can be used for performing such ations. In various instances, the logic for the adders for the M and D state calculations may be deployed for calculating the final sum, which D state adder may be efficiently deployed since it is not otherwise being used in the final processing g to the g values.

In certain ces, these calculations and relevant processes may be ured so as to pond to the output of a given sequencing rm, such as including an ensemble of sequencers, which as a collective may be capable of outputting (on average) a new human genome at 30x coverage every 28 minutes (though they come out of the sequencer ensemble in groups of about 150 genomes every three days). In such an instance, when the present mapping, aligning, and variant calling operations are configured to fit within such a sequencing platform of processing technologies, a portion ofthe 28 minutes (e.g., about 10 minutes) it takes for the sequencing cluster to sequence a genome, may be used by a suitably configured mapper and/or aligner, as herein described, so as to take the WO 14320 PCT/0S2017/036424 image/BCL/FASTQ file s from the sequencer and perform the steps of mapping and/or ng the genome, e.g., post-sequencer processing. That leaves about 18 minutes of the sequencing time period for performing the variant calling step, ofwhich the HMM operation is the main computational component, such as prior to the nucleotide sequencer sequencing the next genome, such as over the next 28 minutes. Accordingly, in such instances, 18 minutes may be ed to computing the 20 trillion HMM cells that need to be processed in accordance with the processing of a genome, such as where each of the HMM cells to be processed includes about twelve mathematical operations (e.g., eight multiplications and/or four addition operations). Such a throughput allows for the following computational dynamics (20 trillion HMM cells) x (12 math ops per cell) / (18 minutes x 60 seconds/minute), which is about 222 billion operations per second ofsustained throughput. sets forth the logic blocks 17 of the sing engine of ing exemplary M, I, and D state update circuits that present a simplification of the circuit provided in The system may be configured so as to not be memory-limited, so a single HMM engine instance 13 (e.g., that computes all of the single cells in the HMM matrix 30 at a rate of one cell per clock cycle, on average, plus overheads) may be replicated multiple times (at least 65-70 times to make the throughput efficient, as described above).

Nevertheless, to minimize the size of the hardware, e.g., the size of the chip 2 and/or its ated resource usage, and/or in a further effort to include as many HMM engine instances 13 on the chip 2 as ble and/or possible, fications may be made with regard to the logic blocks 15a'-c' ofthe processing ce 13 for ing one or more of the transition probabilities to be calculated.

In particular, it may be assumed that the gap open penalty (GOP) and gap continuation penalty (GCP), as described above, such as for inserts and deletes are the same and are known prior to chip configuration. This simplification implies that the I-to-M and D- to-M transition probabilities are identical. In such an instance, one or more ofthe multipliers, e.g., set forth in may be eliminated, such as by pre-adding I and D states before multiplying by a common Indel-to-M transition probability. For ce, in various instances, if the I and D state calculations are assumed to be the same, then the state calculations per cell can be simplified as presented in . Particularly, if the I and D state values are the same, then the I state and the D state may be added and then that sum may be multiplied by a single value, thereby saving a multiply. This may be done because, as seen with respect to , the gap continuation and/or close penalties for the I and D states are the same. However, as indicated above, the system can be configured to calculate different values for both the I and D transition state probabilities, and in such an instance, this fication would not be employed. onally, in a further simplification, rather than dedicate chip or other computing resources configured specifically to perform the final sum operation at the bottom of the HMM matrix, the present HMM accelerator 8 may be configured so as to effectively append one or more additional rows to the HMM matrix 30, with respect to computational time, e.g., overhead, it takes to perform the calculation, and may also be configured to "borrow" one or more adders from the M-state 15a and D-state 15c computation logic such as by MUXing in the final sum values to the existing adders as needed, so as to perform the actual final g ation. In such an ce, the final logic, including the M logic 15a, I logic 15b, and D logic 15c blocks, which blocks together form part of the HMM MID instance 17, may include 7 multipliers and 4 adders along with the various MUXing involved.

Accordingly, sets forth the M, I, and D state update circuits 15a', 15b', and 15c' including the effects of simplifying assumptions d to transition probabilities, as well as the effect of sharing various M, I, and/or D resources, e.g., adder resources, for the final sum operations. A delay block may also be added to the M-state path in the M-state computation block, as shown in . This delay may be added to sate for delays in the actual hardware implementations of the multiply and addition operations, and/or to simplify the control logic, e.g., 15.

] As shown in FIGS. 9 and 10, these respective multipliers and/or adders may be floating point multipliers and adders. However, in s instances, as can be seen with respect to , a log domain configuration may be implemented where in such configuration all of the multiplies tum into adds. presents what log domain calculation would look like if all the multipliers turned into adders, e.g., 15a", 15b", and 15c", such as occurs when ing a log domain computational configuration. Particularly, all of the multiplier logic turns into an adder, but the adder itself turns into or otherwise es a function where the function such as: f(a,b) = max(a,b)- log2(1+2A(-[a-b]), such as where the log portion of the equation may be maintained within a LUT whose depth and physical size is determined by the precision required.

Given the typical read and haplotype sequence lengths as well as the values typically seen for read quality ) scores and for the related transition probabilities, the dynamic range requirements on the internal HMM state values may be quite . For instance, when implementing the HMM module in software, various of the HMM jobs 20 may result in underruns, such as when implemented on single-precision (32-bit) floatingpoint state values. This implies a dynamic range that is greater than 80 powers of 10, thereby requiring the variant call software to bump up to double-precision (64-bit) floating point state values. However, full 64-bit double-precision floating-point representation may, in various instances, have some negative implications, such as if compact, high-speed hardware is to be implemented, both storage and e pipeline resource ements will need to be increased, thereby occupying greater chip space, and/or slowing timing. In such instances, a point-only linear-domain number representation may be implemented. Nevertheless, the dynamic range demands on the state values, in this embodiment, make the bit widths involved in certain circumstances less than desirable. Accordingly, in such instances, fixedpoint-only log-domain number representation may be implemented, as described herein.

] In such a scheme, as can be seen with respect to , instead of representing the actual state value in memory and computations, the -log-base-2 of the number may be represented. This may have several advantages, including employing multiply operations in linear space that translate into add operations in log space; and/or this log domain representation of numbers inherently supports wider dynamic range with only small increases in the number of integer bits. These log-domain M, I, D state update calculations are set forth in FIGS. 11 and 12.

As can be seen when comparing the logic 17 configuration of with that of the multiply ions go away in the main. Rather, they are replaced by add operations, and the add operations are morphed into a function that can be sed as a max operation followed by a correction factor addition, e.g., via a LUT, where the correction factor is a function of the difference n the two values being summed in the log-domain. Such a correction factor can be either computed or ted from the look-uptable.

Whether a correction factor computation or look-up-table implementation is more efficient to be used depends on the required precision (bit width) on the difference between the sum . In particular instances, therefore, the number of main bits for state representation can be in the neighborhood of 8 to 12 integer bits plus 6 to 24 fractional bits, ing on the level of quality desired for any given entation. This implies ere between 14 and 36 bits total for log-domain state value representation. Further, it has been determined that there are log-domain fixed-point representations that can provide acceptable quality and acceptable hardware size and speed.

In various instances, one read sequence is typically processed for each HMM job 20, which as indicated may include a comparison against two haplotype sequences. And like above for the haplotype memory, a ong structure may also be used in the read sequence memory 18 to allow various software implemented functions the ability to write new HMM job information 20b while a current job 20a is still being processed by the HMM engine instance 13. Hence, a read sequence storage requirement may be for a single 1024x32 two-port memory (such as one port for write, one port for read, and/or separate clocks for write and read ports). ularly, as described above, in vanous instances, the architecture employed by the system 1 is configured such that in determining whether a given base in a sequenced sample genome matches that of a ponding base in one or more reference genomes, a virtual matrix 30 is formed, wherein the reference genome is theoretically set across a ntal axis, while the sequenced reads, representing the sample genome, is theoretically set in descending fashion down the vertical axis. Consequently, in performing an HMM calculation, the HMM processing engine 13, as herein described, is configured to traverse this virtual HMM matrix 30. Such processing can be depicted as in as a swath 35 moving diagonally down and across the virtual array performing the various HMM calculations for each cell ofthe virtual array, as seen in More particularly, this theoretical sal involves processmg a first grouping ofrows of cells 35a from the matrix 30 in its entirety, such as for all haplotype and read bases within the grouping, before proceeding down to the next grouping of rows 35b (e.g., the next group of read bases). In such an instance, the M, I, and D state values for the first grouping are stored at the bottom edge ofthat initial grouping ofrows so that these M, I, and D state values can then be used to feed the top row ofthe next ng (swath) down in the matrix 30. In s instances, the system 1 may be ured to allow up to 1008 length ypes and/or reads in the HMM accelerator 8, and since the numerical representation employs W-bits for each state, this implies a 1008word x W-bit memory for M, I, and D state storage. ] ingly, as indicated, such memory could be either a single-port or double-port memory. Additionally, a cluster-level, scratch pad memory, e.g., for storing the results of the swath boundary, may also be provided. For instance, in accordance with the disclosure above, the memories discussed already are configured for a per-engine-instance 13 basis. In particular HMM entations, le engine instances 13a-(n+l) may be grouped into a cluster 11 that is serviced by a single tion, e.g., PCie bus 5, to the PCie interface 4 and DMA 3 via CentCom 9. Multiple clusters lla-(n+l) can be instantiated so as to more efficiently utilize PCie bandwidth using the existing CentCom 9 functionality.

Hence, in a typical configuration, somewhere n 16 and 64 engines 13m are instantiated within a cluster 1ln, and one to four clusters might be tiated in a typical FPGA/ASIC implementation of the HMM 8 (e.g., depending on whether it is a dedicated HMM FPGA image or whether the HMM has to share FPGA real estate with the sequencer/mapper/aligner and/or other modules, as herein disclosed). In particular instances, there may be a small amount of memory used at the cluster-level 11 in the HMM re.

This memory may be used as an elastic First In First Out ("FIFO") to capture output data from the HMM engine instances 13 in the cluster and pass it on to CentCom 9 for further transmittal back to the software of the CPU 1000 via the DMA 3 and PCie 4. In , this FIFO could be very small (on the order of two 32-bit words), as data are typically passed on to CentCom 9 almost immediately after arriving in the FIFO. r, to absorb potential disrupts in the output data path, the size of this FIFO may be made parametrizable. In particular instances, the FIFO may be used with a depth of 512 words. Thus, the cluster-level storage requirements may be a single 512x32 two-port memory (separate read and write ports, same clock domain). sets forth the vanous HMM state transitions 17b depicting the relationship between Gap Open Penalties (GOP), Gap Close Penalties (GCP), and transition probabilities involved in determining whether and how well a given read sequence matches a particular haplotype sequence. In performing such an analysis, the HMM engine 13 includes at least three logic blocks 17b, such as a logic block for determining a match state 15a, a logic block for determining an insert state 15b, and a logic block for ining a delete state 15c.

These M, I, and D state calculation logic 17 when riately ured function efficiently to avoid andwidth bottlenecks, such as of the HMM computational flow.

However, once the M, I, D core computation architecture is determined, other system enhancements may also be configured and implemented so as to avoid the development of other bottlenecks within the system.

Particularly, the system 1 may be configured so as to maximize the process of ently g information from the computing core 1000 to the variant caller module 2 and back again, so as not to produce other bottlenecks that would limit overall throughput.

One such block that feeds the HMM core M, I, D state computation logic 17 is the transition probabilities and priors calculation block. For instance, as can be seen with respect to each clock cycle employs the tation of seven transition probabilities and one Prior at the input to the M, I, D state computation block 15a. However, after the simplifications that result in the ecture of , only four unique transition probabilities and one Prior are employed for each clock cycle at the input of the M, I, D state computation block.

Accordingly, in various instances, these calculations may be simplified and the ing values generated. Thus, increasing throughput, efficiency, and ng the possibility of a bottleneck forming at this stage in the process. onally, as described above, the Priors are values generated via the read quality, e.g., Phred score, of the particular base being investigated and whether, or not, that base matches the hypothesis haplotype base for the current cell being evaluated in the virtual HMM matrix 30. The relationship can be described via the equations bellow: First, the read Phred in question may be expressed as a ility= read Phred/10)). Then the Prior can be computed based on whether the read base matches the hypothesis haplotype base: If the read base and hypothesis haplotype base match: Prior = 1 - read Phred expressed as a probability. Otherwise: Prior= (read Phred expressed as probability)/3. The divide-by-three operation in this last equation reflects the fact that there are only four possible bases (A, C, G, T). Hence, if the read and haplotype base did not match, then it must be one of the three remaining possible bases that does match, and each of the three possibilities is modeled as being equally likely.

] The per-read-base Phred scores are delivered to the HMM hardware accelerator 8 as 6-bit . The equations to derive the Priors, then, have 64 possible outcomes for the "match" case and an additional 64 possible outcomes for the "don'tmatch" case. This may be efficiently implemented in the hardware as a 128 word look-up-table, where the address into the look-up-table is a 7-bit ty formed by concatenating the Phred value with a single bit that indicates whether, or not, the read base matches the hypothesis haplotype base.

Further, with respect to determining the match to insert and/or match to delete probabilities, in various implementations of the architecture for the HMM hardware accelerator 8, separate gap open penalties (GOP) can be specified for the Match-to-Insert state tion, and the Match-to-Delete state transition, as indicated above. This equates to the M2I and M2D values in the state tion diagram of being different. As the GOP values are delivered to the HMM hardware accelerator 8 as 6-bit Phred-like values, the gap open transition probabilities can be computed in accordance with the following equations: M2I transition probability read GOP(I)/10)) and M2D transition probability= lW'(-(read GOP(D)/10)). Similar to the Priors derivation in hardware, a simple 64 word p-table can be used to derive the M2I and M2D values. If GOP(I) and GOP(D) are inputted to the HMM hardware 8 as potentially different values, then two such look-up-tables (or one resource-shared look-up-table, potentially clocked at twice the frequency ofthe rest ofthe circuit) may be utilized.

Furthermore, with respect to determining match to match transition probabilities, in various ces, the match-to-match transition probability may be calculated as: M2M transition probability = 1 - (M2I transition probability + M2D transition probability). Ifthe M2I and M2D transition probabilities can be ured to be less than or equal to a value of½,then in various embodiments the equation above can be implemented in hardware in a manner so as to increase overall efficiency and throughput, such as by reworking the equation to be: M2M transition probability= (0.5 - M2I tion probability) + (0.5 - M2D transition probability). This rewriting of the equation allows M2M to be derived using two 64 element look-up-tables followed by an adder, where the look-up-tables store the results.

Further still, with t to determining the Insert to Insert and/or Delete to Delete transition probabilities, the 121 and D2D transition probabilities are functions of the gap continuation probability (GCP) values inputted to the HMM hardware accelerator 8. In various instances, these GCP values may be 6-bit Phred-like values given on a per-read-base basis. The 121 and D2D values may then be derived as shown: 121 transition probability = lW'(-(read GCP(I)/10)), and D2D transition probability= lW'(-(read GCP(D)/10)). Similar to some of the other transition probabilities sed above, the 121 and D2D values may be efficiently implemented in hardware, and may e two look-up-tables (or one resourceshared p-table), such as having the same form and contents as the to-Indel lookup-tables discussed previously. That is, each look-up-table may have 64 words.

Additionally, with respect to determining the Inset and/or Delete to Match ilities, the I2M and D2M tion probabilities are functions of the gap continuation probability (GCP) values and may be computed as: I2M tion probability = 1 - 121 transition probability, and D2M transition probability= 1 - D2D transition probability, where the 121 and D2D transition probabilities may be derived as discussed above. A simple subtract operation to implement the equations above may be more expensive in hardware resources than simply implementing r 64 word p-table and using two copies of it to implement the I2M and D2M derivations. In such instances, each look-up-table may have 64 words. Of course, in all relevant embodiments, simple or complex subtract ions may be formed with the suitably configured hardware. provides the circuitry 17a for a simplified calculation for HMM transition probabilities and Priors, as described above, which supports the general state transition diagram of . As can be seen with respect to , in s instances, a simple HMM hardware rator architecture 17a is presented, which accelerator may be configured to include separate GOP values for Insert and Delete transitions, and/or there may be separate GCP values for Insert and Delete transitions. In such an instance, the cost of generating the seven unique transition probabilities and one Prior each clock cycle may be ured as set forth below: eight 64 word look-up-tables, one 128 word look-up-table, and one adder.

Further, in various instances, the hardware 2, as ted herein, may be configured so as to fit as many HMM engine ces 13 as possible onto the given chip target (such as on an FPGA, sASIC, or ASIC). In such an instance, the cost to implement the transition probabilities and priors generation logic 17a can be substantially reduced relative to the costs as provided by the below configurations. Firstly, rather than supporting a more general version of the state transitions, such as set forth in , e.g., where there may be separate values for GOP(I) and GOP(D), rather, in various instances, it may be assumed that the GOP values for insert and delete transitions are the same for a given base. This results in l simplifications to the hardware, as indicated above.

In such instances, only one 64 word p-table may be employed so as to generate a single l value, replacing both the M2I and M2D transition probability values, whereas two tables are lly employed in the more general case. Likewise, only one 64 word look-up-table may be used to generate the M2M transition probability value, whereas two tables and an add may typically be employed in the general case, as M2M may now be calculated as 1-2xM2Indel.

Secondly, the assumption may be made that the sequencer-dependent GCP value for both insert and delete are the same AND that this value does not change over the course of an HMM job 20. This means that: a single Indel transition probability may be calculated d ofseparate 121 and D2D values, using one 64 word look-up-table instead of two tables; and single Indel2Match transition probability may be ated instead of separate I2M and D2M values, using one 64 word look-up-table instead oftwo tables.

] Additionally, a further simplifying assumption can be made that assumes the Inset2Insert and Delete2Delete (I2I and D2D) and Insert2Match and Delete2Match (I2M and D2M) values are not only identical between insert and delete transitions, but may be static for the particular HMM job 20. Thus, the four look-up-tables associated in the more general architecture with 121, D2D, I2M, and D2M tion probabilities can be ated altogether. In various of these instances, the static Indel2Indel and Indel2Match probabilities could be made to be entered via software or via an RTL parameter (and so would be bitstream programmable in an FPGA). In certain instances, these values may be made eam-programmable, and in certain instances, a training mode may be implemented employing a training sequence so as to further refine transition probability accuracy for a given sequencer run or genome analysis. sets forth what the new state tion 17b diagram may look like when enting these various simplifying assumptions. Specifically, sets forth the simplified HMM state transition diagram depicting the relationship between GOP, GCP, and tion probabilities with the simplifications set forth above.

Likewise, sets forth the circuitry 17a,b for the HMM transition probabilities and priors generation, which supports the simplified state transition m of . As seen with respect to , a circuit realization of that state transition diagram is provided. Thus, in various instances, for the HMM hardware accelerator 8, the cost of generating the transition ilities and one Prior each clock cycle reduces to: Two 64 word look-up-tables, and One 128 word look-up-table.

As set forth above, the engine control logic 15 is configured for generating the virtual matrix and/or traversing the matrix so as to reach the edge ofthe swath, e.g., via highlevel engine state machines, where result data may be finally summed, e.g., via final sum control logic 19, and stored, e.g., via put/get logic.

Accordingly, as can be seen with respect to , in various embodiments, a method for producing and/or traversing an HMM cell matrix 30 is provided. Specifically, sets forth an example of how the HMM accelerator l logic 15 goes about traversing the virtual cells in the HMM matrix. For instance, assuming for exemplary purposes, a 5 clock cycle latency for each multiply and each add operation, the worst-case y through the M, I, D state update calculations would be the 20 clock cycles it would take to propagate through the M update calculation. There are half as many operations in the I and D state update calculations, implying a 10 clock cycle latency for those operations.

These latency implications of the M, I, and D compute operations can be understood with respect to , which sets forth s examples ofthe cell-to-cell data dependencies. In such instances, the M and D state information of a given cell feed the D state computations of the cell in the HMM matrix that is ately to the right (e.g., having the same read base as the given cell, but having the next haplotype base). Likewise, the M and I state ation for the given cell feed the I state computations ofthe cell in the HMM matrix that is immediately below (e.g., having the same haplotype base as the give cell, but having the next read base). So, in particular instances, the M, I, and D states of a given cell feed the D and I state computations of cells in the next diagonal of the HMM cell matrix.

Similarly, the M, I, and D states of a given cell feed the M state computation of the cell that is to the right one and down one (e.g., having both the next haplotype base AND the next read base). This cell is ly two diagonals away from the cell that feeds it (whereas, the I and D state calculations rely on states from a cell that is one diagonal away).

This quality ofthe I and D state calculations relying on cells one diagonal away, while the M state calculations rely on cells two diagonals away, has a cial result for hardware design.

Particularly, given these configurations, I and D state calculations may be adapted to take half as long (e.g., 10 cycles) as the M state ations (e.g., 20 cycles).

Hence, if M state calculations are started 10 cycles before I and D state calculations for the same cell, then the M, I, and D state ations for a cell in the HMM matrix 30 will all te at the same time. Additionally, if the matrix 30 is traversed in a diagonal fashion, such as having a swath 35 of about 10 cells each within it (e.g., that spans ten read bases), then: The M and D states produced by a given cell at (hap, rd) coordinates (i, j) can be used by cell (i+ 1, j) D state calculations as soon as they are all the way through the compute pipeline ofthe cell at (i, j).

The M and I states produced by a given cell at (hap, rd) coordinates (i, j) can be used by cell (i, j+1) I state calculations one clock cycle after they are all the way through the compute pipeline of the cell at (i, j). Likewise, the M, I and D states produced by a given cell at (hap, rd) coordinates (i, j) can be used by cell (i+ 1, j+1) M state calculations one clock cycle after they are all the way through the compute pipeline of the cell at (i, j). Taken together, the above points establish that very little dedicated storage is needed for the M, I, and D states along the diagonal of the swath path that spans the swath length, e.g., of ten reads. In such an instance, just the registers required to delay cell (i, j) M, I, and D state values one clock cycle for use in cell (i+ 1, j+1) M calculations and cell (i, j+1) I calculations by one clock cycle). Moreover, there is at of a virtuous cycle here as the M state computations for a given cell are begun 10 clock cycles before the I and D state calculations for that same cell, ly outputting the new M, I, and D states for any given cell simultaneously.

In view of the above, and as can be seen with respect to , the HMM accelerator control logic 15 may be configured to process the data within each of the cells of the virtual matrix 30 in a manner so as to traverse the matrix. ularly, in various embodiments, operations start at cell (0,0), with M state calculations beginning 10 clock cycles before I and D state calculations begin. The next cell to traverse should be cell (1,0).

However, there is a ten cycle latency after the start of I and D ations before the results from cell (0,0) will be available. The hardware, therefore, inserts nine "dead" cycles into the compute pipeline. These are shown as the cells with haplotype index less than zero in FIG.

After completing the dead cycle that has an effective cell position in the matrix of (-9,-9), the M, I, and D state values for cell (0,0) are available. These (e.g., the M and D state outputs of cell (0,0)) may now be used straight away to start the D state computations of cell (0, 1). One clock cycle later, the M, I, and D state values from cell (0,0) may be used to begin the I state computations of cell (0, 1) and the M state computations of cell (1,1).

The next cell to be traversed may be cell (2,0). r, there is a ten cycle y after the start of I and D ations before the results from cell (1,0) will be WO 14320 PCT/0S2017/036424 available. The hardware, therefore, s eight dead cycles into the compute pipeline. These are shown as the cells with haplotype index less than zero, as in along the same diagonal as cells (1,0) and (0,1). After completing the dead cycle that has an effective cell position in the matrix of (-8, -9), the M, I, and D state values for cell (1,0) are available.

These (e.g., the Mand D state outputs of cell (1,0)) are now used straight away to start the D state ations ofcell (2,0).

One clock cycle later, the M, I, and D state values from cell (1,0) may be used to begin the I state computations of cell (1,1) and the M state computations of cell (2,1). The M and D state values from cell (0, 1) may then be used at that same time to start the D state calculations of cell (1,1). One clock cycle later, the M, I, and D state values from cell (0,1) are used to begin the I state computations of cell (0,2) and the M state computations of cell (1,2).

Now, the next cell to traverse may be cell (3,0). However, there is a ten-cycle latency after the start of I and D calculations before the s from cell (2,0) will be available. The hardware, therefore, inserts seven dead cycles into the compute pipeline. These are again shown as the cells with haplotype index less than zero in along the same diagonal as cells (2,0), (1,1), and (0,2). After completing the dead cycle that has an effective cell on in the matrix of (-7,-9), the M, I, and D state values for cell (2,0) are available.

These (e.g., the M and D state outputs of cell (2,0)) are now used straight away to start the D state computations of cell (3,0). And, so, computation for another ten cells in the diagonal begins.

Such processing may continue until the end of the last full diagonal in the swath 35a, which, in this example (that has a read length of 35 and haplotype length of 14), will occur after the diagonal that begins with the cell at (hap, rd) coordinates of (13,0) is completed. After the cell (4,9) in Figure 16 is traversed, the next cell to traverse should be cell (13,1). However, there is a ten-cycle latency after the start of the I and D calculations before the results from cell (12,1) will be available.

The hardware may be configured, therefore, to start operations ated with the first cell in the next swath 35b, such as at coordinates (0, 10). Following the processing of cell (0, 10), then cell (13, 1) can be traversed. The whole diagonal ofcells beginning with cell (13, 1) is then traversed until cell (5, 9) is reached. Likewise, after the cell (5, 9) is traversed, the next cell to se should be cell (13, 2). However, as before there may be a ten-cycle y after the start of I and D calculations before the results from cell (12, 2) will be available. Hence, the hardware may be configured to start operations associated with the first cell in the second diagonal of the next swath 35b, such as at coordinates (1, 10), followed by cell(0, 11).

Following the processing of cell (0, 11), the cell (13, 2) can be traversed, in accordance with the methods disclosed above. The whole diagonal 35 of cells beginning with cell (13,2) is then traversed until cell (6, 9) is reached. Additionally, after the cell (6, 9) is traversed, the next cell to be traversed should be cell (13, 3). However, here again there may be a ten-cycle latency period after the start of the I and D calculations before the results from cell (12, 3) will be available. The hardware, therefore, may be configured to start operations associated with the first cell in the third diagonal ofthe next swath 35c, such as at nates (2, 10), followed by cells (1, 11) and (0, 12), and likewise.

This continues as indicated, in accordance with the above until the last cell in the first swath 35a (the cell at (hap, rd) coordinates (13, 9)) is traversed, at which point the logic can be fully dedicated to traversing diagonals in the second swath 35b, starting with the cell at (9, 10). The pattern outlined above repeats for as many swaths of 10 reads as necessary, until the bottom swath 35c (those cells in this example that are ated with read bases having index 30, or greater) is reached.

In the bottom swath 35, more dead cells may be inserted, as shown in FIG 16 as cells with read indices greater than 35 and with haplotype s greater than 13. onally, in the final swath 35c, an additional row of cells may effectively be added.

These cells are indicated at line 35 in , and relate to a dedicated clock cycle in each diagonal of the final swath where the final sum operations are occurring. In these cycles, the M and I states of the cell immediately above are added together, and that result is itself summed with a running final sum (that is initialized to zero at the left edge of the HMM matrix 30).

Taking the discussion above as context, and in view of , it is possible to see that, for this example of read length of 35 and haplotype length of 14, there are 102 dead cycles, 14 cycles associated with final sum operations, and 20 cycles line latency, for a total of 102+14+20 = 146 cycles of overhead. It can also be seen that, for any HMM job with a read length greater than 10, the dead cycles in the upper left comer of are ndent ofread length. It can also be seen that the dead cycles at the bottom and bottom right portion of are dependent on read length, with fewest dead cycles for reads having mod(read length, 10) = 9 and most dead cycles for mod(read length, 10) = 0. It can further be seen that the overhead cycles become smaller as a total percentage ofHMM matrix tion cycles as the haplotype lengths increase (bigger matrix, partially fixed number of overhead cycles) or as the read lengths increase (note: this refers to the percentage of overhead associated with the final sum row in the matrix being reduced as read length -rowcount-increases ). Using such histogram data from representative whole human genome runs, it has been determined that traversing the HMM matrix in the manner described above s in less than 10% overhead for the whole genome processing. r s may be employed to reduce the amount of overhead cycles including: Having dedicated logic for the final sum operations rather than sharing adders with the M and D state calculation logic. This eliminates one row of the HMM matrix 30. Using dead cycles to begin HMM matrix ions for the next HMM job in the queue.

Each grouping of ten rows of the HMM matrix 30 constitutes a "swath" 35 in the HMM accelerator function. It is noted that the length of the swath may be sed or decreased so as to meet the efficiency and/or throughput demands of the system. Hence, the swatch length may be about five rows or less to about fifty rows or more, such as about ten rows to about forty-five rows, for instance, about fifteen or about twenty rows to about forty rows or about thirty-five rows, including about twenty five rows to about thirty rows of cells in length.

With the exceptions noted in the section, above, related to harvesting cycles that would otherwise be dead cycles at the right edge of the matrix of , the HMM matrix may be processed one swath at a time. As can be seen with respect to , the states of the cells in the bottom row of each swath 35a feed the state ation logic in the top row of the next swath 35b. Consequently, there may be a need to store (put) and retrieve (get) the state information for those cells in the bottom row, or edge, ofeach swath.

The logic to do this may include one or more ofthe following: when the M, I, and D state computations for a cell in the HMM matrix 30 complete for a cell with mod(read index, 10) = 9, save the result to the M, I, D state storage . When M and I state computations (e.g., where D state computations do not require information from cells above them in the matrix) for a cell in the HMM matrix 30 begin for a cell with mod(read index, 10) = 0, retrieve the previously saved M, I, and D state information from the appropriate place in the M, I, D state e memory. Note in these instances that M, I, and D state values that feed row 0 (the top row) M and I state calculations in the HMM matrix 30 are simply a predetermined constant value and do not need to be recalled from memory, as is true for the Mand D state values that feed column 0 (the left column) D state calculations.

As noted above, the HMM rator may or may not include a dedicated summing resource in the HMM hardware accelerator such that exist simply for the purpose of the final sum operations. However, in particular instances, as described herein, an additional row may be added to the bottom ofthe HMM matrix 30, and the clock cycles associated with this extra row may be used for final summing operations. For instance, the sum itself may be achieved by borrowing (e.g., as per ) an adder from the M state computation logic to do the M+I operation, and further by borrowing an adder from the D state computation logic to add the newly formed M+I sum to the running final sum accumulation value. In such an instance, the control logic to activate the final sum operation may kick in whenever the read index that guides the HMM traversing operation is equal to the length of the inputted read sequence for the job. These operations can be seen at line 34 toward the bottom ofthe sample HMM matrix 30 of.

Hence, as can be seen above, in one implementation, the variant caller may make use of the mapper and/or aligner engines to determine the likelihood as to where s reads originated, such as with t to a given location, e.g., chromosomal location.

In such ces, the variant caller may be configured to detect the underlying sequence at that location, such as ndently of other regions not immediately adjacent to it. This is particularly useful and works well when the region of interest does not resemble any other region of the genome over the span of a single read (or a pair of reads for paired-end sequencing). However, a significant fraction of the human genome does not meet this criterion, which can make variant calling, e.g., the process of reconstructing a subject's genome from the reads that an NGS produces, challenging.

Particularly, though DNA sequencing has ed dramatically, variant calling remains a difficult m, largely due to the genome's redundant structure. As disclosed herein, r, the complexities presented by the genome's redundancy may be overcome, at least in part, from a perspective driven by short read data. More ularly, the devices, systems, and methods of employing the same as sed herein may be configured in such a manner so as to focus on Homologous or Similar s that may otherwise have been characterized by low variant calling cy. In certain instances, such low variant calling accuracy may stem from difficulties ed in read mapping and alignments with respect to homologous regions that typically may result in very low read MAPQs.

Accordingly, presented herein are strategic entations that accurately call variants (SNPs, Indels, and the like) in homologous regions, such as by y considering the information present in these homologous regions.

For instance, many regions of the genome are homologous, e.g., they have near-identical copies located elsewhere in the genome, e.g., in multiple locations, and as a result, the true source location of a read may be subject to considerable uncertainty. ically, if a group of reads is mapped with low confidence, e.g., due to apparent homology, a typical variant caller may ignore and not s the reads, even though they may contain useful information. In other instances, if a read is mis-mapped (e.g., the primary alignment is not the true source of the read), detection errors may result. More specifically, previously implemented short-read sequencing technologies have been tible to these problems, and conventional detection methods often leaves large regions ofthe genome in the dark.

] In some instances, long-read sequencing can be employed to mitigate these problems, but it typically has much higher cost and/or higher error rates, takes longer, and/or suffers from other shortcomings. Therefore, in various instances, it may be beneficial to perform a region joint ion operation as herein described. For instance, instead of considering each region in isolation and/or instead of performing and analyzing long read sequencing, multi-region joint ion (MRJD) methodologies may be employed, such as where the MRJD protocol considers multiple, e.g., all, locations from which a group of reads may have originated, and attempts to detect the underlying sequences together, e.g., jointly, using all available information, which may be regardless of low or abnormal confidence and/or certainty scores.

For example, for a diploid organism with statistically uniform coverage, a brute force Bayesian calculation, as described above, may be performed in a variant call analysis. However, in a brute force MLRD computation, the xity of the calculation grows rapidly with the number of regions N, and the number of candidate haplotypes K to be considered. Particularly, to consider all combinations of candidate haplotypes, the number of candidate solutions for which to calculate probabilities may often times be exponential. For instance, as described in greater detail below, in a brute force implementation, the number of candidate ypes includes the number of active positions, which if a graph-assembly WO 14320 PCT/0S2017/036424 technique is used to generate the list of candidate haplotypes in a variant call operation, such as in the building of a De Brujin graph as disclosed herein, then the number of active positions is the number of independent "bubbles" in the graph. Hence, such a brute-force calculation can be prohibitively expensive to implement, and as such brute force Bayesian calculations can be prohibitively complex.

Accordingly, in one aspect, as set forth in A, a method to reduce the complexity of such brute force calculations is herein provided. For instance, as disclosed above, though the speed and accuracy of DNA/RNA sequencing has improved dramatically, especially with t to the methods disclosed herein, variant calling, e.g., the process of reconstructing a subject's genome from the reads a sequencer produces, remains a difficult problem, largely due to the genome's redundant structure. The devices, systems, and methods disclosed herein therefore are ured to reduce the complexities presented by the genome's ancy from a perspective driven by short read data in contrast to long read sequencing. In ular, provided herein are methods for performing very long read detection that accounts for homologous and/or similar s of the genome that are usually characterized by low t calling accuracy without necessarily having to perform long read sequencmg.

For instance, in one ment, a system and method for performing multi region joint detection is provided. Specifically, in a first instance, a general variant calling operation may be performed such as employing the s disclosed herein. Particularly, a general variant caller may employ a reference genome sequence, which reference genome presents all the bases in a model genome. This reference forms the backbone of an analysis by which a subject's genome is compared to the reference genome. For instance, as discussed above, employing a Next Gen cer, a subject's genome may be broken down into subsequences, e.g., reads, typically about 100 - 1,000 bases each, which reads may be mapped and aligned to the reference, much like putting a jigsaw puzzle together.

Once the subject's genome has been mapped and/or aligned, usmg this reference genome in ison to the subject's actual genome, it may be determined to what extent, and how the subject'sgenome differs from the reference genome, e.g., on a base by base basis. Particularly, in comparing the subject's genome to one or more reference genomes, such as on a base by base basis, the analysis moves iteratively along the sequences comparing the one with the other(s) to determine ifthey agree or ee. Accordingly, each base within the sequences represents a position to be called, such as ented by position A in A.

Specifically, for every position A of the reference to be called with respect to the subject's genome, a pile up of sequences, e.g., reads, will be mapped and aligned in such a manner that a large sample set ofreads may all overlap one another at any given position A.

Particularly, this oversampling can include a number ofreads, e.g., from one to a hundred or more, where each of the reads in the pileup have tides overlapping the region being called. The calling of these reads from base to base, therefore, involves the formation of a processing window that slides along the sequences making calls, where the length of the window, e.g., the number of bases under examination at any given time, forms the active region of determination. Hence, the window ents the active region of bases in the sample being called, where the calling involves comparing each base at a given on, e.g., A, in all of the reads of the pile up within the active region, where the identity of the base at that position in the number of pile up of reads, es evidence for the true identity of the base at that position being called.

For this e, based on the relevant MAPQ confidence score derived for each read segment, it may be generally ined, within a certain confidence score, that the mapping and aligning was med accurately. However, the question still remains, no matter how slight, as to whether or not the mapping and aligning ofthe reads is accurate, ofif one or more of the reads really belong to someplace else. Accordingly, in one aspect, provided herein are devices and methods for improving the confidence in performing variant calling.

Particularly, in vanous instances, the variant caller can be ured to perform one or more multi-region joint detection ions, as herein described, which may be employed to give greater confidence in the achievable results. For instance, in such an instance, the variant caller may be configured to analyze the various regions in the genome so as to determine particular regions that appear to be similar. For example, as can be seen with respect to A, there may be a nce region A, and a reference region B, where the referenced sequences are very similar to one r, e.g., but with a few regions of dissimilar base pair matching, such as where example RefA has an "A," and example Ref B has a "T", but outside of these few dissimilates, everyplace else within the region in question may appear to match. Because ofthe extent of similarities, these two regions, e.g., RefA and RefB, will typically be considered homologous, or paralogous, regions.

As depicted, the two reference regions A and B are 99% similar. There may be other regions, e.g., Ref's C and D, which are relatively r, e.g., about 93% similar, but as compared to the 99% similarity between reference regions A and B, the nce regions C and D would not be considered homologous, or at least would have a lessor chance of actually being homologous. In such an instance, the variant calling procedures may be able to adequately call out the differences between reference regions C and D, but may, in certain instances, have difficulties calling out the differences between the highly homologous regions of reference regions A and B, e.g., because of their high homology. ularly, because of the extent ofthe dissimilarity between reference sequences A and B to reference sequences C and D, it would not be expected that reads that map and align to either Ref Seq A or B, would mistakenly be mapped to Ref Seq C or D. However, it might be expected that reads that map and align to Ref Seq A may be mis-mapped to Ref Seq B.

Given the extent ofthe homology, mis-mapping between regions A and B may be quite likely. Accordingly, to increase accuracy it may be ble for the system to be able to guish and/or t for the difference between homologous regions, such as when performing a mapping, ng, and/or variant calling procedure. Specifically, when generating a pile up of reads that map and align to a region within Ref A, and generating a pile up of reads that map and align to a region within Ref B, any of the reads may in fact be mis-mapped to the wrong place, and as such, to effectuate better accuracy, when performing the variant g operations disclosed , these homologous regions, and the reads mapped and aligned o, should be ered together, such as in a joint detection protocol, e.g., a multi-region joint detection protocol, as described herein.

Accordingly, presented herein, are devices, systems, as well as the methods of their use, which are directed to multi-region joint detection (MRJD), such as where a plurality, e.g., all, ofthe reads from the s pileups ofthe various identified homologous regions are considered together, such as where instead of making a single call for each location, a joint call is made for all locations that appear to be homologous. Making such joint calls is advantageous e before attempting to make a call for each reference individually, it would first have to be determined to which region, of which reference, the various reads in question actually map and align, and that is inherently uncertain, and the very problem being solved by the proposed joint detection. Hence, e the regions ofthe two references are so similar, it is very difficult to determine which reads map to which regions. However, if these regions are called jointly, it is not necessary to make an upfront decision about which homologous reads map to which reference . Therefor, when making a joint call, the assumption may be made that any reads in a pileup ofa region on one reference, e.g., A, that is homologous to another region on a second nce, e.g., B, could belong to either Ref. A or Ref. B. uently, where desired, an MRJD protocol may be implemented on addition to the variant call algorithm implemented in the s, systems, and methods herein. For instance, in one iteration, a variant call algorithm takes the evidence presented in the mapped and/or aligned reads for a given region in the sample and reference genomes, es the possibility that what appears to be in the sample's genome is in fact present, based on a comparison with the reference genome, and makes a decision given the evidence as to how the sample actually differs from the reference, e.g., given this evidence the variant caller algorithm determines the most likely answer of what's different between the read and the reference. However, MRJD is a further algorithm that may be implemented along with the VC algorithm, where the MRJD is configured to help the variant caller to more accurately determine if an observed ence, e.g., in the subject's read, is in fact a true deviation from the reference.

] Accordingly, the first step in an MJRD analysis involves the identification of homologous s, based on a tage of correspondence between the sequence in a plurality of regions of one or more references, e.g., Ref. A and Ref. B, and the pileup sequences in one or more regions of the subject's reads. Particularly, Ref. A and Ref. B may ly be diploid forms of the same genetic material, such as where there are two copies of a given region of the chromosome. Hence, where diploid references are being analyzed, at various positions RefA may have one particular nucleotide, and at that same position in Ref.

B, another nucleotide may be present. In this e, Ref. A and Ref. B, are homozygous at position A for "A". However, as can be seen in A, the DNA of the subject is heterozygous at this position A, such as where with respect to the reads of the pile up of Ref.

A, one allele of the subject's chromosome has an "A", but the other allele has a "C", yet with respect to Ref. B, another copy of the subject's chromosome has an "A" for both s at position A. This also becomes more complicated, where the sample being analyzed contains a mutation, e.g., at one of those naturally occurring variable positions, such as a heterozygous SNP at position A (not shown).

As can be seen with respect to Ref. A of B, at position A, the subject's sample may include reads that indicate there is zygosity at position A, such as where some ofthe reads include a "C" at this position, and some ofthe reads indicate an "A" at this position (e.g., Haplotypea1 = "A", Ha2 = "C"); while with respect to Ref. B, the reads at position A indicate homozygosity, such as where all the reads in the pileup have an "A" at that position (e.g., Hb1 = "A", Hb2 = "A"). However, MRJD overcomes these difficulties by making a joint call aneously, by ing all of the reads that get mapped to both regions ofthe reference, while considering the possibility that any one of the reads may be in the wrong location. After the various homologous regions are identified, the next step is to determine the correspondence n the homologous nce regions, and then, with respect to MRJD, the mapper and/or aligners determination as to where the various applicable reads are "supposed to map" between the two gous regions may be discarded, and rather, all of the reads in any of the pileups in these homologous regions may be ered collectively together, knowing that any of these reads may belong to any of the homologous regions being compared. Hence, the calculations for determining these joint calls, as set forth in detail below, considers the possibility that any of these reads came from any of the homologous reference regions, and, where applicable, from either haplotype of either of the reference regions.

It is to be noted, although the preceding was with reference to multiple s ofhomology within a reference, the same analysis may be applied for single region detection as well. For instance, as can be seen with respect to B, even for a single region, for any given region, there may be two separate haplotypes present, e.g., H 1 and H2, that the subjects genetic sample may have for a particular region, and because they are haplotypes, they are likely to be very similar to one another. Consequently, ifthese positions are analyzed one in isolation of the other, it may be hard to determine if there are true variations being considered. Thus, the calculations being performed with respect to gous regions are useful for non-homologous regions as well, e any specific region is likely to be diploid, e.g., having both a first haplotype (H1) and a second haplotype (H2), and so being analyzing the regions jointly will enhance the accuracy of the system. Likewise, for a tworeference region, e.g., a homologous region, as described above, what is being called is an HA1 and HA2 for the first region, and an HA1 and HA2 for the second region (which is equivalent two strands for each chromosome and two regions for each strand = 4 dtypes, generally.

Accordingly, MRJD may be employed to determine an initial , with respect to one or more, e.g., all, gous regions, and then single region detection may be applied back to one or more, e.g., all, single or non-homologous regions, e.g., employing the same basic analysis, and thus, better accuracy may be achieved. Hence, single region nonjoint ion may also be performed. For instance, with respect to single region detection, for the candidate haplotypes, HA1, in current iterations the reference region may be about 300-500 base pairs long, and on top of the reference a graph, e.g., a De Bruijn graph, as set forth in C, is built, such as from K-mers from the reads, where any location that differs from the reference forms a divergent pathway or "bubble" in the graph, from which haplotypes are extracted, where each ted haplotype, e.g., divergent pathway, forms a potential hypothesis for what might be on one of the two strands of the chromosomes at a particular location ofthe active region under examination.

However, if there are a lot of divergent pathways, e.g., a lot of bubbles through the graph are formed, as seen with respect to C, and a large number of haplotypes are extracted, then a maximum cutoff may be introduced to keep the calculations manageable. The cutoff can be at any statistically significant number, such as 35, 50, 100, 125-128, 150, 175, 200, or more, etc. Nevertheless, in certain instances, substantially a greater number, e.g., all, ofthe ypes may be considered.

In such an instance, instead of extracting complete source to sink haplotypes from start to finish, e.g., from the ing of the ce to the end, only the sequences associated with the individual bubbles need be ted, e.g., only the bubbles need to be aligned to the reference. Accordingly, the bubbles are extracted from the DBG, the sequences aligned to the reference, and from these alignments, specific SNPs, insertions, deletions, and the like may be determined, with respect as to why the sequences of the various bubbles differ from the reference. Hence, in this regard, all of the different etical haplotypes for analysis may be derived from mixing and matching the sequences pertaining to all of the various s in different combinations. In a manner such as this, all of the haplotypes to be extracted do not need to be enumerated. These methods for ming multi-region joint detection, are described in greater detail herein below. r, abstractly, even though all of these candidate haplotypes may be tested, a growing the tree algorithm may be performed where the graph being produced begins to look like a growing tree. For instance, a branching tree graph of joint haplotypes/diplotypes may be built in such a manner that as the tree grows, the underlying algorithm functions to both grow and prune the tree at the same time as more and more ations are made, and it becomes apparent that various different candidate hypotheses are simply too improbable. Hence, as the tree grows and is pruned, not all ofthe esized haplotypes need to be calculated.

Specifically, with respect to the growing of the tree function, when there is disagreement between two references, or between the references and the reads, as to what base is present at given positions being resolved, it must be determined which base actually belongs in which position, and in view of such disagreements it must be determined which differences may be caused by SNPs, Indels, or the like, versus which are machine errors.

Accordingly, when growing the tree, e.g., extracting bubbles from the De Bruijn graph, such as via SW or NW aligning, and positioning them within the ng tree graph, each bubble to be extracted becomes an event in the tree graph, which represents possible SNPs, Indels, and/or other ences from the reference. See C.

Particularly, in a DBG, the bubbles represent mismatches from the reference, e.g., entative of Indels (which bases have been added or deleted), SNPs (which bases are different), and the like. Consequently, as the bubbles are aligned to the reference(s), the various differences between the two are categorized as events, and a list of the various events, e.g., bubbles, is generated, Therefore, the determination then becomes: what combination of the possible events, e.g., of possible SNPs and Indels, has led to the actual variations in the subject's genetic sequence, e.g., is the truth in each of the actual various haplotypes, e.g., 4, based on probability. More particularly, any one candidate, e.g., joint diplotype candidate, g a root Go senting events for a given segment) may have 4 haplotypes, and each ofthe four haplotypes will form an identified subset ofthe events.

However, as can be seen with respect to D, when performing a growing and/or pruning ofthe tree function, a full list of the entire subset of all combinations of events can be, but need not be, determined all at once. Instead, the determination begins at a single position Go, e.g., one event, and the tree is grown from there one event at a time, which through the pruning function, may leave various low probability events unresolved.

Hence, with t to a growing the tree function, as can be seen with respect to D, the calculation begins with determining the ypes, e.g., HA1, HA2, HB 1, HB2 (for a d organism), where the initial haplotypes are considered to all be unresolved with t to their respective references, e.g., Ref. A and Ref. B, basically with none ofthe events present.

Accordingly, the initial starting point is with the root ofthe tree being G0, and the joint diplotype having all events lved. Then a particular event, e.g., an initial bubble, is selected as the origin for determination, y the initial event is to be resolved for all of the haplotypes, where the event may be a first point of divergence from the reference, such as with respect to the potential presence of an SNP or Indel at position one.

As exemplified in E, at position one, there is an event or bubble, such as an SNP, where a "C" has been substituted for an "A", such that the reference has an "A" at position one, but the read in on has a "C". In such an instance, since for this position in the pileup there are 4 haplotypes, and each may have either an "A", as in the reference, or the event "C", there are potentially 24 = 16 possibilities for resolving this position. Hence, the calculation moves immediately from the root to 16 branches, representing the potential tions for the event at position one.

Therefore, as can be seen with respect to D, all of the ial sequences for all of the four haplotypes may be set forth, e.g., HA1, HA2, HB 1, HB2, where at position one there is either the "A", as in accordance with the reference, or event "C", indicating the presence of an SNP, for that one event, where the event "C" is determined by the examining the various bubble pathways through the graph. So, for each branch or child node, each branch may differ based on the likelihood of the base at on one according to or diverging from the reference, while the rest of the events remain unresolved. This process then will be ed for each branch node, and for each base within the variation bubbles, so as to resolve all events for all haplotypes. Hence, the probabilities may be recalculated for ing any particular read given the various potential haplotypes.

Particularly, for each node, there may be four haplotypes, and each haplotype may be ed against each read in the pileup. For instance, in one embodiment, the SW, NW, and/or HMM , analyzes each node and considers each of the four haplotypes for each node. Consequently, ting each node activates the SW and/or HMM engine to analyze that node by considering all ofthe haplotypes, e.g., 4, for that node in comparison for each of the reads, where the SW and/or HMM engine considers one haplotype for one read for each ofthe haplotypes and each ofthe reads for all ofthe viable nodes.

Hence, if for exemplary purposes of this example, it is the case that there is a heterozygous SNP "C" for the one region of one haplotype, e.g., one strand of one chromosome has a "C", but all ofthe other bases at this position for the other strands do not, e.g., they all match the reference "A", then it would be expected that all of the reads in the pile up support this finding, such as by having a majority of "A"s at position one, and a minority, e.g., about ¼, of the reads having a "C" at position one, for the true node. Thus, if any later observable reads at a different node, show a multiplicity of "Cs" at position one, then that node will be unlikely to be the true node, e.g., will have a low ility, e there will not be enough reads with Cs at this position in the pileup to make their occurrence likely. Specifically, it will be more probable that the nce of a "C" at this position in the reads in question is evidence of a sequencing or other scientific error, rather than being a true haplotype candidate. Consequently, if certain nodes end up having small probabilities, as compared to the true node, it is because they are not supported by a majority of the reads, e.g., in the pileup, and thus, these nodes may be pruned off, thereby discarding the nodes of low probabilities, but in a manner that preserves the true node(s).

Accordingly, once the event one position has been determined, the next event position may be determined, and the processes herein described may then be repeated for that new position with respect to any ofthe surviving nodes that have not heretofore been pruned.

Particularly, event two may be selected from the existing available nodes, and that event can serve as the G1 root for determining the likely identity of the base at on two, such as by once again defining the new haplotypes, e.g., 4, as well as their various branches, e.g., 16, explaining the possible variations with respect to position 2. Hence, through repeating this same process, event 2 may now be ed. Therefore, as can be seen with respect to D, once position 1 has been determined, a new node for position 2 may be selected, and its 16 potential haplotype candidates may be considered. In such an instance, the candidates for each of HA1, HA2, HB 1, HB2 may be determined, but in this instance, since position 1 has y been resolved, with t to determining the nucleotide identify for each of the haplotypes at position 1, it is position 2, that will now be resolved, for each ofthe haplotypes at position 2, as set forth in D, showing the resolution ofposition 2.

Once this process is finished, once all of the events have been processed and ed, e.g., including all children nodes and en of children nodes that have not been pruned, then the nodes ofthe tree that have not been pruned may be examined, and it may be determined based on the probability scores, which tree ents the joint diplotype, e.g., which sequence has the highest probability of being true. Therefore, in this manner, because ofthe g function, the entire tree does not need to be built, e.g., most ofthe tree will end up being pruned as the analysis continues, so the overall amount of calculations is greatly reduced over non-pruning functions, albeit substantially more than ming non-joint diplotype calling, e.g., single region g. Accordingly, the t analytics modules are able to determine and resolve two or more regions of high homology with a high degree of WO 14320 PCT/0S2017/036424 accuracy, e.g., employing joint diplotype analysis, where traditional methods are simply not capable ofresolving such regions at all, e.g., because offalse positives and irresolution.

Particularly, various variant caller implementations may be configured to simply not perform an analysis on regions ofhigh homology. The present iterations overcome these and other such problems in the field. More particularly, the t devices, systems, and their methods ofuse may be ured so as to consider a greater proportion, e.g., all of the haplotypes, e the occurrence of regions of high homology. Of course, the speed of these calculations may further be increased, by not performing certain calculations where it can be determined that the results of such calculations have a low probability of being true, such as by implementing a pruning on, as herein described.

A benefit of these configurations, e.g., diplotype resolution and pruning, is that now the size of the active region window, e.g., of bases being analyzed, may be increased from about a few d of bases being processed to a few thousands, or even tens or hundreds of thousands of bases can be processed together, such as in one contiguous active region. This increase in size ofthe active window of analysis allows for more evidence to be considered when determining the identity of any ular nucleotide at any given position, thereby allowing for a greater context within which a more accurate determination of the identity of the nucleotide may be made. Likewise, a greater context allows for supporting evidence to better be chained together when comparing one or more reads covering one or more regions having one or more deviations from the reference. Hence, in such a manner, one event can be connected to another event, which itself may be connected to another event, etc., and from these connections a more accurate call with t to a given particular event presently under consideration may be made, thereby ng evidence from farther away, e.g., hundreds to thousands s or more away, to be ative in making a present variant call (despite the fact that any given read is only typically hundreds of bases long), thereby further making the processes herein much more accurate.

] Particularly, in a manner such as this, the active region can further be made to include thousands, to tens of thousands, even hundreds of nds of bases or more, and consequently, the method of forming a De Bruijn graph by extracting all of the haplotypes can be avoided, as only a limited number of haplotypes, those with bubbles that may be viable, need be explored, and even ofthose that are viable, once it becomes clear they are no longer viable they may be , and for those that remain viable, chaining may be ed so as to improve the accuracy of the eventual variant calls being made. This is all made possible by quantum and/or Hardware ing. It may also be performed in software by a CPU or a GPU, but it will be slower.

It is to be noted that with respect to the above examples, it is the probability of the input data, e.g., the reads, that are being determined, given these haplotype theories produced by the De Bruijn graph. r, it may also be useful to employ Bayes theorem, such as for determining the probability of reads given a joint diplotype, down to the opposite probability of determining from the theory of a joint diplotype a best fit given the reads and the evidence ed. ingly, as can be seen with respect to C, from the generated De Bruijn graph, once multi-region joint detection, and/or pruning has occurred, a set of potential haplotypes will , and then these haplotypes will be tested against the actual reads of the subject. Specifically, each horizontal cross section represents a ype, e.g., BI, that may then be subjected to another HMM protocol so as to be tested against the reads so as to determine the probability ofa particular read given the haplotype B 1.

However, in certain instances, the haplotype, e.g., BI, may not yet be fully determined, but HMM may still be useful to be performed, and in such an instance, a modified HMM calculation, e.g., a partially determined (PD)-HMM operation, discussed below, may be performed where the haplotype is allowed to have undetermined variants, e.g., SNPs and/or indels, in it that have yet to be determined, and as such, the calculation is similar to calculating the best possible probability for an achievable answer given any combination ants in the unresolved positions. Therefore, this further facilitates the ive g of the tree function, where the actual growing of the tree, e.g., the performing of PD-HMM operations, need not be restricted to only those calculations where all the possible variants are known. Hence, in this manner, a number of PD-HMM ations may be performed, in an iterative fashion, to grow the tree of nodes, despite the fact there are still un-determined regions ofunknown possible events in particular candidate haplotypes, and where it becomes possible to trim the tree, PD-HMM resources may be shifted, fluidly, from calculating pruned nodes so as to process only those possibilities that have the st probability for successful characterizing the true genotype.

Accordingly, when determining the probability of a specific base ly being present at any one position, the identity of the base at that position may be determined based on the identity at that on on each region of each chromosome, e.g., each ype, that represents a viable candidate. Hence, for any candidate, what is being determined is the identity of the given base at the position in question in each of the four haplotypes simultaneously. Particularly, what is being determined is the probability of ing the reads of each of the s given the determined likelihood. Specifically, each candidate represents a joint diplotpye, and so being each candidate includes about four haplotypes, which may be set forth in the following equation as G = genotype, where G = the four haplotypes of a single d region of a chromosome of the genome e.g., a joint diplotype. In such an instance, what is to be calculated is the probability of actually observing each of the identified candidate read bases of the ces in the pileups assuming that they are in fact the truth. This initial determination may be performed by an HMM ype calculation, as set forth herein above.

For instance, for a candidate "Joint Diploidtype" = 4 Haplotypes: (Region A: HA1HA2, and Region B: HB1HB2) = G ➔ P(R/G) as determined by an HMM (Error Model) = II P(r/G) = P(r/HA1) + ... + P(r/Hn) Hence, if it is assumed that the ic haplotype Ha1 is the true sequence in this region, and the read came from there, then what are the odds that this read sequence Ha1 was actually observed. Accordingly, the HMM calculator functions to determine, assuming that the Ha1 haplotype is the truth, what is the likelihood of actually observing the given read sequence in on.

Specifically, if the read actually matches the haplotype, this will be a very high probability, of course. However, if the particular read in question does not match the haplotype, then any deviation from there should be explainable by a scientific error, such as a cing or sequencing machinery error, and not an actual variation. Hence, the HMM calculation is a on ofthe error models. Specifically, it asks what is the probability ofthe necessary combination of errors that would have had to occur so as to observe the particular reads being analyzed. Consequently, in this model not only one region is being considered, but a multiplicity ofpositions at a multiplicity ofregions at a multiplicity ofstrands are being considered simultaneously (e.g., d of considering at most possibly two haplotypes at one , now what is being considered is simultaneously the possibility offour ypes for any given position at any given region, aneously, using all ofthe reads data from all of the regions in question. These processes, e.g., pruning the tree, multi-region joint detection, and PD-HMM, will now be described in greater detail.

WO 14320 PCT/0S2017/036424 Specifically, as can be seen with respect to FIGS. 17 and 18, a high-level processing chain is provided, such as where the processing chain may include one or more of the following steps: Identifying and inputting homologous regions, performing cessing of the input homologous regions, performing a pruned very long read (VLRD) or multi region joint ion (MJRD),S and outputting a variant call file. Particularly with respect to identifying homologous regions, a mapped, aligned, and/or sorted SAM and/or BAM file, e.g., a CRAM, may be used as the primary input to a multi-region joint detection processing engine implementing an MRJD algorithm, as described . The MJRD processing engine may be part of an integrated circuit such as a CPU and/or GPU and/or Quantum computing rm, g software, e.g., a quantum algorithm, or implemented within an FPGA, ASIC, or the like. For instance, the above sed mapper and/or aligner may be used to generate a CRAM file, e.g., with settings to output N secondary alignments for each read along with the primary alignments. These primary and secondary reads may then be used to identify a list of homologous regions, which gous regions may be computed based on a user defined similarity threshold between the N regions ofthe reference genome. This list of identified homologous regions may then be fed to the pre-processing stage ofa suitably ured MRJD module.

] Accordingly, in the pre-processing stage, for every set ofhomologous regions, a pileup may first be generated such as by using the primary alignments from one or more, e.g., every, region in the set. See, for instance, . Using this joint pileup, a list of active/candidate variant positions (SNPS/INDELs) may then be generated whereby each of these ate variants may be processed and ted by the MRJD pre-processing engine(s). To reduce computation complexity, a connection matrix may be computed that may be used to define the order ofprocessing ofthe candidate variants.

In such implementations, the multi-region joint detection algorithm evaluates each identified candidate variant based on the processing order defined in the generated connection matrix. Firstly, one or more candidate joint diplotypes (Gi) may be generated and given a candidate variant. Next, the a-posteriori probabilities of each of the joint diplotypes (P(GilR)) may be calculated. From these a-posteriori probabilities a genotype matrix may be computed. Next, N diplotypes with the lowest a-posteriori probabilities may be pruned so as to reduce the ational complexity of the calculations. Then the next candidate variant that provides evidence for the current candidate variant being evaluated may be included and the above process repeated. Having included information such as from one or more, e.g., all, the candidate variants from one or more, e.g., all, regions in the homologous region set for the current variant, a variant call may be made from the final genotyping matrix. Each of the active positions, therefore, may all be evaluated in the manner above thereby resulting in a final VCF file.

] Particularly, as can be seen with respect to B, a MJRD preprocessing step may be implemented, such as including one or more of the following steps or blocks: The identified and assembled joint pile-up is loaded, a ate variant list is then created from the assembled joint pile up, and a connection matrix is computed. Particularly, m various ces, a preprocessing methodology may be performed, such as prior to performing one or more variant call operations, such as a multiple read joint detection operation. Such operations may include one or more preprocessing blocks, including: steps ning to the loading of joint pile-ups, generating a list of t candidates from the joint pileups, and computing a connection matrix. Each of the blocks and potential steps associated therewith will now be discussed in greater detail.

] Specifically, a first joint pile up pre-processing block may be included in the analysis procedure. For example, various reference regions for an identified span may be extracted, such as from the mapped and/or aligned reads. Particularly, using the list of homologous regions, a joint pileup for each set of homologous regions may be generated.

Next, a user-defined span may be used to extract the N reference regions corresponding to N homologous regions within a set. Subsequently, one or more, e.g., all, of the nce regions may be aligned, such as by using a Smith-Waterman alignment, which may be used to generate a universal coordinate system of all the bases in the N reference regions. r, all the primary reads corresponding to each region may then be ted from the input SAM or BAM file and be mapped to the universal nates. This mapping may be done, as described herein, such as by using the alignment information (CIGAR) t in a CRAM file for each read. In the scenario where some reads pairs were not previously mapped, the reads may be mapped and/or aligned, e.g., Smith-Waterman aligned, to its respective reference region.

More particularly, once a joint pile up has been generated and loaded, see for instance, , a ate variant list may be created, such as from the joint pile up. For instance, a De Bruijn graph (DBG) or other assembly graph may be produced so as to extract s candidate variants (SNPs/Indels) that may be identified from the joint pileup. Once WO 14320 PCT/0S2017/036424 the DBG is produced the various bubbles in the graph can be mined so as to derive a list of variant candidates.

Particularly, given all the reads, a graph may be generated usmg each reference region as a ne. All of the identified candidate variant positions can then be aligned to sal coordinates. A tion matrix may then be computed, where the matrix defines the order ofprocessing of the active positions, which may be a function of the read length and/or insert size. As nced herein, shows an example of a joint pileup oftwo homologous regions in chromosome 1. gh this pileup is with reference to two homologous regions of some 1, this is for exemplary purposes only as the production of the pileup process may be used for any and all homologous regions regardless of chromosome.

As can be seen with respect to , a candidate variant list may be created as follows. First, a joint pileup may be formed and a De Bruijn graph (DBG) or other assembly graph may be constructed, in accordance with the methods disclosed herein. The DBG may then be used to extract the candidate variants from the joint pileups. The construction of the DBG is performed in such a manner as to generate bubbles, indicating variations, representing alternate pathways through the graph where each alternate path is a candidate haplotypes. See, for instance, FIGS. 20 and 21.

Accordingly, the various bubbles in the graph represent the list of candidate variant haplotype positions. Hence, given all of the reads, the DBG may be generated using each reference region as a backbone. Then all of the candidate variant positions can be d to universal coordinates. Specifically, illustrates a flow chart setting forth the process of generating a DBG and using the same to e ate haplotypes. More specifically, the De Bruijn graph may be employed in order to create the candidate variant list of SNPs and INDELs. Given that there are N regions that are being jointly processed by MRJD, N de-bruijn graphs may be constructed. In such an instance, every graph may use one reference region as a backbone and all ofthe reads ponding to the N regions.

For instance, in one methodological implementation, after the DBG is constructed, the candidate haplotypes may be extracted from the De Bruijn graph based on the candidate . However, when employing an MRJD ocessing protocol, as described herein, N regions may be jointly processed, such as where the length of the regions can be a few thousand bases or more, and the number ofhaplotypes to be extracted can grow exponentially very quickly. Accordingly, in order to reduce the computational complexity, instead of extracting entire ypes, only the bubbles need be extracted from the graphs that are entative ofthe ate variants.

An e ofbubble structures formed in a De Bruijn graph is shown in . A number of regions to be processed jointly are identified. This determines one of two processing pathways that may be followed. Ifjoint regions are identified all the reads may be used to form a DBG. Bubbles showing possible variants may be extracted so as to identify the various candidate haplotypes. Specifically, for each bubble a SW alignment may be performed on the alternate paths to the reference backbone. From this the candidate variants may be extracted and the events from each graph may be stored.

However, in other ces, once the first process has been performed, so as to generate one or more DBGs, and/or i is now equal to 0, then the union of all candidate events from all of the DBGs may be generated, where any duplicates may be removed. In such an instance, all candidate variants may be mapped, such as to a universal coordinate system, so as to produce the candidate list, and the candidate variant list may be sent as an input to a pruning module, such as the MJRD module. An example of only performing bubble extraction, instead of ting the entire haplotypes, is shown in . In this instance, it is only the bubble region showing possible variants that is ted and processed, as described herein.

Specifically, once the representative s have been extracted, the global alignment, e.g., Waterman alignment, of the bubble path and the corresponding reference backbone may be performed to get the ate t(s) and its position in the reference. This may be done for all extracted bubbles in all ofthe De Bruijn graphs. Next, the union of all the extracted candidate variants may be taken from the N graphs, the ate candidates, if any, may be removed, and the unique candidate variant positions may be mapped to the universal coordinate system obtained from the joint pile-up. This results in a final list of candidate variant positions for the N regions that may act as an input to a "Pruned" MRJD algorithm.

In particular preprocessing blocks, as bed herein above, a connection matrix may be computed. For instance, a connection matrix may be used to define the order ofprocessing of active, e.g., candidate, positions, such as a function ofread length and insert size. For example, to further reduce computational complexity, a connection matrix may be computed so as to define the order of sing of identified candidate variants that are obtained from the De Bruijn graph. This matrix may be constructed and employed in conjunction with or as a sorting function to determine which candidate variants to s first. This connection , therefore, may be a function of the mean read length and the insert size ofthe paired-end reads. ingly, for a given candidate variant, other candidate variant positions that are at integral multiples of the insert size or within the read length have higher weights compared to the ate variants at other positions. This is because these candidate ts are more likely to provide ce for the current t being evaluated.

An exemplary sorting function, as implemented herein, is shown in for mean read length of 101 and insert-size of300.

With respect to a MJRD pruning function, exemplary steps of a pruned MRJD algorithm, as referenced above, is set forth in . For instance, the input to the MRJD platform and algorithm is the joint pileup ofN regions, e.g., all the candidate variants (SNPs/ INDELs), the a-priori probabilities based on a mutation model, and the connection matrix.

Accordingly, the input into the pruned MRJD processing platform may be the joint pile-up, the identified active positions, the generated connection matrix, and the a-posteriori ility model, and/or the results thereof.

Next, each candidate variant in the list can be processed and other variants can be successively added as evidence for a current candidate being processed using the connection matrix. Accordingly, given the current candidate variant and any supporting candidates, candidate joint diplotypes may be ted. For instance, a joint diplotype is a set of 2N haplotypes, where N is the number of regions being jointly sed. The number of candidate joint diplotypes M is a function of the number of regions being jointly processed, number of /candidate ts being considered, and the number of phases.

An example for generating joint diplotypes is shown below.

For: P = 1, Number ofactive/candidate variant positions being considered; N = 2, Number ofregions being jointly processed; M = 22-N.P = 24 = 16 candidate joint-diplotypes Hence, for a single candidate active position, given all the reads and both the reference regions, let the two ypes be 'A'and 'G'.

Unique haplotypes = 'A'and 'G' Candidate Diplotypes = 'AA','AG','GA'and 'GG',(4 candidates for 1 region).

Candidate Joint Diplotypes = 'AAAA', 'AAAG', 'AAGA', 'AAGG' 'AGAA', 'AGAG', 'AGGA', 'AGGG' 'GAAA', 'GAAG', 'GAGA','GAGG' 'GGAA', 'GGAG', 'GGGA','GGGG' Accordingly, using the candidate joint diplotypes, the read likelihoods can be calculated given a haplotype for each haplotype in every candidate joint diplotype set. This may be done using a HMM thm, as bed herein. However, in doing so the HMM algorithm may be modified from its standard use case so as to allow for candidate variants (SNPs/INDELs) in the haplotype, which have not yet been processed, to be considered.

Subsequently, the read likelihoods can be calculated given a joint diplotype (P(rilGm)) using the results from the ed HMM. This may be done using the formula below.

For the case of2-regionjoint ion: Gm= [Sll,m, B12,m, B21,m, B22,m], wherein Sij,m, i is the region and j is the phase P(rilGm) = 11,m)+ P(rilB12,m)+ P(rilB21,m)+ P(rilB22,m) ) = IL P(rilGm). Given P(rilGm), it is straightforward to calculate P(RIGm) for all the reads. Next, using Bayes' formula, the a-posteriori probability (P(GilR)) may be computed from P(RIGi) and the ri probabilities (P(Gi)).

P(GilR) = ) P(Gi) I Lk P(RIGk) P(Gk).

Further, an intermediate genotype matrix may be calculated for each region given the a-posteriori probabilities for all the candidate joint diplotypes. For each event combination in the pe matrix the a-posteriori probabilities of all joint diplotypes supporting that event may be summed up. At this point, the genotype matrix may be considered as "intermediate" because not all the candidate variants supporting the current candidate have been included. However, as seen r, the number of joint diplotype candidates grows exponentially with the number of candidate variant positions and number of regions. This in-tum exponentially increases the computation required to calculate the riori probabilities. Therefore, in order to reduce the computational complexity, at this stage, the number ofjoint diplotypes based on the a-posteriori probabilities may be pruned so that the number ofjoint diplotypes to keep may be user defined and programmable. y, the final genotype matrix may be updated based on a user-defined confidence metric of variants which is computed using the intermediate genotype matrix. The various steps of these processes are set forth in the process flow diagram of.

The process above may be repeated until all the candidate variants are included as ce for the t candidates being sed using the connection matrix.

Once all of the candidates have been included, the processing of the current candidate is done. Other stopping criteria for processing candidate variants are also possible. For example, the process may be stopped when the confidence has stopped sing as more candidates variants are added. This analysis, as exemplified in , may be restarted and repeated in the same manner for all other candidate variants in the list thereby resulting in a final variant call file at the output of MRJD. Accordingly, instead of ering each region in isolation, a Multi-Region Joint Detection protocol, as described herein, may be employed so as to consider all locations from which a group of reads may have originated as it attempts to detect the underlying sequences jointly using all available information.

Accordingly, for Region Joint Detection, an exemplary MRJD protocol may employ one or more of the following equations in accordance with the methods sed herein. Specifically, instead of considering each region to be assessed in ion, MRJD considers a plurality of locations from which a group of reads may have been originated and attempts to detect the underlying sequences jointly, such as by using as much as, e.g., all, the available information that is useful. For instance, in one exemplary embodiment: Let N be the number of regions to be jointly sed. And let H1c be a ate haplotype, k = 1...K, each of which may e various SNPs, insertions and/or deletions relative to a reference sequence. Each haplotype H1c represents a single region along a single strand (or "phase", e.g., maternal or paternal), and they need not be contiguous (e.g., they may include gaps or "don'tcare" sequences).

Let Gm be a candidate solution for both phases <D 1,2 (for a d organism) and all regions n = l ...N: G = [Gm, 1,1 .. . Gm, 1, N] m Gm,2,1 .. . Gm,2,N where each element Gm,<D,n is a haplotype chosen from the set ofcandidates {H1.•.H1c}.

First, the probability of each read may be calculated for each candidate ype P(rilH1c), for example, by using a Hidden Markov Model (HMM). In the case of WO 14320 PCT/0S2017/036424 datasets with paired reads, ri indicates the pair {ri,1, ri,2}, and P(rilH1c) = P(ri,1IH1c) P(ri,2IH1c). In the case of datasets with linked reads (e.g., barcoded reads), ri indicates the group of reads {ri,1 .. ,ri,Nd that came from the same long molecule, and P(rilH1c) = TI~~1 P(ri, nlHk).

Next, for each candidate solution Gm, m=l ...M, we calculate the conditional probability of each read P(rilGm) = ~ I~=l I~=l P(rilGm, <D, n) and conditional probability ofthe entire pileup R ={r1 }: P(RIGm) = Tif=Rl P(rilGm).

Next, the a-posteriori probability is calculated of each candidate solution given the observed pileup: P(GmlR) = P(RIGm)P(Gm)/ I~1 P(RIGi)P(Gi) where P(Gm) indicates the a-priori probability ofthe candidate solution, which is set forth in detail here below.

Finally, the relative probability of every candidate variant Vi is calculated ~;:: ~~) = LnlGm=>vj P(GmlR) I LmlGm=>ref P(GmlR) , such as where Gm -+ Vj indicates that Gm supports variant Vj, and Gm -+ ref indicates that Gm supports the reference. In a VCF file, this may be ed as a quality score on a phred scale: QUAL(Vj) = -10log10 P(VilR) .

P(ref I R) An exemplary process for performing various t calling operations is set forth herein with respect to where a tional and MRJD detection s are compared. Specifically, illustrates a joint pileup of paired reads for two regions whose reference sequences differ by only 3 bases over the range of interest. All the reads are known to come from either region #1 or region #2, but it is not known with certainty from which region any individual read originated. Note, as described above, that the bases are only shown for the positions where the two references differ, e.g., bubble regions, or where the reads differ from the nce. These regions are referred to as the active positions. All other positions can be ignored, as they ffect the calculation.

Accordingly, as can be seen with t to , in a conventional detector, the read pairs 1-16 would be mapped to region #2, and these alone would be used for variant calling in region #2. All of these reads match the nce for region #2, so no variants would be called. Likewise, read pairs 17-23 would be mapped to region #1, and these alone would be used for variant calling in region #1. As can be seen, all of these reads match the reference for region #1, so no ts will be . However, read pairs 24-32 map equally well to region #1 and region #2 (each has a one-base difference to ref#1 and to ref #2), so the mapping is indeterminate, and a typical variant caller would simply ignore these reads. As such, a conventional variant caller would make no variant calls for either region, as seen in .

However, with MRJD, illustrates that the result is completely different than that received employing conventional s. The relevant calculations are set forth below. In this instance N = 2 s. Additionally, there are three positions, each with 2 candidate bases (one can safely ignore bases whose count is sufficiently low, and in this example the count is zero on all but 2 bases in each on). If all combinations are considered, this will yield K = 23 = 8 candidate ypes: H1 = CAT, H2 = CAA, H3 = CCT, H4 = CCA, H5= GAT, H6 = GAA, H7 = GCT, Hs = GCA.

In a force calculation where all combinations of all candidate haplotypes are considered, the number of candidate ons is M = K2N = g2·2 = 4096, and P(Gm/R) may be calculated for each ate solution Gm, The following illustrates this calculation for two candidate solutions: CAT GCA] [CAT GCA] Gm1= [CAT GCA ' Gm2 = CCT GCA Where Gm1 has no variants (this is the solution found by a conventional detector), and Gm2 has a single heterozygous SNP A➔c in position #2 ofregion #1.

The probability P(rilH1c) depends on various factors including the base quality and other parameters of the HMM. It may be d that only base call errors are present and all base call errors are equally likely, so P(rilH1c) = (1-Petp(i)-Ne(i)(pJ3te(i), where Pe is the probability of a base call error, Np(i) is the number of active base position(s) overlapped by read i, and Ne(i) is the number of errors for read i, assuming haplotype H1c. ingly, it may be assumed that Pe = 0.01, which corresponds to a base quality ofphred 20. The table set forth in shows P(rilH1c), for all read pairs and all candidate haplotypes. The two far right columns show P(rilGm1) and m2), with the product at the bottom. shows that P(RIGm1) = 3S 30 15 and P(RIGm2) = 2.2- , a difference of 15 orders of magnitude in favor ofGm2.

The a-posteriori probabilities P(GmlR) depend on the a-priori probabilities P(Gm), To complete this example, a simple independent identically distributed (IID) model may be assumed, such that the a-priori probability of a candidate on with Nv variants is (1 - Pvt·Np-N\pvl9tv, where Np is the number of active positions (3 in this case) and Pv is the probability of a variant, assumed to be 0.01 in this example. This yields P(Gm) = 7.22e-13, and P(Gm2) = 0.500. It is noted that Gm2 is heterozygous over region #1, and all heterozygous pairs of haplotypes have a mirror-image representation with the same probability (obtained by simply swapping the phases). In this case, the sum of the probabilities for Gm2 and its mirror image sum to 1.000. Calculating probabilities of dual variants, a heterozygous A-+C SNP at position #2 ofregion #1, with quality score ofphred 50.4 can be seen.

Accordingly, as can be seen, there is an immense computational xity for performing a brute force variant calling operation, which complexity can be reduced by performing multiple region joint detection, as described . For instance, the complexity of the above calculations grows rapidly with the number of regions N and the number of candidate haplotypes K. To consider all combinations ofcandidate haplotypes, the number of ate ons for which to calculate probabilities is M = K2N. In a brute force implementation, the number of candidate haplotypes is K = 2Np' where Np is the number of active positions (e.g., as exemplified above, if assembly techniques are used to generate the list ofcandidate haplotypes, then Np is the number ofindependent bubbles in the graph). Hence, a mere brute-force calculation can be prohibitively ive to implement.

For example, if N = 3 and Np =10, the number of candidate solutions is M = 2310 = 260 = 1018. However, in practice, it'snot on to have values ofNp much higher than this.

Consequently, because a brute force Bayesian calculation can be prohibitively complex, the following ption sets forth further methods for reducing the xity of such calculations. For instance, in a first step of another embodiment, starting with a small number of positions Ni (or even a single on Ni= 1), the Bayesian calculation may be performed over those positions. At the end of the calculation, the candidates whose probability falls below a predefined threshold may be eliminated, such as in a pruning of the tree function, as described above. In such an instance, the threshold may be adaptive.

] Next, in a second step, the number of positions Ni may be increased by a small number ~NP (such as one: Ni+i = Ni + ~Np), and the surviving candidates can be combined with one or more, e.g., all, possible candidates at the new position(s), such as in a growing the tree on. These steps of (1) performing the Bayesian calculation, (2) pruning the tree, and (3) growing the tree, may then be repeated, e.g., sequentially, until a stopping criteria is met. The threshold history may then be used to determine the confidence of the result (e.g., the probability that the true solution was or was not found). This process is illustrated in the flow chart set forth in .

It is to be understood that there are a variety of le variations to this approach. For ce, as indicated, the pruning old may be adaptive, such as based on the number of surviving candidates. For instance, a simple implementation may set the threshold to keep the number of candidates below a fixed number, while a more sophisticated implementation may set the old based on a cost-benefit analysis ofincluding additional candidates. Further, a simple stopping criteria may be that a result has been found with a sufficient level of confidence, or that the confidence on the initial position has stopped increasing as more positions are added. Further still, a more ticated implementation may perform some type of cost-benefit analysis of continuing to add more positions.

Additionally, as can be seen with respect to , the order in which new positions are added may depend on several criteria, such as the distance to the initial position(s) or how highly connected these positions are to the already-included positions (e.g., the amount of overlap with the paired .

A useful feature of this algorithm is that the probability that the true solution wasn't found can be quantified. For instance, a useful te is obtained by simply summing the ilities of all pruned branches at each step: Ppruned = Ppruned + Lmi;pruned set P(G/n IR). Such an te is useful for calculating the ence of the resulting t calls: P(vj I R) LmlGm=>vj P(GmlR) + Ppruned I P(ref I R) LmlGm=>ref P(GmlR) + Ppruned. Good confidence estimates are essential for producing good Receiver Operating Characteristic (ROC) curves. This is a key advantage of this pruning method over other ad hoe complexity reductions.

] Returning to the example pileup of , and starting from the left-most position (position #1) and working toward the right one base position at a time, using a g threshold ofphred 60 on each iteration: Let {cfn, m=1...Mi} represent the candidate solutions on the j-th iteration. shows the candidate solutions on the first iteration, representing all combinations ofbases C and G, listed in order of decreasing probability. For any solution with equivalent mirror-image representations (obtained by swapping the phases), only a single representation is shown here. The probabilities for all candidate solutions can be calculated, and those probabilities beyond the pruning threshold (indicated by the solid line in the ) can be dropped. As can be seen with respect to , as a result of the pruning methods disclosed herein, six candidates survive.

Next, as can be seen with respect to , the tree can be grown by finding all combinations ofthe surviving candidates from iteration #1 and candidate bases (C and A) in the position #2. A partial list of the new ates is shown in , again shown in order of decreasing probability. Again, the probabilities can be calculated and compared to the pruning threshold, and in this instance 5 candidates survive.

Finally, all combinations of the surviving candidates from iteration #2 and the candidate bases in position #3 (A and T) can be determined. The final candidates and their associated probabilities are shown in . Accordingly, when calculating the probabilities of individual variants, it is determined that a heterozygous A-+C SNP at on #2 of region #1, with quality score ofphred 50.4, which is the same result found in the brute-force calculation. In this example, pruning had no significant effect on the end , but in general pruning may affect the calculation, often resulting in a more confidence score.

There are many le variations to the implementations of this approach, which may affect the performance and complexity ofthe system, and different variations may be appropriate for different scenarios. For instance, there can be ions in deciding which regions to include. For example, prior to running a Multi-Region Joint Detection, the variant caller may be configured to ine r a given active region should be processed individually or jointly with other regions, and ifjointly, it may then determine which regions to e. In other instances, some implementations may rely on a list of secondary alignments provided by the mapper so as to inform or otherwise make this decision. Other implementations may use a database ofhomologous regions, computed e, such as based on a search ofthe reference .

Accordingly, a useful step in such operations is in deciding which positions to include. For instance, it is to be noted that various regions of interest may not be selfcontained and/or isolated from adjacent regions. Hence, information in the pileup can nce the probability of bases separated by far more than the total read length (e.g., the paired read length or long molecule length). As such, it must be decided which positions to include in the MRJD calculation, and the number ofpositions is not unconstrained (even with g). For e, some implementations may process pping blocks of positions and update the results for a subset of the positions based on the confidence levels at those positions, or the completeness of the evidence at those positions (e.g., positions near the middle ofthe block typically have more complete evidence than those near the edge).

Another ining factor may be the order in which new positions may be added. For instance, for pruned MRJD, the order of adding new positions may affect performance. For example, some implementations may add new positions based on the distance to the already-included positions, or the degree of connectivity with these ons (e.g., the number of reads overlapping both positions). Additionally, there are also many variations on how pruning may be performed. In the example set forth above, the pruning was based on a fixed probability old, but in general the pruning threshold may be adaptive or based on the number of surviving candidates. For instance, a simple implementation may set the threshold to keep the number of candidates below a fixed number, while a more sophisticated entation may set the threshold based on a costbenefit analysis ofincluding onal candidates.

Various implementations may perform pruning based on probabilities P(RIGm) instead of the a-priori probabilities P(GmlR). This has the advantage of allowing the elimination of equivalent -image representations across regions (in addition to phases).

This advantage is at least partially offset by the disadvantage of not pruning out candidates with very low a-priori probabilities, which in various instances may be beneficial. As such, a useful solution may depend on the scenario. Ifpruning is done, such as based on the P(RIGm), then the bayesian calculation would be performed once after the final iteration.

Further in the example above, the process was stopped after processing all base positions in the pileup shown, but other stopping criteria are also possible. For ce, if only a subset of the base positions (e.g. when processing overlapping ) is being solved for, the process may stop when the result for the subset has been found with a sufficient level of ence, or when the confidence has stopped increasing as more positions are added. A more sophisticated implementation, however, may m some type of cost-benefit is, weighing the computational cost against the potential value of adding more positions.

A-priori probabilities may also be useful. For instance, in the examples above, a simple IID model was used, but other models may also be used. For example, it is to be noted that clusters ofvariants are more common than would be predicted by an IID model. It is also to be noted that variants are more likely to occur at ons where the references . ore, incorporating such knowledge into the a-priori probabilities P(Gm) can improve the detection performance and yield better ROC curves. Particularly, it is to be noted that the a-priori probabilities for homologous regions are not well-understood in the genomics community, and this knowledge is still evolving. As such, some implementations may update the a-priori models as better information s available. This may be done automatically as more results are ed. Such updates may be based on other biological samples or other s of the genome for the same sample, which learnings can be applied to the methods herein to further promote a more rapid and accurate analysis.

Accordingly, in some instance, an iterative MJRD process may be implemented. ically, the methodology described herein can be extended to allow message passing between related regions so as to further reduce the complexity and/or increase the detection performance of the . For instance, the output of the ation at one location can be used as an input a-priori ility for the calculation at a nearby location. Additionally, some implementations may use a combination ofpruning and iterating to achieve the desired performance/complexity ff.

Further, sample preparation may be ented to optimize the MRJD process. For instance, for paired-end sequencing, it may be useful to have a tight distribution on the insertion size when using conventional detection. However, in various instances, introducing ion in the insertion size could significantly improve the performance for MRJD. For example, the sample may be prepared to ionally introduce a bimodal distribution, a multi-modal distribution, or bell-curve-like distribution with a higher variance than would typically be implemented for conventional detection. ] illustrates the ROC curves for MRJD and a conventional detector for human sample NA12878 over selected regions of the genome with a single homologous copy, such that N = 2, with varying degrees of reference sequence similarity. This dataset used paired-end sequencing with a read length of 101 and a mean insertion size of . 400. As can be seen with respect to , MRJD offers dramatically improved sensitivity and specificity over these regions than conventional detection methods. illustrates the same results displayed as a function of the sequence similarity of the references, measured over a window of 1000 bases (e.g. if the references differ by 10 bases out of 1000, then the similarity is 99.0 percent). For this dataset, it may be seen that conventional detection starts to perform badly at a sequence similarity -0.98, while MRJD performs quite well up to 0.995 and even beyond.

Additionally, in various instances, this methodology may be extended to allow message passing between related regions to further reduce the complexity and/or increase the detection performance. For instance, the output ofthe calculation at one location can be used as an input a-priori probability for the calculation at a nearby location, and in some implementations may use a combination of pruning and iterating to achieve the desired performance/complexity tradeoff. In particular instances, as ted above, prior to g multi-region joint detection, the variant caller may ine whether a given active region should be processed individually or jointly with other regions. Additionally, as indicated above, some implementations may rely on a list of secondary alignments provided by the mapper to make such a on. Other implementations may use a database of homologous s, computed offline based on a search ofthe reference genome.

In view of the above, a Pair-Determined Hidden Markov Model (PD-HMM may be implemented in a manner so as to take advantage of the benefits of MRJD. For instance, MRJD can tely estimate the probability of observing a portion or all of the reads given each possible joint diplotype, which comprises one haplotype per ploidy per homologous reference region, e.g., for two gous regions in diploid chromosomes, each joint diplotype will include four haplotypes. In such instances, all or a n of the possible haplotypes may be considered, such as by being constructed, for ce, by ing each reference region with every possible subset of all the variants for which there is nontrivial evidence. However, for long homologous reference regions, the number of possible variants is large, so the number of haplotypes nations of variants) becomes exponentially large, and the number ofjoint diplotypes (combinations ofhaplotypes) may be astronomical.

Consequently, to keep MRJD calculations tractable, it may not be useful to test all possible joint diplotypes. Rather, in some instances, the system may be configured in such a manner that only a small subset of "most likely" joint diplotypes is tested. These "most likely" joint diplotypes may be determined by incrementally constructing a tree of partially-determined joint diplotypes. In such an ce, each node of the tree may be a partially determined joint diplotype that includes a partially determined haplotype per ploidy per homologous reference . In this instance, a partially determined haplotype may include a reference region modified by a partially determined subset of the possible variants. Accordingly, a partially determined subset of the possible variants may e an indication, for each possible variant, of one of three states: that the variant is determined and present, or the variant is determined and absent, or the variant is not yet ined, e.g., it may be present or absent. At the root of the tree, all variants are undetermined in all haplotypes; tree nodes branching successively r from the root have successively more variants determined as t or absent in each haplotype ofeach node'sjoint diplotype.

Further, in the context of this joint diplotype tree, as described above, the amount of MRJD calculations is kept limited and tractable by trimming branches of the tree in which all joint diplotype nodes are unlikely, e.g., moderately to extremely ly, relative to other more likely branches or nodes. Accordingly, such trimming may be performed on branches at nodes that are still only partially determined; e.g., several or many variants are still not determined as present or absent from the haplotypes of a trimmed node's joint diplotype. Thus, in such an instance, it is useful to be able to te or bound the likelihood of observing each read assuming the truth of a partially determined haplotype. A modified pair hidden Markov model (pHMM) calculation, denoted "PD-HMM" for "partially determined pair hidden Markov model" is useful to estimate the ility P(RIH) of ing read R assuming the true haplotype H* is consistent with partially determined haplotype H. tent in this context means that some specific true ype H* agrees with partially determined haplotype H with respect to all ts whose presence or e are determined in H, but for variants undetermined in H, H* may agree with the reference sequence either modified or unmodified by each undetermined variant.

] Note that it is not generally adequate to run an ordinary pHMM calculation for some shorter sub-haplotype ofH chosen to encompass only determined variant positions. It is generally important to build the joint diplotype tree with undetermined variants being resolved in an efficient order, which is generally quite different than their geometric order, so that a partially determined haplotype H will typically have many undetermined variant ons interleaved with determined ones. To properly er PCR indel errors, it is useful to use a pHMM-like calculation spanning through all determined variants and significant radius around them, which may not be compatible with attempts to avoid undetermined variant positions.

Accordingly, the inputs to PD-HMM may include the called nucleotide sequence ofread R, the base y scores (e.g., phred scale) ofthe called nucleotides ofR, a baseline haplotype HO, and a list ofundetermined variants (edits) from HO. The undetermined variants may e single-base substitutions , multiple-base substitutions (MNPs), insertions, and deletions. Advantageously, it may be adequate to support undetermined SNPs and deletions. An undetermined MNP may be imperfectly but adequately represented as multiple independent SNPs. An undetermined insertion may be represented by first editing the insertion into the baseline haplotype, then indicating the corresponding undetermined on which would undo that ion.

Restrictions may be placed on the undetermined deletions, to facilitate hardware engine implementation with d state memory and logic, such as that no two undetermined deletions may overlap e the same baseline haplotype bases). If a partially ined haplotype must be tested with undetermined ts violating such restrictions, this may be resolved by converting one or more undetermined variants into determined variants in a larger number of PD-HMM operations, ng cases with those variants present or absent. For example, iftwo undetermined deletions A and B violate by overlapping each other in baseline haplotype HO, then deletion B may be edited into HO to yield HOB, and two PD-HMM operations may be performed using undetermined deletion A only, one for baseline haplotype HO, and the other for baseline haplotype HOB, and the maximum probability output ofthe two PD-HMM operations may be retained.

The result of a PD-HMM operation may be an estimate of the maximum P(RIH*) among all haplotypes H* that can be formed by g HO with any subset of the undetermined variants. The maximization may be done locally, contributing to the pHMM- like dynamic programming in a given cell as if an adjacent undetermined variant is present or absent from the haplotype, whichever scores better, e.g., contributes the greater partial probability. Such local maximization during dynamic programming may result in larger estimates of the maximum P(RIH*) than true zation over individual pure H* haplotypes, but the difference is generally inconsequential.

Undetermined SNPs may be orated into PD-HMM by allowing one or more matching nucleotide values to be specified for each haplotype position. For example, if base 30 of HO is 'C'and an undetermined SNP replaces this 'C'with a 'T',then the PDHMM operation's haplotype may indicate position 30 as matching both bases 'C'and 'T'.In the usual pHMM dynamic programming, any tion to an 'M'state results in multiplying the path probability by the probability ofa correct base call (ifthe haplotype position matches the read position) or by the ility of a specific base call error (if the haplotype position mismatches the read on); for PD-HMM this is modified by using the correct-call probability if the read position matches either possible haplotype base (e.g. 'C'or 'T'),and the base-call-error probability otherwise.

Undetermined haplotype deletions may be incorporated into PD-HMM by flagging optionally-deleted haplotype positions, and modifying the dynamic programming of pHMM to allow alignment paths to skip horizontally across rmined deletion haplotype segments without probability loss. This may be done in various manners, but with the common property that probability values in M, I, and/or D states can transmit horizontally (along the haplotype axis) over the span of an undetermined deletion t being reduced by ordinary gap-open or tend probabilities.

] In one particular embodiment, haplotype positions where undetermined deletions begin are flagged "FI", and positions where undetermined deletions end are flagged "F2". In addition to the M, I, and D "states" (partial probability representations) for each cell of the HMM matrix (haplotype horizontal/ read vertical), each PD-HMM cell may further e BM, BI, and BD "bypass" states. In FI-flagged haplotype columns, BM, BI, and BD states receive values copied from M, I, and D states of the cell to the left, respectively. In non-F2-flagged haplotype columns, particularly columns starting with an FI flagged column end ing into the interior of an undetermined deletion, BM, BI, and BD states transmit their values to BM, BI, and BD states of the cell to the right, respectively. In F2-flagged haplotype columns, in place ofM, I, and D states used to calculate states cent cells, the maximum ofM and BM is used, and the maximum of I and BI is used, and the maximum of D and BD is used, respectively. This is exemplified in an F2 column as multiplexed selection of signals from M and BM, from I and BI, and from D and BD registers.

Note that although BM, BI, and DB state registers may be ented in FI through F2 s, and maximizing M/BM, I/BI, and D/BD multiplexers may be shown in an F2 , these components may be t for all cell calculations, enabling an undetermined deletion to be handled in any position, and enabling multiple undetermined deletions with corresponding FI and F2 flags throughout the haplotype. Note also that FI and F2 flags may be in the same column, for the case of a single-base undetermined deletion. It is further to be noted that the PD-HMM matrix of cells may be depicted as a schematic representation of the logical M, I, D, BM, BI, and BD state calculations, but in a re implementation, a smaller number of cell calculating logic elements may be present, and pipelined appropriately to calculate M, D, I, BM, BI, and BD state values at high clock frequencies, and the matrix cells may be calculated with various degrees of hardware parallelism, in various orders consistent with the inherent l dependencies of the PDHMM calculation.

Thus, in this embodiment, the pHMM state values in one column may be ately left of an undetermined deletion which may be captured and transmitted rightward, unchanged, to the ost column of this undetermined deletion, where they substitute into pHMM ations whenever they beat normal-path scores. Where these maxima are chosen, the "bypass" state values BM, BI, and BD represent the local dynamic mming results where the undetermined on is taken to be present, while "normal" state values M, I, and D represent the local dynamic programming results where the undetermined deletion is taken to be absent.

In r embodiment, a single bypass state may be used, such as a BM state receiving from an M state in FI d columns, or receiving a sum of M, D, and/or I states. In another embodiment, rather than using s" states, gap-open and/or tend penalties are eliminated within columns of undetermined deletions. In another embodiment, bypass states contribute additively to dynamic programming rightward of undetermined deletions, rather than local maximization being used. In a further ment, more or fewer or differently defined or differently located ype position flags are used to trigger bypass or similar behavior, such as a single flag indicating membership in an undetermined deletion. In an additional embodiment, two or more overlapping undetermined deletions may participate, such as with the use of additional flags and/or bypass states. Additionally, undetermined insertions in the ype are supported, rather than, or in addition to, undetermined deletions. Likewise, undetermined insertions and/or deletions on the read axis are supported, rather than or in addition to undetermined deletions and/or insertions on the haplotype axis. In another embodiment, undetermined multiple-nucleotide tutions are supported as atomic variants (all present or all absent). In a further embodiment, undetermined length-varying substitutions are supported as atomic variants. In another embodiment, undetermined variants are penalized with fixed or configurable probability or score adjustments.

This PD-HMM calculation may be implemented as a hardware engine, such as in FPGA or ASIC technology, by extension of a hardware engine architecture for "ordinary" pHMM ation or may be ented by one or more quantum circuits in a quantum computing platform. In addition to an engine pipeline logic to calculate, transmit, and store M, I, and D state values for various or successive cells, parallel pipeline logic can be constructed to calculate, transmit, and store BM, BI, and BD state values, as described herein and above. Memory resources and ports for e and retrieval of M, I, and D state values can be accompanied by similar or wider or deeper memory resources and ports for storage and retrieval of BM, BI, and BD state . Flags such as FI and F2 may be stored in memories along with associated ype bases.

Multiple matching nucleotides for e.g. undetermined SNP haplotype positions may be encoded in any manner, such as using a vector of one bit per possible nucleotide value. Cell calculation dependencies in the pHMM matrix are unchanged in PD-HMM, so order and pipelining of le cell calculations can remain the same for PD-HMM.

However, the y in time and/or clock cycles for complete cell ation increases somewhat for PD-HMM, due to the requirement to compare "normal" and "bypass" state values and select the larger ones. Accordingly, it may be advantageous to include one or more extra pipeline stages for PD-HMM cell calculation, resulting in additional clock cycles of latency. Additionally, it may further be advantageous to widen each "swath" of cells calculated by one or more rows, to keep the longer pipeline filled without dependency issues.

This PD-HMM calculation tracks twice as many state values (BM, BI, and BD, in addition to M, I, and D), as an ordinary pHMM calculation, and may e about twice the hardware ces for an equivalent throughput engine embodiment. However, a PD-HMM engine has exponential speed and efficiency ages for increasing numbers of undetermined variants, versus an ordinary pHMM engine run once for each haplotype representing a distinct combination ofthe undetermined variants being present or absent. For example, if a lly ined ype has 30 undetermined ts, each of which may be ndently present or absent, there are 2A30, or more than 1 billion, distinct specific haplotypes that pHMM would otherwise need to process.

Accordingly, these and other such operations herein disclosed may be performed so as to better understand and accurately predict what happened to the subject's genome such that the reads varied in relation to nce. For instance, even though the occurrence of mutations may be random, there are instances wherein the likelihood of their occurrence appears to be potentially table to some extent. Particularly, in some instances when mutations occur, they may occur in certain defined locations and in certain forms. More particularly, mutations, ifthey occur, will occur on one allele or another or both, and will have a tendency to occur in certain locations over others, such as at the ends of the chromosomes. Consequently, this and other associated information may be used to develop mutation models, which may be generated and employed to better assess the likely presence of a mutation in one or more regions of the genome. For instance, by taking account of various a priori knowledge, e.g., one or more mutation models, when ming c variation es, better and more accurate genomic analysis results may be obtained, such as with more accurate demarcations of genetic mutation.

Such mutation models may give an account for the frequency and/or on of various known ons and/or mutations that appear to happen in ction with one another or otherwise non-randomly. For instance, it has been ined that toward the ends of a given chromosome variations occur more predominantly. Thus, known models of ons can be generated, stored in a database herein, and used by the system to make a better prediction of the presence of one or more variations within the genomic data being analyzed. Additionally, a machine learning process, as described in greater detail herein below, may also be implemented such that the various results data derived by the analyses performed herein may be analyzed and used to better inform the system as to when to make a specific variance call, such as in ance with the machine learning principles disclosed herein. Specifically, machine learning may be implemented on the collective data sets, especially with respect to the variations determined, and this learning may be used to better generate more comprehensive mutation models that in tum may be employed to make more accurate variance determinations.

Hence, the system may be ured to observe all the various variation data, mine that data for various correlations, and where correlations are found, such information may be used to better weight and ore more tely determine the ce of other variations in other genome samples, such as on an ongoing basis. Accordingly, in a manner such as this, the system, especially the variant calling mechanism, may constantly be updated with respect to the learned variant correlation data so as to make better variant calls moving forward, so as to get better and more accurate results data.

Specifically, telemetry may be employed to update the growmg mutation model so as to achieve better analysis in the system. This may be of particular usefulness when analyzing samples that are in some way connected with one another, such as from being within the same geographical population, and/or may be used to determine which reference genome out of a multiplicity of reference genomes may be a better reference genome by which a particular sample is to be analyzed. Further, in various instances, the mutation model and/or telemetry may be employed so as to better select the reference genome to be employed in the system processes, and thereby enhance the cy and efficiency of the results of the system. Particularly, where a plurality of reference genomes may be employed in one or more of the analyses herein, a particular reference genome may be selected for use over the others such as by applying a mutation model so at select the most appropriate reference genome to apply.

It is to be noted that when performing secondary analysis, the fundamental structure for each region of the genome being mapped and aligned may include one or more underlying genes. Accordingly, in various instances, this understanding of the underlying genes and/or the functions ofthe proteins they code for may be informative when performing secondary analysis. Particularly, tertiary indications and/or results may be useful in the secondary analysis protocols being run by the present , such as in a process of biological contextually sensitive mutation model. More particularly, since DNA codes for genes, which genes code for ns, information about such proteins that result in mutations and/or abhorrent functions can be used to inform the mutation models being employed in the performance ofsecondary and/or tertiary analyses on the t's genome.

For example, tertiary analysis, such as on a sample set of genes coding for mutated proteins, may be ative when performing secondary analysis of genomic regions known to code for such mutations. Hence, as set forth above, various tertiary processing results may be used to inform and/or update the mutation models used herein for achieving better accuracy and efficiency when performing the various secondary analysis operations disclosed herein. Specifically, ation about mutated proteins, e.g., contextual ry analysis, can be used to update the mutation model when performing secondary analysis of those s known to code for the proteins and/or to potentially e such mutations ] Accordingly, in view of the above, for embodiments involving FPGA- accelerated mapping, ent, sorting, and/or variant calling applications, one or more of these functions may be implemented in one or both of software and hardware (HW) processing components, such as software running on a traditional CPU, GPU, QPU, and/or firmware such as may be embodied in an FPGA, ASIC, sASIC, and the like. In such instances, the CPU and FPGA need to be able to communicate so as to pass results from one step on one device, e.g., the CPU or FPGA, to be processed in a next step on the other device.

For instance, where a mapping on is run, the building of large data structures, such as an index of the nce, may be implemented by the CPU, where the running of a hash function with respect thereto may be implemented by the FPGA. In such an instance, the CPU may build the data structure, store it in an associated memory, such as a DRAM, which memory may then be accessed by the processing engines running on the FPGA.

For ce, in some embodiments, communications n the CPU and the FPGA may be implemented by any suitable onnect such as a peripheral bus, such as a PCie bus, USB, or a networking interface such as Ethernet. However, a PCie bus may be a comparatively loose integration n the CPU and FPGA, whereby transmission latencies between the two may be relatively high. Accordingly, although one device e.g., (the CPU or FPGA) may access the memory attached to the other device (e.g., by a DMA transfer), the memory region(s) accessed are non-cacheable, because there is no facility to maintain cache ncy between the two devices. As a consequence, transmissions between the CPU and FPGA are constrained to occur between large, high-level processing steps, and a large amount of input and output must be queued up between the devices so they don't slow each other down waiting for high y operations. This slows down the various processing operations disclosed . Furthermore, when the FPGA accesses non-cacheable CPU memory, the full load of such access is imposed on the CPU's external memory interfaces, which are bandwidth-limited compared to its internal cache interfaces.

] Accordingly, because of such loose GA integrations, it is generally necessary to have "centralized" software control over the FPGA ace. In such instances, the various software threads may be sing various data units, but when these threads te work for the FPGA engine to perform, the work must be aggregated in "central" buffers, such as either by a single aggregator software thread, or by multiple threads locking ation access via semaphores, with ission of aggregated work via DMA packets managed by a central software module, such as a kernel-space driver. Hence, as results are produced by the HW engines, the reverse process occurs, with a software driver receiving DMA packets from the HW, and a de-aggregator thread distributing results to the various waiting software worker threads. However, this centralized software control of communication with HW FPGA logic is cumbersome and expensive in resource usage, reduces the ency of software threading and HWI software communication, limits the practical HWI software communication bandwidth, and dramatically increases its latency.

Additionally, as can be seen with respect to A, a loose integration between the CPU 1000 and FPGA 7 may require each device to have its own dedicated external memory, such as DRAMs 1014, 14. As depicted in A, the CPU(s) 1000 has its own DRAM 1014 on the system motherboard, such as DDR3 or DDR4 DIMMs, while the FPGA 7 has its own dedicated DRAMs 14, such as four 8GB SODIMMs, that may be directly connected to the FPGA 7 via one or more DDR3 busses 6, such as a high latency PCie bus. se, the CPU 1000 may be icably coupled to its own DRAM 1014, such as by a suitably configured bus 1006. As indicated above, the FPGA 7 may be configured to include one or more processing s 13, which processing engines may be configured for performing one or more functions in a bioinformatics pipeline as herein described, such as where the FPGA 7 includes a mapping engine 13a, an alignment engine 13b, and a variant call engine 13c. Other engines as described herein may also be ed. In various embodiments, one or both of the CPU may be configured so as to include a cache 1014a, 14a respectively, that is capable of storing data, such as result data that is transferred thereto by one or more of the various components of the system, such as one or more es and/or processing engines.

Many of the operations disclosed herein, to be performed by the FPGA 7 for genomic processing, e large memory accesses for the performance of the underlying operations. Specifically, due to the large data units involved, e.g. 3+ billion nucleotide reference genomes, 100+ n nucleotides of sequencer read data, etc., the FPGA 7 may need to access the host memory 1014 a large number oftimes such as for accessing an index, such as a 30GB hash table or other reference genome index, such as for the purpose of g the seeds from a sequenced DNA/RNA query to a 3Gbp reference genome, and/or for fetching candidate ts, e.g., from the reference genome, to align against. ingly, in s implementations ofthe system herein disclosed, many rapid random memory accesses may need to occur by one or more of the hardwired processing engines 13, such as in the performance of a mapping, aligning, and/or variant calling operation. However, it may be prohibitively impractical for the FPGA 7 to make so many small random accesses over the peripheral bus 3 or other networking link to the memory 1014 attached to the host CPU 1000. For instance, in such ces, latencies of return data can be very high, bus efficiency can be very low, e.g., for such small random accesses, and the burden on the CPU external memory interface 1006 may be prohibitively great.

Additionally, as a result of each device needing its own dedicated external memory, the typical form factor of the full CPU 1000 + FPGA 7 platform is forced to be larger than may be desirable, e.g., for some applications. In such instances, in addition to a standard system motherboard for one or more CPUs 1000 and supporting chips 7 and memories, 1014 and/or 14, room is needed on the board for a large FPGA e (which may even need to be larger so as to have enough pins for several external memory busses) and l memory modules, 1014, 14. Standard motherboards, however, do not include these components, nor would they easily have room for them, so a practical embodiment may be configured to e an expansion card 2, containing the FPGA 7, its memory 14, and other supporting components, such as power supply, e.g. connected to the PCie expansion slot on the CPU motherboard. To have room for the expansion card 2, the system may be fabricated to be in a large enough chassis, such as a 1U or 2U or larger rack-mount server.

] In view ofthe above, in various instances, as can be seen with respect to B, to overcome these factors, it may be desirable to configure the CPU 1000 to be in a tight coupling arrangement with the FPGA 7. Particularly, in various instances, the FPGA 7 may be tightly coupled to the CPU 1000, such as by a low latency interconnect 3, such as a quick path interconnect (QPI). Specifically, to establish a tighter CPU+FPGA integration, the two devices may be connected by any suitable low latency interface, such as a "processor interconnect" or similar, such as ® Quick Path Interconnect (QPI) or HyperTransport (HT).

Accordingly, as seen with respect to B, a system 1 is provided wherein the system includes both a CPU 1000 and a processor, such as an FPGA 7, n both s are associated with one or more memory modules. For ce, as ed, the CPU 1000 may be coupled, such as via a suitably configured bus 1006, to a DRAM 1014, and likewise, the FPGA 7 is icably coupled to an associated memory 14 via a DDR3 bus 6. However, in this instance, instead ofbeing coupled to one another such as by a typical high latency onnect, e.g., PCie interface, the CPU 1000 is coupled to the FPGA 7 by a low latency, hyper ort interconnect 3, such as a QPI. In such an instance, due to the inherent low latency nature of such interconnects, the associated memories 1014, 14 of the CPU 1000 and the FPGA 7 are readily accessible to one another. Additionally, in various instances, due to this tight coupling configuration, one or more cashes 1114a/14a associated with the s may be configured so as to be coherent with respect to one another.

Some key properties of such a y coupled CPU/FPGA interconnect include a high bandwidth, e.g., 12.8GB/s; low latency, e.g., 100-300ns; an adapted protocol designed for allowing efficient remote memory accesses, and efficient small memory transfers, e.g., on the order of 64 bytes or less; and a supported protocol and CPU integration for cache access and cache coherency. In such instances, a natural interconnect for use for such tight integration with a given CPU 1000 may be its native CPU-to-CPU interconnect 1003, which may be employed herein to enable multiple cores and multiple CPUs to operate in parallel in a shared memory 1014 space, thereby allowing the accessing of each other's cache stacks and external memory in a cache-coherent manner.

Accordingly, as can be seen with respect to FIGS. 34A and 34B, a board 2 may be provided, such as where the board may be configured to receive one or more CPUs 1000, such as via a plurality of onnects 1003, such as native CPU-CPU onnects 1003a and 1003b. However, in this instance, as depicted in A, a CPU 1000 is ured so as to be coupled to the interconnect 1003a, but rather than another CPU being coupled therewith via interconnect 1003b, an FPGA 7 of the disclosure is configured so as to be coupled therewith. Additionally, the system 1 is configured such that the CPU 1000 may be coupled to the associated FPGA 7, such as by a low latency, tight coupling interconnect 3.

In such ces, each memory 1014, 14 ated with the respective devices 1000, 7 may be made so as to accessible to each other, such as in a andwidth, cache coherent manner.

] Likewise, as can be seen with respect to B, the system can also be configured so as to receive packages 1002a and/or 1002b, such as where each ofthe packages include one or more CPUs 1000a, 1000b that are tightly coupled, e.g., via low latency interconnects 3a and 3b, to one or more FPGAs 7a, 7b, such as where given the system architecture, each package 2a and 2b may be coupled one with the other such as via a tight coupling onnect 3. Further, as can be seen with respect to , in various ces, a package 1002a may be provided, wherein the package 1002a includes a CPU 1000 that has been ated in such a manner so as to be closely coupled with an integrated circuit such as an FPGA 7. In such an instance, because of the close coupling of the CPU 1000 and the FPGA 7, the system may be constructed such that they are able to directly share a cache 1014a in a manner that is consistent, coherent, and readily accessible by either device, such as with respect to the data stored therein.

] Hence, in such instances, the FPGA 7, and or package 2a/2b, can, in effect, masquerade as another CPU, and thereby operate in a cache-coherent shared-memory environment with one or more CPUs, just as multiple CPUs would on a socket motherboard 1002, or multiple CPU cores would within a mutli-core CPU device. With such an FPGA/CPU interconnect, the FPGA 7 can efficiently share CPU memory 1014, rather than having its own dedicated external memory 14, which may or may not be included or accessed. Thus, in such a configuration, rapid, short, random accesses are supported efficiently by the interconnect 3, such as with low latency. This makes it cal and efficient for the various processing engines 13 in the FPGA 7 to access large data structures in CPU memory 1000.

For instance, as can be seen with respect to , a system for performing one or more of the methods disclosed herein is provided, such as where the method includes one or more steps for performing the functions of the disclosure, such as one or more mapping and/or aligning and/or variant calling on, as described herein, in a shared manner. Particularly, in one step (1) a data ure may be generated or otherwise provided, such as by an NGS and/or CPU 1000, which data structure may then be stored in an associated memory (2), such as a DRAM 1014. The data structure may be any data structure, such as with respect to those described herein, but in this instance, may be a plurality ofreads of sequenced data and/or a reference genome and/or an index of the nce genome, such as for the performance ofmapping and/or aligning and/or variant calling functions.

] In a second step (2), such as with respect to mapping and/or aligning, etc. functions, an FPGA 7 associated with the CPU 1000, such as by a tight coupling interface 3, may access the CPU associated memory 1014, so as to perform one or more actions with respect to the stored sequenced reads, reference (s), and/or an index thereof.

Particularly, in a step (3), e.g., in an exemplary g operation, the FPGA 7 may access the data structure, e.g., the sequenced reads and/or reference sequences, so as to e one or more seeds there from, such as where the data structure includes one or more reads and/or genome reference sequences. In such an instance, the seeds, e.g., or the reference and/or read sequences may be employed for the purposes of performing a hash function with respect thereto, such as to produce one or more reads that have been mapped to one or more positions with respect to the reference genome.

In a further step (3), the mapped result data may be stored, e.g., in either the host memory 1014 or in an associated DRAM 14. Additionally, once the data has been mapped, the FPGA 7, or a processing engine 13 thereof, may be reconfigured, e.g., partially figured, as an alignment engine, which may then access the stored mapped data structure so as to perform an aligning function thereon, so as to produce one or more reads that have been aligned to the reference genome. In an additional step (4), the host CPU may then access the mapped and/or aligned data so as to perform one or more functions thereon, such as for the tion of a De Brujin Graph ("DBG"), which DBG may then be stored in its associated memory. Likewise, in one or more additional steps, the FPGA 7 may once again access the host CPU memory 1014 so as to access the DBG and perform an HMM analysis thereon so as to produce one or more variant call files.

In ular instances, the CPU 1000 and/or FPGA 7 may have one or more memory cache'swhich due to the tight coupling ofthe interface between the two devices will allow the separate caches to be coherent, such as with respect to the transitionary data, e.g., results data, stored thereon, such as results from the performance of one or more ons herein. In a manner such as this, data may be shared substantially seamlessly between the tightly coupled devices, y allowing a pipeline of functions to be weaved together such as in a bioinformatics ne. Thus, in such an instance, it may no longer be necessary for the FPGA 7 to have its own dedicated al memory 14 attached, and hence, due to such a tight ng configuration, the stored reads, the reference , and/or reference genomic index, as herein described, may be intensively shared, e.g., in a cache coherent manner, such as for read mapping and alignment, and other genomic data processing operations.

Additionally, as can be seen with respect to , the low latency and cache coherency configurations, as well as other component configurations discussed herein, allow r, level operations to be performed in one device (e.g., in a CPU or FPGA), before handing back a data ure or processing thread 20 to the other device, such as for further processing. For example, in one instance, a CPU thread 20a, may be configured toque up large amounts of work for the FPGA hardware logic 13 to s, and the same or another thread 20b, may be configured to then process the large queue of results data generated y, such as at a substantially later time. However, in various instances, it may be more efficient, as presented herein, for a single CPU thread 20 to make a blocking "function call" to a coupled FPGA hardware engine 13, which CPU may be set to resume software execution as soon as the hardware function ofthe FPGA is completed. Hence, rather than packaging up data structures in packets to stream by DMA 14 into the FPGA 7, and unpacking results when they return, a software thread 20 could simply provide a memory pointer to the FPGA engine 13, which could access and modify the shared memory 1014/14 in place, in a cache-coherent manner.

Particularly, given the relationship between the structures ed herein, the granularity of the software/hardware cooperation can be much finer, with much smaller, lower level operations being ted so as to be performed by various hardware engines 13, such as function calls from various allocated software s 20. For e, in a loose CPU/FPGA interconnect platform, for efficient acceleration of DNA/RNA read g, ent, and/or variant calling, a full g/aligning/variant calling ne may be constructed as one or more software and/or FPGA engines, with unmapped and unaligned reads being streamed from software to re, and the fully mapped and aligned reads streamed from the hardware back to the software, where the process may be repeated, such as for variant calling. With respect to the urations herein described, this can be very fast.

However, in various instances, such a system may suffer from limitations of flexibility, complexity, and/or programmability, such because the whole map/align and/or variant call pipeline is implemented in hardware circuitry, which although reconfigurable in an FPGA, is generally much less flexible and programmable than software, and may therefore be limited to less algorithmic complexity.

By contrast, using a tight CPU/FPGA onnect, such as a QPI or other interconnect in the configurations disclosed herein, l resource expensive discrete ions, such as seed generation and/or g, rescue scannmg, s alignment, gapped, e.g., Smith-Waterman, alignment, etc., can be implemented as distinct separately accessible hardware engines 13, e.g., see , and the overall mapping/alignment and/or variant call algorithms can be ented in software, with low-level acceleration calls to the FPGA for the specific expensive processing steps. This framework allows full software programmability, outside the specific acceleration calls, and enables greater algorithmic complexity and flexibility, than standard hardware implemented ions.

Furthermore, in such a framework of software execution accelerated by discrete low-level FPGA hardware acceleration calls, hardware acceleration functions may more easily be shared for multiple purposes. For instance, when hardware engines 13 form large, monolithic pipelines, the individual pipeline subcomponents may generally be specialized to their environment, and interconnected only within one pipeline, which unless tightly coupled may not generally be accessible for any purpose. But many genomic data processing operations, such as Smith-Waterman alignment, gapless alignment, De Bruijn or assembly graph construction, and other such operations, can be used in various higher level parent algorithms. For example, as bed herein, Smith-Waterman alignment may be used in DNA/RNA read mapping and aligning, such as with respect to a reference genome, but may also be configured so as to be used by haplotype-based variant callers, to align candidate haplotypes to a reference genome, or to each other, or to sequenced reads, such as in a HMM is and/or variant call function. Hence, exposing various discrete low-level hardware acceleration functions via general software function calls may enable the same acceleration logic, e.g., 13, to be leveraged throughout a genomic data processing application, such as in the performance ofboth alignment and variant calling, e.g. HMM, operations.

It is also practical, with tight CPU/FPGA interconnection, to have distributed rather than centralized CPU 1000 software control over communication with the various FPGA re engines 13 bed herein. In widespread practices of multi-threaded, core, and multi-CPU software design, many software threads and processes communicate and cooperate seamlessly, without any central software modules, s, or threads to manage intercommunication. In such a format, this is practical because of the cache-coherent shared , which is visible to all s in all cores in all of the CPUs; while physically, coherent memory sharing between the cores and CPUs occurs by intercommunication over the processor onnect, e.g., QPI or HT.

In a similar manner, as can be seen with respect to FIGS. 36 - 38, the systems provided herein may have a number of CPUs and/or FPGAs that may be in a tight CPU/FPGA interconnect configuration that incorporates a multiplicity ofthreads, e.g., 20a, b, c, and a licity of processes g on one or the multiple cores and/or CPUs, e.g., 1000a, 100b, and 1000c. As such, the system components are configured for communicating and cooperating in a distributed manner with one another, e.g., between the various different CPU and/or FPGA hardware acceleration engines, such as by the use of cache-coherent memory sharing between the various CPU(s) and FPGA(s). For instance, as can be seen with respect to , a multiplicity of CPU cores 1000a, 1000b, and 1000c can be coupled together in such a manner as to share one or more memories, e.g., DRAMs 1014, and/or one or more caches having one or more layers, e.g., LI, L2, L3, etc., or levels associated therewith. Likewise, with respect to , in another embodiment, a single CPU 1000 may be configured to include multiple cores 1000a, 1000b, and 1000c that can be coupled together in such a manner so as to share one or more memories, e.g., DRAMs 1014, and/or one or more caches, 1014a, having one or more layers or levels associated therewith.

] Hence, in either embodiment, data to be passed from one or more software threads 20 from one or more CPU cores 1000 to a re engine 13, e.g., of an FPGA, or vice versa, may be continuously and/or seamlessly updated in the shared memory 1014, or a cache and/or layer thereof, which is visible to each device. Additionally, requests to process data in the shared memory 1014, or notification of results updated therein, can be signaled between the software and/or hardware, such as over a suitably configured bus, e.g., DDR4 bus, such as in queues that may be ented within the shared memory itself. Standard software mechanisms for control, transfer, and data protection, such as semaphores, mutexes, and atomic integers, can also be implemented similarly for software/hardware coordination.

Consequently, in some embodiments, as exemplified in , with no need for the FPGA 7 to have its own dedicated memory 14, or other external resources, due to cache coherent memory-sharing over a tight CPU/FPGA interconnect, it becomes much more practical to package the FPGA 7 more compactly and natively within traditional CPU 1000 motherboards, without the use of expansion cards. See, for example FIGS. 34A and 34B and . Several packaging alternatives are available. Specifically, an FPGA 7 may be installed onto a CPU motherboard in a CPU socket, as shown in FIGS. 34A and 34B, such as by use of an appropriate interposer, such as a small PC board 2, or alternative wirebond packaging of the FPGA die within the CPU chip package 2a, where the CPU socket pins are appropriately routed to the FPGA pins, and include power and ground connections, a processer interconnect 3 (QPI, HT, etc.), and other system connections. Accordingly, an FPGA die and CPU die may be included in the same multi-chip package (MCP) with the necessary connections, including power, , and CPU/FPGA interconnect, made within the package 2a. Inter-die connections may be made by -die wire-bonding, or by connection to a common ate or interposer, or by bonded pads or through-silicon vias between stacked dice.

Additionally, m vanous implementations, FPGA and CPU cores may be fabricated on a single die, see , using a system-on-a-chip (SOC) methodology. In any of these cases, custom logic, e.g., 17, may be instantiated inside the FPGA 7 to both communicate over the GA interconnect 3, e.g., by properly ted protocols, and to e, convert, and/or route memory access requests from internal FPGA engines 13 to the CPU/FPGA interconnect 3, via appropriate ols, to the shared memory 1014a.

Additionally, some or all of this logic may be ed into custom silicon, to avoid using up FPGA logic real estate for this purpose, such as where the hardened logic may reside on the CPU die, and/or the FPGA die, or a separate die. Also, in any of these cases, power supply and heat dissipation requirements may be appropriately achieved, such as within a single e (MCP or SOC). Further, the FPGA size and CPU core count may be selected to stay within a safe power envelope, and/or dynamic methods (clock frequency management, clock gating, core ing, power islands, etc.) may be used to regulate power consumption according to changing the CPU and/or the FPGA computation demands.

All e packaging options share several advantages. The tightly-integrated CPU/FPGA platform becomes compatible with rd motherboards and/or system chassis, of a variety of sizes. If the FPGA is installed via an interposer in a CPU socket, see FIGS. 34A and 34B, then at least a dual-socket motherboard1002 may be employed. In others instances, a quad-socket motherboard may be employed so as to allow 3 CPUs+ 1 FPGA, 2 CPUs + 2 FPGAs, or 1 CPU + 3 FPGAs, etc. configurations to be implemented. If each FPGA resides in the same chip package as a CPU (either MCP or SOC), then a single-socket motherboard may be employed, potentially in a very small s (although a dual socket motherboard is depicted); this also scales upward very well, e.g. 4 FPGAs and 4 multi-core CPUs on a 4-socket server motherboard, which nevertheless could operate in a compact chassis, such as a 1U rack-mount server.

Accordingly, in various ces, therefore, there may be no need for an expansion card to be led so as to integrate the CPU and FPGA acceleration, because the FPGA 7 may be integrated in to the CPU socket 1003. This implementation avoids the extra space and power requirements of an ion card, and avoids various additional failure points expansion cards sometimes have with respect to relatively low-reliability components.

Furthermore, rd CPU cooling solutions (head sinks, heat pipes, and/or fans), which are efficient yet low-cost since they are manufactured in high volumes, can be applied to FPGAs or CPU/FPGA packages in CPU sockets, whereas cooling for ion cards can be ive and inefficient.

Likewise, an FPGA/interposer and/or CPU/FPGA package may include the full power supply of a CPU socket, e.g. 150W, whereas a standard expansion card may be power limited, e.g. 25W or 75W from the PCie bus. In various instances, for genomic data processing applications, all these packaging options may tate easy installation of a tightly-integrated CPU+FPGA compute platform, such as within a DNA sequencer. For instance, typical modem "next-generation" DNA sequencers contain the sequencing apparatus (sample and reagent storage, fluidics tubing and l, sensor arrays, primary image and/or signal processing) within a chassis that also contains a standard or custom server motherboard, wired to the sequencing apparatus for sequencing control and data acquisition. A y-integrated GA platform, as herein described, may be ed in such a sequencer such as by simply installing one or more FPGA/interposer and/or PU packages m CPU sockets of its existing motherboard, or atively by installing a new motherboard with both CPU(s) and ), e.g., tightly coupled, as herein sed. r, all of these packaging options may be configured to facilitate easy deployment of the tightly-integrated CPU+FPGA platform such as into a cloud accessible and/or datacenter server rack, which include compact/dense s with very high reliability/availability.

] Hence, in accordance with the ngs herein, there are many processing stages for data from DNA (or RNA) sequencing to mapping and aligning to sorting and/or de-duplicating to variant calling, which can vary depending on the primary and/or secondary and/or tertiary processing technologies employed and their applications. Such processing steps may include one or more of: signal processing on electrical measurements from a sequencer, an image processing on optical measurements from the sequencer, base calling using processed signal or image data to determine the most likely nucleotide sequence and confidence scores, filtering sequenced reads with low quality or onal clusters, detecting and trimming adapters, key sequences, barcodes, and low quality read ends, as well as De nova sequence assembly, generating and/or utilizing De Bruijn graphs and/or sequence graphs, e.g., De Bruijn and sequence graph construction, editing, trimming, p, repair, coloring, annotation, comparison, transformation, splitting, splicing, analysis, subgraph selection, traversal, iteration, ion, ing, filtering, import, export, including mapping reads to a reference genome, aligning reads to candidate mapping locations in the reference genome, local assembly of reads mapped to a reference region, sorting reads by aligned position, marking and/or removing duplicate reads, including PCR or optical duplicates, re-alignment of multiple overlapping reads for indel consistency, base quality score recalibration, variant calling (single sample or joint), structural variant analysis, copy number t analysis, c variant calling (e.g., tumor sample only, d tumor/normal, or tumor/unmatched normal, etc.), RNA splice junction detection, RNA alternative splicing analysis, RNA transcript assembly, RNA transcript expression analysis, RNA differential expression analysis, RNA variant g, DNA/RNA difference analysis, DNA methylation analysis and calling, variant quality score recalibration, variant filtering, variant annotation from known variant databases, sample ination detection and estimation, phenotype prediction, disease testing, treatment response prediction, custom treatment design, ry and mutation history analysis, population DNA analysis, genetic marker identification, encoding genomic data into standard formats and/or compression files (e.g. PASTA, FASTQ, SAM, BAM, VCF, BCF), decoding genomic data from standard formats, querying, selecting or filtering genomic data s, general compression and decompression for genomic files (gzip, BAM compression), specialized compression and decompression for genomic data , genomic data tion and decryption, statistics calculation, comparison, and presentation from genomic data, c result data companson, accuracy analysis and reporting, genomic file storage, archival, retrieval, backup, recovery, and transmission, as well as genomic database construction, querying, access management, data extraction, and the like.

All ofthese operations can be quite slow and ive when implemented on traditional compute rms. The sluggishness of such exclusively software ented operations may be due in part to the complexity of the algorithms, but is typically due to the very large input and output datasets that results in high latency with respect to moving the data. The devices and systems disclosed herein overcome these ms, in part due to the configuration of the various hardware processmg engmes, acceleration by the s hardware implementations, and/or in part due to the CPU/FPGA tight coupling configurations. Accordingly, as can be seen with respect to , one or more, e.g., all of these operations, may be accelerated by cooperation ofCPUs 1000 and FPGAs 7, such as in a distributed processing model, as described herein. For instance, in some cases (encryption, general compression, read mapping, and/or alignment), a whole operational function may be substantially or entirely implemented in custom FPGA logic (such as by hardware design methodology, e.g. RTL), such as where the CPU software mostly serves the function of ing large data packets for preprocessing via worker threads 20, such as aggregating the data into s jobs to be processed by one or more hardware implemented sing engines, and feeding the s data , such as in a first in first out format, to one or more ofthe FPGA engine(s) 13, and/or receives results therefrom.

For instance, as can be seen with respect to , in various embodiments, a worker thread generates various packets ofjob data that may be compiled and/or streamed into larger job s that may be queued up and/or further aggregated in preparation for transfer, e.g., via a DDR3 to the FPGA 7, such as over a high bandwidth, low y, point to point interconnect protocol, e.g., QPI 3. In particular instances, the data may be buffered in accordance with the particular data sets being transferred to the FPGA. Once the packaged data is received by the FPGA 7, such as in a cache coherent manner, it may be processed and sent to one or more specialized clusters 11 whereby it may further be directed to one or more sets of processing engines for processing thereby in accordance with one or more of the pipeline operations herein bed.

Once processed, results data may then be sent back to the cluster and queued up for being sent back over the tight coupling point to point interconnect to the CPU for post processing. In certain embodiments, the data may be sent to a de-aggregator thread prior to post processing. Once post processing has occurred, the data may be sent back to the initial worker thread 20 that may be waiting on the data. Such distributed processing is particularly beneficial for the functions herein sed above. Particularly, these functions are distinguishable by the facts that their thmic complexity (although having a very high net computational burden) are pretty limited, and they each may be configured so as to have a fairly m compute cost across their various erations.

However, in various cases, rather than processing the data in large packets, smaller sub-routines or discrete function protocols or elements may be performed, such as pertaining to one or more functions ofa pipeline, rather than performing the entire processing functions for that pipeline on that data. Hence, a useful strategy may be to identify one or more critical e-intensive sub-functions in any given operation, and then implement that sub-function in custom FPGA logic are acceleration), such as for the intensive sub-function(s), while implementing the e of the operation, and ideally much or most of the algorithmic xity, in software to run on CPUs/GPUs/QPUs, as described herein, such as with respect to . lly, it is typical of many genomic data processing operations that a small tage of the algorithmic complexity ts for a large percentage of the overall computing load. For instance, as a typical example, 20% of the algorithmic complexity for the performance of a given function may account for 90% of the compute load, while the remaining 80% of the algorithmic complexity may only account for 10% of the compute load. Hence, in various instances, the system components herein described may be configured so as to ent the high, e.g., 20% or more, complexity portion so as to be run very efficiently in custom FPGA logic, which may be a tractable and maintainable in a hardware design, and thus, may be configured for executing this in FPGA; which in tum may reduce the CPU compute load by 90%, thereby enabling 1Ox overall acceleration. Other typical examples may be even more extreme, such as where 10% of the algorithmic complexity may account for 98% of the e load, in which case applying FPGA acceleration, as herein described, to the 10% complexity portion be even easier, but may also enable up to 50x net acceleration. In various instances, where extreme accelerated sing is desired, one or more ofthese functions may be performed by a quantum processing unit.

However, such a meal" or distributed processing acceleration approaches may be more practical when implemented in a tightly integrated CPU/GPU+FPGA platform, rather than on a loosely integrated CPU/GPU+FPGA platform.

Particularly, in a loosely integrated platform, the portion, e.g., the functions, to be implemented in FPGA logic may be selected so as to minimize the size of the input data to the FPGA engine(s), and to minimize the output data from the FPGA engine(s), such as for each data unit processed, and additionally may be configured so as to keep the software/hardware boundary tolerant of high latencies. In such instances, the boundary between the hardware and software portions may be forced, e.g., on the loosely-integrated platform, to be drawn h certain low-bandwidth/high-latency cut-points, which divisions may not otherwise be ble when zing the partitioning ofthe algorithmic complexity and computational loads. This may often result either in enlarging the ries of the hardware portion, assing an undesirably large portion of the algorithmic complexity in the hardwired format, or in shrinking the boundaries of the hardware portion, rably excluding portions with dense compute load.

By st, on a tightly integrated CPU/GPU+FPGA platform, due to the cache-coherent shared memory and the high-bandwidth/low-latency CPU/GPU/FPGA interconnect, the low-complexity/high-compute-load portions of a genomic data sing operation can be selected very precisely for implementation in custom FPGA logic (e.g., via the hardware engine(s) bed herein), with optimized software/hardware boundaries. In such an instance, even if a data unit is large at the desired software/hardware boundary, it can still be efficiently handed off to an FPGA hardware engine for processing, just by passing a pointer to the particular data unit. Particularly, in such an instance, as per B, the hardware engine 13 of the FPGA 7, may not need to access every element of the data unit stored within the DRAM 1014; rather, it can access the necessary elements, e.g., within the cache 1014a, with efficient small accesses over the low-latency onnect 3' ed by the CPU/GPU cache, thereby consuming less aggregate bandwidth than ifthe entire data unit had to be accessed and/or transferred to the FPGA 7, such as by DMA of the DRAM 1014, over a loose interconnect 3, as per A.

In such instances, the hardware engine 13 can annotate processing results into the data unit ce in CPU/GPU memory 1014, without streaming an entire copy of the data unit by DMA to CPU/GPU . Even if the desired software/hardware boundary is not appropriate for a software thread 20 to make a high-latency, non-blocking queued handoff to the hardware engine 13, it can potentially make a blocking function call to the hardware engine 13, sleeping for a short latency until the hardware engine completes, the latency being dramatically d by the cache-coherent shared memory, the low-latency/high-bandwidth interconnect, and the distributed software/hardware coordination model, as in B.

In particular instances, e the specific algorithms and requirements of signal/image processing and base calling vary from one sequencer technology to another, and because the quantity of raw data from the sequencer's sensor is typically gargantuan (this being reduced to enormous after signal/image processing, and to merely huge after base calling), such signal/image processing and base calling may be efficiently performed within the sequencer itself, or on a nearby compute server ted by a high bandwidth transmission channel to the sequencer. However, DNA sequencers have been achieving increasingly high throughputs, at a rate of increase exceeding Moore's Law, such that existing Central Processing Unit ("CPU") and/or Graphics Processing Unit "GPU" based signal/image processing and base calling, when implemented individually and alone, have become singly inadequate to the task. Nevertheless, since a tightly ated CPU + FPGA and/or a GPU + FPGA and/or a GPU/CPU + FPGA platform can be configured to be compact and easily instantiated within such a sequencer, e.g., as CPU and/or GPU and/or FPGA chip oned on the sequencer's motherboard, or easily installed in a server adjacent to the sequencer, or a based server system accessible remotely from the sequencer, such a sequencer may be an ideal rm to offer the e e acceleration offered by the custom FPGA/ASIC hardware engines described herein.

For instance, the system provided herein may be configured so as to perform primary, secondary, and/or tertiary processing, or portions f so as to be implemented by an accelerated CPU, GPU, and/or FPGA; a CPU+ FPGA; a GPU+ FPGA; a GPU/CPU+ FPGA; QPU; CPU/QPU; U; CPU and/or GPU and/or QPU + FPGA platform.

Further, such accelerated platforms, e.g., including one or more FPGA and/or QPU hardware engines, are useful for implementation in cloud-based systems, as described herein. For e, signal/image processing, base calling, mapping, aligning, sorting, de-duplicating, and/or variant calling algorithms, or portions thereof, generally require large amounts of floating point and/or fixed-point math, notably additions and multiplications. These functions can also be configured so as to be performed by one or more quantum processing circuits such as to be implemented in a quantum processing platform. ularly, large modern FPGAs/quantum circuits contain thousands ofhighspeed multiplication and addition resources. More particularly, these circuits may include custom engines that may be implemented on or by them, which custom engines may be configured to perform parallel arithmetic operations at rates far ing the capabilities of simple general CPUs. Likewise, simple GPUs, have more comparable parallel arithmetic resources. However, GPUs often have d architectural tions and programming restrictions that may prevent them from being fully ed. Accordingly, these FPGA and/or quantum sing and/or GPU arithmetic resources can be wired up or ise configured by design to operate in exactly the designed manner with near 100% efficiency, such as for performing the calculations necessary to execute the ons herein.

Accordingly, GPU cards may be added to expansion slots on a motherboard with a tightly integrated CPU and/or FPGA, thereby allowing all three processor types to cooperate, although the GPU may still cooperate with all of its own limitations and the limitations of loose integration.

More particularly, in various instances, with respect to Graphics sing Units (GPUs), a GPU can be configured so as to implement one or more of the ons, as herein described, so as to accelerate the processing speed of the underlying ations necessary for preforming that function, in whole or in part. More particularly, a GPU may be configured to perform one or more tasks in a mapping, aligning, sorting, de-duplicating, and/or variant calling protocol, such as to accelerate one or more of the computations, e.g., the large amounts of floating point and/or fixed-point math, such as additions and multiplications involved therein, so as to work in conjunction with a server's CPU and/or FPGA to accelerate the application and processing performance and shorten the ational cycles required for performing such ons. Cloud servers, as herein described, with GPU/CPU/FPGA cards may be configured so as to easily handle computeintensive tasks and deliver a smoother user experience when leveraged for virtualization.

Such e-intensive tasks can also be offloaded to the cloud, such as to be med by a quantum processing unit.

Accordingly, if a tightly integrated CPU+FPGA or GPU+FPGA and/or CPU/GPU/FPGA with shared memory rm is employed within a sequencer, or attached or cloud based server, such as for signal/image processing, base calling, mapping, aligning, sorting, de-duplicating, and/or variant calling functions, there may be an advantage achieved such as in an incremental development process. For instance, initially, a limited portion ofthe compute load, such as a dynamic programming function for base calling, mapping, ng, sorting, de-duplicating, and/or variant calling may be implemented in one or more FPGA engines, where as other work may be done in the CPU and/or GPU expansion cards.

However, the tight CPU/GPU/FPGA integration and shared memory model, herein presented, may be further ured, later, so as to make it easy to incrementally select additional compute-intensive functions for GPU, FPGA, and/or quantum acceleration, which may then be ented as processing engines, and various of their functions may be offloaded for ion into the FPGA(s) and/or in some instances may be offloaded onto the cloud, e.g., for performance by a QPU, thereby accelerating signal/image/base calling/mapping/aligning/variant processing. Such incremental advances can be implemented as needed to keep up with the increasing throughput of various primary and/or secondary and/or tertiary processing technologies.

Hence, read mapping and alignment, e.g., of one or more reads to a reference genome, as well as sorting, de-duplicating, and/or variant calling may be ted from such GPU and/or FPGA and/or QPU acceleration. Specifically, mapping and ent and/or t calling, or portions thereof, may be implemented partially or entirely as custom FPGA logic, such as with the "to be mapped and/or aligned and/or variant called" reads streaming from the CPU/GPU memory into the FPGA map/align/variant calling engines, and mapped and/or aligned and/or variant called read records streaming back out, which may further be streamed back on-board, such as in the performance of sorting and/or variant calling. This type of FPGA acceleration works on a loosely-integrated CPU/GPU+FPGA rm, and in the configurations described herein may be extremely fast. Nevertheless, there are some onal advantages that may be gained by moving to a tightly-integrated CPU/GPU/QPU + FPGA platform.

Accordingly, with t to mappmg and aligning and variant calling, in some embodiments, a shared advantage of a tightly-integrated CPU/GPU+FPGA and/or quantum processing platform, as described , is that the map/align/variant calling acceleration, e.g., hardware acceleration, can be efficiently split into several discrete compute-intensive operations, such as seed generation and/or mapping, seed chain ion, paired end rescue scans, gapless alignment, and gapped alignment (Smith-Waterman or man-Wunsch), De Bruijn graph formation, performing a HMM computation, and the like, such as where the CPU and/or GPU and/or quantum computing re performs r (but not necessarily less complex) tasks, and may make acceleration calls to discrete hardware and/or other quantum computing engines as needed. Such a model may be less efficient in a typical loosely-integrated CPU/GPU+FPGA platform, e.g., due to large amounts of data to transfer back and forth between steps and high ies, but may be more efficient in a tightly-integrated CPU+FPGA, GPU+ FPGA, and/or quantum computing platform with cache-coherent shared memory, high-bandwidth/low-latency interconnect, and distributed software/hardware coordination model. Additionally, such as with respect to t calling, both Hidden Markov model (HMM) and/or dynamic programming (DP) algorithms, including Viterbi and forward algorithms, may be implemented in association with a base calling/mapping/aligning/sorting/de-duplicating operation, such as to compute the most likely original ce ning the observed sensor measurements, in a configuration so as to be well suited to the el cellular layout ofFPGAs and quantum circuits described herein. ically, an efficient ation ofhardware and/or software resources in a distributed processing configuration can result from reducing re and/or quantum computing acceleration to discrete compute-intensive functions. In such ces, several of the functions disclosed herein may be performed in a monolithic pure-hardware engine so as to be less compute intensive, but may nevertheless still be algorithmically x, and therefore may consume large quantities of physical FPGA resources (lookup-tables, flipflops , block-RAMs, etc.). In such instances, moving a portion or all of various te functions to software could take up available CPU cycles, in return for relinquishing substantial amounts of FPGA area. In certain of these instances, the freed FPGA area can be used for establishing greater parallelism for the e intensive map/align/variant call subfunctions , thus increasing acceleration, or for other genomic acceleration functions. Such benefits may also be achieved by implementing compute intensive functions in one or more dedicated quantum circuits for implementation by a quantum computing platform.

Hence, in various embodiments, the algorithmic complexity of the one or more functions disclosed herein may be somewhat lessened by being configured in a pure hardware or pure quantum computing implementation. However, some ions, such as comparing pairs of candidate alignments for paired-end reads, and/or performing subtle mapping quality (MAPQ) estimations, represent very low e loads, and thus could benefit from more complex and accurate processing in CPU/GPU and/or quantum ing software. Hence, in general, ng the hardware processing to specific compute-intensive ions would allow more complex and accurate algorithms to be employed in the CPU/GPU portions.

Furthermore, m vanous embodiments, the whole or a part of the map/align/sorting/de-duplicating/variant g ions, disclosed herein, could be configured in such a manner that the more algorithmically x computations may be employed at high levels in hardware and/or via one or more quantum circuits, such as where the called compute-intensive hardware and/or quantum functions are configured to be performed in a dynamic or ive order. Particularly, a monolithic pure-hardware/quantum processing design may be ented in a manner so as to on more efficiently as a linear pipeline. For example, if during processing one Smith-Waterman alignment displayed evidence ofthe true alignment path escaping the scoring band, e.g., swath as described above, another Smith-Waterman alignment could be called to t this. Hence, these configurations could essentially reduce the FPGA hardware/quantum acceleration to discrete functions, such as a form of procedural abstraction, which would allow higher level complexity to be built easily on top of it.

] Additionally, in various instances, flexibility within the map/align/variant calling algorithms and features thereof may be ed by reducing hardware and/or quantum acceleration to discrete compute-intensive functions, and configuring the system so as to perform other, e.g., less intensive parts, in the software of the CPU and/or GPU. For instance, gh hardware algorithms can be modified and igured in FPGAs, generally such changes to the hardware designs, e.g., via firmware, may require several times as much design effort as similar changes to software code. In such instances, the computeintensive portions of g and alignment and sorting and de-duplicating, and/or variant calling, such as seed mapping, seed chain formation, paired end rescue scans, gapless alignment, gapped alignment, and HMM, which are relatively well-defined, are thus stable functions and do not require frequent algorithmic changes. These functions, therefore, may be suitably zed in hardware, whereas other ons, which could be executed by CPU/GPU software, are more appropriate for incremental improvement of algorithms, which is significantly easier in software. However, once fully developed could be implemented in hardware. One or more ofthese functions may also be configured so as to be implemented in one or more m circuits ofa quantum processing machine.

Accordingly, in various instances, variant calling (with respect to DNA or RNA, single sample or joint, germline or somatic, etc.) may also benefit from FPGA and/or quantum acceleration, such as with respect to its various compute intensive functions. For instance, haplotype-based callers, which call bases on evidence derived from a context provided within a window around a potential variant, as described above, is often the most compute-intensive operation. These operations include comparing a ate haplotype (e.g., a single-strand nucleotide sequence representing a theory ofthe true sequence ofat least one of the sampled strands at the genome locus in question) to each sequencer read, such as to estimate a conditional probability rving the read given the truth ofthe haplotype.

Such an ion may be performed via one or more of an MRJD, Pair Hidden Markov Model (pair-HMM), and/or a Pair-Determined Hidden Markov Model (PDHMM ) calculation that sums the probabilities of possible combinations of errors in cing or sample preparation (PCR, etc.) by a dynamic programming algorithm. Hence, with respect thereto, the system can be configured such that a pair-HMM or PD-HMM calculation may be accelerated by one or more, e.g., parallel, FPGA hardware or quantum processing engines, whereas the CPU/GPU/QPU software may be configured so as to execute the remainder of the parent haplotype-based variant calling thm, either in a looselyintegrated or tightly-integrated GA, or GPU+FPGA or CPU and/or GPU+FPGA and/or QPU platform. For instance, in a loose ation, software threads may construct and e a De Bruijn and/or assembly graph from the reads overlapping a chosen active region (a window or contiguous subset of the reference genome), extract candidate haplotypes from the graph, and queue up ype-read pairs for DMA transfer to FPGA hardware engines, such as for pair-HMM or PD-HMM comparison. The same or other re threads can then receive the pair-HMM results queued and DMA-transferred back from the FPGA into the CPU/GPU memory, and m genotyping and Bayesian probability calculations to make final variant calls. Of course, one or more ofthese functions can be configured so as to be run on one or more quantum computing platforms.

For ce, as can be seen with respect to , the CPU/GPU 1000 may include one or more, e.g., a ity, of threads 20a, 20b, and 20c, which may each have access to an associated DRAM 1014, which DRAM has work space 1014a, 1014b, and 1014c, within which each thread 20a, 20b, and 20c, may have access, respectively, so as to perform one or more operations on one or more data structures, such as large data structures.

These memory portions and their data structures may be accessed, such as via respective cache portions 1014a',such as by one or more processing engines 13a, 13b, 13c ofthe FPGA 7, which processing engines may access the referenced data structures such as in the performance of one or more of the operations herein bed, such as for mapping, ng, sorting, and/or variant calling. Because of the high bandwidth, tight ng interconnect 3, data pertaining to the data structures and/or related to the sing results may be shared substantially seamlessly between the CPU and/or GPU and/or QPU and/or the associated FPGA, such as in a cache coherent manner, so as to optimize processing efficiency.

] Accordingly, in one aspect, as herein disclosed, a system may be provided wherein the system is configured for sharing memory resources amongst its component parts, such as in relation to performing some computational tasks or sub-functions via software, such as run by a CPU and/or GPU and/or QPU, and performing other computational tasks or sub functions via firmware, such as via the hardware of an associated chip, such as an FPGA and/or ASIC or structured ASIC. This may be achieved in a number of different ways, such as by a direct loose or tight coupling between the CPU/GPU/QPU and the chip, e.g., FPGA.

Such configurations may be particularly useful when distributing operations related to the processing of large data structures, as herein described, that have large functions or subfunctions to be used and accessed by both the CPU and/or GPU and/or QPU and the integrated circuit. ularly, in various embodiments, when processing data through a genomics pipeline, as herein described, such as to rate overall processing function, timing, and efficiency, a number of ent operations may be run on the data, which operations may involve both software and hardware processing components.

Consequently, data may need to be shared and/or otherwise communicated, between the software component running on the CPU and/or GPU and/or the QPU and the hardware component embodied in the chip, e.g., an FPGA or ASIC. ingly, one or more of the various steps in the sing pipeline, or a portion thereof, may be performed by one device, e.g., the CPU/GPU/QPU, and one or more of the various steps may be performed by the other device, e.g., the FPGA or ASIC. In such an instance, the CPU and the FPGA need to be communicably coupled, such as by a point to point interconnect, in such a manner to allow the efficient transmission of such data, which coupling may involve the shared use of memory resources. To achieve such distribution of tasks and the g of ation for the performance of such tasks, the CPU and/or GPU and/or QPU may be loosely or y coupled to each other and/or to an FPGA, or other chip set, and a workflow management system may be included so as to distribute the workload efficiently.

Hence, in particular embodiments, a genomics analysis platform is provided.

For instance, the platform may include a motherboard, a memory, and plurality of integrated ts, such as forming one or more of a CPU/GPU/QPU, a mapping module, an alignment module, a sorting module, and/or a variant call module. Specifically, in particular ments, the platform may include a first integrated circuit, such as an integrated circuit forming a central processing unit (CPU) and/or a graphics processing unit (GPU) that is responsive to one or more software algorithms that are configured to instruct the CPU/GPU to perform one or more sets of cs analysis ons, as described herein, such as where the CPU/GPU includes a first set of physical electronic interconnects to connect with the motherboard. In other embodiments, a quantum processing unit is provided, n the QPU includes one or more quantum circuits that are configured for performing one or more of the ons disclosed herein. In various instances, a memory is provided where the memory may also be attached to the motherboard and may further be onically connected with the CPU and/or GPU and/or QPU, such as via at least a portion of the first set of physical electronic interconnects. In such instances, the memory may be configured for storing a plurality of reads of genomic data, and/or at least one or more genetic reference ces, and/or an index, e.g., such as a hash table, of the one or more genetic reference sequences.

Additionally, the platform may include one or more of a second integrated circuit(s), such as where each second integrated circuit forms a field programmable gate array (FPGA) or ASIC, or structured ASIC having a second set ofphysical electronic interconnects to connect with the CPU and the memory, such as via a point-to-point interconnect protocol.

In such an instance, the FPGA (or ured ASIC) may be programmable by firmware to configure a set of hardwired l logic circuits that are interconnected by a plurality of physical interconnects to perform a second set of genomics analysis functions, e.g., mapping, aligning, sorting, de-duplicating, t calling, e.g., an HMM function, etc. Particularly, the hardwired digital logic circuits of the FPGA may be arranged as a set of processing engines to m one or more pre-configured steps in a ce analysis pipeline of the genomics analysis platform, such as where the set(s) of processing engines include one or more of a mapping and/or aligning and/or g and/or de-duplicating and/or variant calling module, which modules may be formed ofthe separate or the same subsets ofprocessing engines.

For instance, with t to variant calling, a pair-HMM or PD-HMM calculation is one of the most compute-intensive steps of a haplotype-based variant calling protocol. Hence, variant calling speed may be greatly improved by accelerating this step in one or more FPGA or quantum processing engines, as herein described. However, there may be additional t in accelerating other compute-intensive steps in additional FPGA and/or QP engines, to e a greater speed-up of variant calling, or a n thereof, or reduce CPU/GPU load and the number of CPU/GPU cores required, or both, as seen with respect to .

Additional compute-intensive functions, with respect to variant calling, that may be implemented in FPGA and/or m processing s include: le-region detection, where reference genome regions covered by adequate depth and/or quality of aligned reads are selected for processing; active-region detection, where reference genome loci with nontrivial evidence of possible variants are identified, and windows of sufficient t around these loci are selected as active regions for r processing; De-Bruijn or other assembly graph construction, where reads overlapping an active region and/or K-mers from those reads are assembled into a graph; assembly graph preparation, such as trimming low-coverage or low-quality paths, ing dangling head and tail paths by joining them onto a reference backbone in the graph, transformation from K-mer to sequence representation of the graph, merging similar branches and otherwise simplifying the graph; extracting ate haplotypes from the assembly graph; as well as aligning candidate haplotypes to the reference genome, such as by Smith-Waterman alignment, e.g., to determine variants (SNPs and/or indels) from the reference represented by each ype, and synchronize its nucleotide positions with the reference.

All of these functions may be implemented as high-performance hardware engines within the FPGA, and/or by one or more quantum circuits of a quantum computing platform. However, calling such a y of hardware ration functions from many integration points in the variant calling software may become inefficient on a loosely-coupled CPU/GPU/QPU+FPGA platform, and therefore a tightly-integrated U/QPU+FPGA platform may be desirable. For instance, various stepwise processing methods such as: constructing, preparing, and extracting haplotypes from a De Bruijn graph, or other assembly graph, could strongly benefit from a tightly-integrated CPU/GPU/QPU+FPGA platform.

Additionally, assembly graphs are large and complex data structures, and g them repeatedly between the CPU and/or GPU and the FPGA could become resource expensive and inhibit significant acceleration.

Hence, an ideal model for such graph processmg, employing a tightlyintegrated U/QPU and/or FPGA platform, is to retain such graphs in cache-coherent shared memory for alternating sing by CPU and/or GPU and/or QPU software and FPGA hardware functions. In such an instance, a software thread processing a given graph may iteratively command various compute-intensive graph processing steps by a hardware engine, and then the re could inspect the results and determine the next steps between the hardware calls, such as exemplified in the process of . This processing model, may be controlled by a suitably configured workflow management system, and/or may be ured to correspond to software paradigms such as a data-structure API or an objectoriented method interface, but with compute intensive functions being accelerated by custom hardware and/or quantum processing engines, which is made practical by being implemented on a tightly-integrated CPU and/or GPU and/or QPU +FPGA platform, with cache-coherent shared memory and high-bandwidth/low-latency CPU/GPU/QPU/FPGA interconnects.

Accordingly, in on to mapping and aligning sequenced reads to a reference genome, reads may be assembled "de novo," e.g., without a reference genome, such as by detecting apparent overlap between reads, e.g., in a pileup, where they fully or mostly agree, and g them into longer sequences, contigs, scaffolds, or graphs. This assembly may also be done locally, such as using all reads determined to map to a given chromosome or portion thereof. Assembly in this manner may also incorporate a reference genome, or segment ofone, into the assembled structure.

In such an instance, due to the complexity ofjoining together read sequences that do not completely agree, a graph structure may be employed, such as where pping reads may agree on a single sequence in one segment, but branch into multiple ces in an adjacent t, as explained above. Such an assembly graph, therefore, may be a sequence graph, where each edge or node represents one nucleotide or a sequence of nucleotides that is ered to adjoin contiguously to the sequences in connected edges or nodes. In particular instances, such an assembly graph may be a k-mer graph, where each node represents a k-mer, or nucleotide sequence of (typically) fixed length k, and where connected nodes are considered to overlap each other in longer observed sequences, typically pping by k-1 nucleotides. In various methods there may be one or more ormations performed between one or more sequence graphs and k-mer . gh assembly graphs are employed in haplotype-based variant g, and some of the graph processing methods employed are similar, there are important differences. De novo assembly graphs are generally much larger, and employ longer .

Whereas variant-calling assembly graphs are constrained to be fairly structured and relatively simple, such as having no cycles and flowing source-to-sink along a reference sequence backbone, de novo ly graphs tend to be more unstructured and x, with cycles, dangling paths, and other anomalies not only permitted, but subjected to special analysis. De novo assembly graph coloring is sometimes employed, ing "colors" to nodes and edges signifying, for example, which biological sample they came from, or matching a nce sequence. Hence, a wider variety of graph analysis and processing ons need to be employed for de novo assembly , often iteratively or recursively, and especially due to the size and complexity of de novo assembly graphs, processing functions tend to be extremely e intensive.

Hence, as set forth above, an ideal model for such graph processing, on a tightly-integrated CPU/GPU/QPU+FPGA platform, is to retain such graphs in cache-coherent shared memory for alternating processing between the CPU/GPU/QPU software and FPGA hardware functions. In such an instance, a software thread sing a given graph may iteratively command various compute-intensive graph sing steps to be performed by a hardware engine, and then inspect the results to thereby determine the next steps to be performed by the hardware, such as by making appropriate hardware calls. Like above, this processing model, is greatly benefitted by implementation on a tightly-integrated CPU+FPGA platform, with cache-coherent shared memory and high-bandwidth/low-latency CPU/FPGA interconnect.

Additionally, as described herein below, tertiary analysis includes genomic sing that may follow graph assembly and/or variant calling, which in clinical applications may include variant annotation, phenotype tion, disease testing, and/or treatment response prediction, as described . Reasons it is beneficial to perform tertiary analysis on such a tightly-integrated CPU/GPU/QPU+FPGA platform are that such a platform configuration enables efficient acceleration of primary and/or secondary sing, which are very compute intensive, and it is ideal to continue with tertiary analysis on the same platform, for convenience and reduced ound time, and to minimize transmission and copying of large genomic data files. Hence, either a loosely or tightly-integrated CPU/GPU/QPU+FPGA rm is a good choice, but a tightly coupled platform may include additional benefits because tertiary analysis steps and methods vary widely from one application to another, and in any case where compute-intensive steps slow down tertiary analysis, custom FPGA acceleration of those steps can be implemented in an optimized fashion.

For instance, a particular benefit to tertiary analysis on a tightly-integrated CPU/GPU/QPU and/or FPGA platform is the ability to re-analyze the c data iteratively, leveraging the CPU/GPU/QPU and/or FPGA acceleration of secondary smg, in response to partial or intermediate tertiary results, which may benefit additionally from the tight ation configuration. For example, after tertiary is s a possible phenotype or disease, but with limited confidence as to whether the detection is true or false, focused secondary re-analysis may be performed with extremely high effort on the particular reads and reference regions impacting the ion, thus improving the cy and confidence of relevant variant calls, and in tum improving the confidence in the detection call. Additionally, if ry analysis determines information about the ancestry or structural variant genotypes of the analyzed individual, secondary analysis may be repeated using a different or modified reference genome, which is more appropriate for the specific individual, thus enhancing the accuracy of variant calls and improving the accuracy of further tertiary analysis steps.

] However, if tertiary analysis is done on a CPU-only platform after primary and secondary processing bly accelerated on a separate platform), then re-analysis with secondary processing tools is likely to be too slow to be useful on the tertiary analysis platform , and the alternative is transmission to a faster platform, which is also prohibitively slow. Thus, in the absence of any form ofhardware or quantum acceleration on the tertiary analysis platform, primary and secondary processing must generally be ted before tertiary analysis , without the possibility of easy re-analysis or iterative secondary analysis and/or pipelining of analytic functions. But on an FPGA and/or quantumaccelerated platform, and ally a y-integrated CPU and/or GPU and/or QPU and/or FPGA platform where secondary processing is maximally efficient, iterative analysis becomes practical and useful.

Accordingly, as indicated above, the s herein disclosed may be implemented in the hardware of the chip, such as by being hardwired therein, and in such instances their implementation may be such that their oning may take place at a faster speed, with greater accuracy, as compared to when implemented in software, such as where there are minimal instructions to be fetched, read, and/or executed. Additionally, in various instances, the functions to be performed by one or more ofthese modules may be distributed such that various of the functions may be configured so as to be ented by the host CPU and/or GPU and/or QPU software, whereas in other instances, various other functions may be performed by the hardware of an associated FPGA, such as where the two or more devices perform their respective functions with one another such as in a seamless fashion. For such purposes, the CPU, GPU, QPU, and/or FPGA or ASIC or ured ASIC may be tightly d, such as via a low latency, high bandwidth interconnect, such as a QPI, CCVI, CAPI, and the like. Accordingly, in some instances, the high computationally intensive functions to be performed by one or more of these modules may be performed by a quantum processor implemented by one or more quantum circuits.

Hence, given the unique hardware and/or quantum processing implementation, the modules of the disclosure may function ly in accordance with their operational parameters, such as without needing to fetch, read, and/or execute instructions, such as when implemented solely in CPU software. Additionally, memory requirements and processing times may be further reduced, such as where the communications within chip is via files, e.g., stored locally in the FPGA/CPU/GPU/QPU cache, such as a cache coherent manner, rather than through extensive accessing an external . Of , in some instances, the chip and/or card may be sized so as to include more memory, such as more on board memory, so as to enhance parallel processing capabilities, thereby resulting in even faster processing speeds. For instance, in certain embodiments, a chip of the disclosure may include an embedded DRAM, so that the chip does not have to rely on external memory, which would therefore result in a further increase in processing speed, such as where a s-Wheeler algorithm or De Brujin Graph may be employed, d of a hash table and hash function, which may in s instances, rely on external, e.g., host memory. In such ces, the running of a portion or an entire pipeline can be accomplished in 6 or 10 or 12 or 15 or 20 minutes or less, such as from start to finish.

As indicated above, there are various different points where any given module can be positioned on the hardware, or be positioned remotely therefrom, such as on a server accessible on the cloud. Where a given module is positioned on the chip, e.g., hardwired into the chip, its function may be performed by the hardware, r, where desired, the module may be positioned remotely from the chip, at which point the platform may include the necessary instrumentality for sending the relevant data to a remote location, such as a server, e.g., quantum server, ible via the cloud, so that the particular 's functionality may be engaged for further processing of the data, in ance with the user selected desired protocols. Accordingly, part of the platform may include a web-based interface for the performance of one or more tasks pursuant to the functioning of one or more of the modules disclosed herein. For ce, where mapping, alignment, and/or sorting are all modules that may occur on the chip, in various instances, one or more of local realignment, duplicate marking, base quality core bration, and/or variant calling may take place on the cloud.

Particularly, once the c data has been generated and/or processed, e.g., in one or more primary and/or secondary processing protocols, such as by being mapped, aligned, and/or , such as to produce one or more variant call files, for instance, to determine how the genetic sequence data from a subject differs from one or more reference sequences, a further aspect ofthe disclosure may be directed to performing one or more other analytical functions on the ted and/or processed genetic data such as for further, e.g., tertiary, processing, as depicted in FIGS. 40. For example, the system may be configured for further processing of the generated and/or secondarily processed data, such as by running it through one or more tertiary sing pipelines 700, such as one or more of a micro-array analysis pipeline, a genome, e.g., whole genome analysis pipeline, ping analysis pipeline, exome analysis pipeline, epigenome analysis pipeline, metagenome is pipeline, microbiome analysis ne, genotyping analysis pipeline, including joint genotyping, variants analyses pipeline, including structural variants pipelines, somatic variants pipelines, and GATK and/or 2 pipelines, as well as RNA sequencing pipelines and other genetic analyses pipelines.

Additionally, in various ces, an additional layer of processing 800 may be provided, such as for e diagnostics, therapeutic treatment, and/or prophylactic prevention, such as ing NIPT, NICU, Cancer, LDT, AgBio, and other such disease diagnostics, prophylaxis, and/or treatments employing the data generated by one or more of the present primary and/or secondary and/or tertiary pipelines. For example, particular bioanalytic pipelines include genome pipelines, epigenome pipelines, metagenome nes, ping nes, variants, e.g., GATK/MuTect2 pipelines, and other such pipelines.

Hence, the devices and methods herein disclosed may be used to generate genetic sequence data, which data may then be used to generate one or more t call files and/or other associated data that may further be subject to the execution of other tertiary processing pipelines in accordance with the devices and methods disclosed herein, such as for particular and/or general disease diagnostics as well as for prophylactic and/or therapeutic ent and/or developmental modalities. See, for instance, FIGS. 41 B, C and 43.

As described above, the methods and/or systems herein presented may include the generating and/or the otherwise acquiring of genetic sequence data. Such data may be generated or otherwise acquired from any suitable source, such as by a NGS or "sequencer on a chip technology." Once generated and/or acquired, the methods and systems herein may include subjecting the data to further processing such as by one or more secondary processing protocols 600. The secondary processing protocols may include one or more of mapping, aligning, and sorting of the generated c sequence data, such as to produce one or more variant call files, for example, so as to determine how the genetic sequence data from a subject s from one or more reference sequences or genomes. A further aspect of the disclosure may be directed to performing one or more other analytical ons on the generated and/or processed genetic data, e.g., secondary result data, such as for additional processing, e.g., tertiary sing 700/800, which processing may be performed on or in association with the same chip or chipset as that hosting the aforementioned sequencer technology.

Accordingly, in a first instance, such as with respect to the generation, ition, and/or transmission of genetic sequence data, as set forth in FIGS. 37 - 41, such data may be produced either locally or remotely and/or the results thereof may then be directly processed, such as by a local computing resource 100, or may be transmitted to a remote location, such as to a remote ing resource 300, for further processing, e.g. for secondary and/or tertiary processing, see FIGS. 42. For instance, the ted genetic sequence data may be processed locally, and directly, such as where the sequencing and secondary sing functionalities are housed on the same chipset and/or within the same device on-site 10. se, the generated genetic sequence data may be processed locally, and indirectly, such as where the sequencing and secondary processing functionalities occur separately by distinct apparatuses that share the same facility or location but may be separated by a space albeit icably ted, such as via a local network 10. In a r ce, the c sequence data may be derived remotely, such as by a remote NGS, and the resultant data may be transmitted over a cloud based network 30/50 to an offsite remote location 300, such as separated geographically from the sequencer.

Specifically, as illustrated in A, in various embodiments, a data generation apparatus, e.g., nucleotide sequencer 110, may be provided on site, such as where the sequencer is a "sequencer on a chip" or a NGS, wherein the sequencer is associated with a local computing resource 100 either directly or indirectly such as by a local network connection 10/30. The local computing resource 100 may include or otherwise be ated with one or more of a data generation 110 and/or a data acquisition 120 ism(s). Such mechanisms may be any mechanism configured for either generating and/or otherwise acquiring data, such as analog, digital, and/or electromagnetic data related to one or more c sequences of a subject or group of subjects, such as where the genetic sequence data is in a BCL or FASTQ file format.

For e, such a data generating mechanism 110 may be a pnmary processor such as a sequencer, such as a NGS, a cer on a chip, or other like mechanism for generating genetic sequence information. Further, such data acquisition mechanisms 120 may be any mechanism configured for receiving data, such as ted genetic sequence information; and/or together with the data generator 110 and/or computing resource 100 is capable of subjecting the same to one or more secondary processing protocols, such as a secondary sing pipeline apparatus configured for running a mapper, aligner, sorter, and/or variant caller protocol on the ted and/or acquired sequence data as herein described. In various instances, the data generating 110 and/or data acquisition 120 apparatuses may be ked together such as over a local network 10, such as for local storage 200; or may be networked together over a local and/or cloud based network 30, such as for transmitting and/or receiving data, such as digital data related to the primary and/or secondary processing of genetic sequence information, such as to or from a remote location, such as for remote sing 300 and/or storage 400. In various embodiments, one or more of these components may be communicably coupled together by a hybrid network as herein described.

The local computing resource 100 may also include or otherwise be associated with a compiler 130 and/or a processor 140, such as a compiler 130 configured for compiling the generated and/or acquired data and/or data associated therewith, and a processor 140 configured for processing the generated and/or acquired and/or compiled data and/or controlling the system 1 and its components, as herein bed, such as for ming y, secondary, and/or tertiary sing. For instance, any suitable er may be employed, however, in certain instances, further efficiencies may be achieved not only by implementing a tight-coupling configuration, such as discussed above, for the efficient and coherent transfer of data between system components, but may further be achieved by implementing a just-in-time (JIT) computer language er configuration. Further, in certain ces, the processor 140 may include a workflow management system for controlling the functioning of the various system components with respect to generated, received, and/or data to be processed through the s stages ofthe platform pipelines.

Specifically, as used herein just-in-time (JIT) refers to a device, system, and/or method for converting acquired and/or generated file formats from one form to r. In a broad usage structure, the JIT system disclosed herein may include a compiler 130, or other computing architecture, e.g., a processing program, that may be implemented in a manner so as to convert s code from one form into another. For instance, in one implementation, a JIT compiler may function to convert bytecode, or other program code that contains instructions that must be interpreted, into instructions that can be sent directly to an associated processor 140 for near immediate ion, such as without the need for interpretation of the ctions by the particular machine language. Particularly, after a coding program, e.g., a Java program, has been written, the source language statements may be compiled by the compiler, e.g., Java compiler, into bytecode, rather than compiled into code that contains ctions that match any given particular hardware platform'sprocessing language. This bytecode compiling action, therefore, is rm-independent code that can be sent to any platform and run on that platform less of its underlying processor.

Hence, a suitable compiler may be a compiler that is configured so as to compile the bytecode into platform-specific able code that may then be executed immediately. In this instance, the JIT compiler may function to immediately convert one file format into another, such as "on the fly".

Hence, a ly ured compiler, as herein described, is capable of overcoming various deficiencies in the art. Specifically, past compiling programs that were written in a specific language had to be recompiled and/or re-written dependent on each ic computer platform on which it was to be implemented. In the present compiling system, the compiler may be configured so as to only have to write and compile a program once, and once written in a particular form, may be converted into one or more other forms nearly immediately. More specifically, the compiler 130 may be a JIT, or in another similar dynamic translation compiler format, which is capable of writing instructions in a platform agnostic language that does not have to be recompiled and/or re-written dependent on the ic computer platform on which it is implemented. For instance, in a particular use model, the compiler may be configured for interpreting compiled bytecode, and/or other coded instructions, into instructions that are understandable by a given particular processor for the conversion of one file format into another, regardless of computing platform.

Principally, the JIT system herein is capable of receiving one genetic file, such as representing a genetic code, for example, where the file is a BCL or FASTQ file, e.g., generated from a c sequencer, and rapidly converting it into another form, such as into a SAM, BAM, and/or CRAM file, such as by using the methods disclosed herein.

Particularly, in various instances, the system herein disclosed may include a first and/or a second compiler 130a and 130b, such as a l compiling machine, that handles one or a plurality ofbytecode instruction conversions at a time. For instance, using a Java type just-in-time compiler, or other suitably configured second compiler, within the present system platform, will allow for the compiling of ctions into bytecode that may then be converted into the particular system code, e.g., as though the program had been compiled initially on that platform. Accordingly, once the code has been compiled and/or (re- )compiled, such as by the JIT compiler(s) 130, it will run more quickly in the computer processor 140. Hence, in various embodiments, just-in-time (JIT) compilation, or other dynamic translation compilation, may be configured so as to be performed during execution of a given program, e.g., at run time, rather than prior to execution. In such an instance, this may include the ) of translation to machine code or ation into another format, which may then be executed directly, thereby ng for one or more of ahead-of-time ation (AOT) and/or interpretation.

More particularly, as implemented within the present system, a typical genome cing dataflow lly produces data in one or more file formats, derived from one or more computing platforms, such as in a BCL, FASTQ, SAM, BAM, CRAM, and/or VCF file format, or their equivalents. For instance, a typical DNA sequencer 110, e.g., an NGS, produces raw signals enting called bases that are designated herein as reads, such as in a BCL and/or FASTQ file, which may optionally be further processed, e.g., enhanced image processing, and/or compressed 150. se, the reads of the generated BCL/FASTQ files may then be further sed within the system, as herein described, so as to produce mapping and/or alignment data, which produced data, e.g., of the mapped and aligned reads, may be in a SAM or BAM file format, or alternatively a CRAM file format. Further, the SAM or BAM file may then be processed, such as through a variant g procedure, so as to produce a variant call file, such as a VCF file or gVCF file. Accordingly, all of these produced BCL, FASTQ, SAM, BAM, CRAM, and/or VCF files, once produced are (extremely) large files that all need to be stored such as in system memory architecture locally 200 or remotely 400. The storage ofany one ofthese files is expensive. The storage of all ofthese file formats is extremely expensive.

As indicated, just-in-time (JIT) or other dual compiling or dynamic translation compilation is, may be configured and deployed herein so as to reduce such high storage costs. For instance, a JIT analysis scheme may be implemented herein so as to store data in only one format (e.g., a compressed FASTQ or BAM, etc., file format), while providing access to one or more file formats (e.g., BCL, FASTQ, SAM, BAM, CRAM, and/or VCF, etc.). This rapid file conversion process may be effectuated by rapidly processing the genomic data utilizing the herein disclosed respective hardware and/or quantum acceleration platforms, e.g., such as for mapping, aligning, sorting, and/or t calling (or component functions thereof, such as de-duplicating, HMM and Smith-Waterman, ssion and decompression, and the like), in hardware engines on an integrated circuit, such as an FPGA, or by a quantum processor. Hence, by implementing JIT or r analysis along with such acceleration, the genomic data can be processed in a manner so as to generate d file formats on the fly, at speeds comparable to normal file . Thus, considerable storage savings may be realized by JIT-like sing with little or no loss of access speed.

Particularly, two general options are useful for the ying storage of the genomic data produced herein so as to be accessible for JIT-like processing, these include the storage of ned reads (e.g., that may include compressed FASTQ, or unaligned compressed SAM, BAM, or CRAM files), and the storage of aligned reads (e.g., that may include compressed BAM or CRAM files). However, since the accelerated processing sed herein allows any of the nced file formats to be derived rapidly, e.g., on the fly, the underlying file format for storage may be selected so as to achieve the smallest compressed file size, thereby decreasing the expense of storage. Hence, because of the comparatively smaller file size for unprocessed, e.g., raw gned, read data, there is an advantage to storing unaligned reads so that the data fields are zed. Likewise, there is an advantage to g the processed and compressed data, such as in a CRAM file format.

More particularly, in view of the rapid processing speeds achievable by the devices, systems, and methods of their use disclosed herein, in many instances, there may be no need to store mapped and/or alignment information for each and every read, because this information may be rapidly derived upon need, such as on the fly. Further, although a compressed FASTQ (e.g. FASTQ.gz) file format is commonly used for storage of genetic sequence data, such unaligned read data may be stored in more advanced compressed formats as well, such as post mapping and/or ng in SAM, BAM, or CRAM files, which may further reduce the file size, such as by use of compact binary representation and/or more targeted compression methods. Hence, these file formats may be compressed prior to storage, be decompressed after storage, and processed rapidly, such as on the fly, so as to convert one file format from another.

An advantage to storing d reads is that much or all of each read's ce content can be omitted. Specifically, system efficiency can be enhanced and e space saved by only storing the ences between the read sequences and the selected nce genome, such as at indicated variant alignment positions of the read. More specifically, since differences from the reference are usually sparse, the d position and list of differences can often be more tly stored than the al read sequence. ore, in various instances, the storage of an aligned read format, e.g., when storing data related to the differences of d reads, may be preferable to the storage ofunaligned read data. In such an instance, if an aligned read and/or variant call format is used as the underlying storage format, such as in a JIT procedure, other formats, such as a SAM, BAM, and/or CRAM, compressed file formats, may also be used.

Along with the aligned and/or unaligned read file data to be stored, a wide variety of other data, such as metadata derived from the various computations determined herein, may also be stored. Such computated data may include read mapped, alignment and/or subsequent processing data, such as alignment scores, mapping confidence, edit distance from the reference, etc. In certain instances, such metadata and/or other extra information need not be retained in the underlying storage for JIT analysis, such as in those instances where it can be reproduced on the fly, such as by the accelerated data processing herein described.

With respect to metadata, this data may be a small file that instructs the system as to how to go backwards or forwards from one file format into conversion to r file format. Hence, the metadata file allows the system to create a bit-compatible version of any other file type. For ce, proceeding forward from an originating data file, the system need only access and implement the instructions ofthe metadata. Along with rapid file format conversion, JIT also enables rapid compression and/or decompression and/or storage, such as in a genomics dropbox memory cache.

As discussed in r detail below, once sequence data is generated 110, it may be stored locally 200, and/or may be made accessible for storage remotely, such as in a cloud accessible dropbox-like memory cache 400. For example, once in the genomic dropbox, the data may appear as accessible on the cloud 50, and may then be further processed, e.g., substantially immediately. This is ularly useful when there is a plurality of g/aligning/sorting/variant g systems 100/300, such as with one on either side of the cloud 50 interface facilitating the automatic ing and processing of the data, which can be further processed such as using the JIT technology herein described.

For instance, an underlying storage format for JIT compiling and/or sing may contain only minimal data , such as read name, base quality scores, alignment position, and/or orientation in the reference, and a list of differences from the reference, such as where each field may be compressed in an optimal manner for its data type. Various other metadata may be included and/or otherwise associated with the storage file. In such an ce, the underlying storage for JIT is may be in a local file system 200, such as on hard disk drives and solid state drives, or a network storage resource such as a NAS or object or Dropbox like storage system 400. Particularly, when various file formats, such as BCL, FASTQ, SAM, BAM, CRAM, VCF, etc., have been produced for a genomic dataset, which may be submitted for JIT processing and/or storage, the JIT or other similar compiling and/or analysis system may be configured so as to convert the data to a single underlying storage format for storage. Additional data, such as metadata and/or other information (which may be small) necessary to reproduce all other desired formats by accelerated genomic data processing, may also be associated with the file and stored. Such additional information may include one or more of: a list of file s to be reproduced, data processing commands to uce each format, unique ID (e.g., URL or MD5/SHA hash) of reference genome, various parameter settings, such as for g, alignment, sorting, variant calling, and/or any other processing, as bed herein, randomization seeds for processing steps, e.g., utilizing pseudo-randomization, to deterministically reproduce the same results, user Interface, and the like.

In various instances, the data to be stored and/or retrieved in a JIT or similar dynamic translation sing and/or analysis system may be presented to the user, or other applications, in a variety of manners. For instance, one option is to have the JIT analysis storage in a standard or custom "JIT object" file format, such as for storage and/or retrieval as a SAM, BAM, CRAM, or other custom file format, and provide user tools to rapidly convert WO 14320 PCT/0S2017/036424 the JIT object into the desired format (e.g., in a local temporary storage 200) using the accelerated processing disclosed herein. r option is to t the ance of multiple file formats, such as BCL, FASTQ, SAM, BAM, CRAM, VCF, etc. to the user, and the user ations, in such a manner that the file system access to various file formats utilizes a JIT procedure, thus only one file type needs be saved, and from these file type, all other files can be ted on the fly. A further option is to make user tools that otherwise accept specific file formats (BCL, FASTQ, SAM, BAM, CRAM, VCF, etc.) that are able to be presented as a JIT object instead, and may automatically call for JIT analysis to obtain the data in the desired data format, e.g., BCL, FASTQ, SAM, BAM, CRAM, VCF, etc. automatically when called.

] Accordingly, JIT procedures are useful for providing access to multiple file formats, e.g., BCL, FASTQ, SAM, BAM, CRAM, VCF, and the like, from a single file format by rapidly processing the underlying stored compressed file format. onally, JIT remains useful even ifonly a single file format is to be accessed, because compression is still achieved relative to storing the ed format directly. In such an instance, the underlying file storage format may be different than the accessed file format, and/or may contain less metadata, and/or may be compressed more efficiently than the accessed format. Further, in such an instance, as discussed above, the file is ssed prior to storage, and ressed upon retrieval, e.g., automatically.

In various instances, the methods ofJIT analysis, as provided herein, may also be used for transmission of genomic data, over the internet or another network, to minimize transmission time and lessen ed network bandwidth. Particularly, in one storage application, a single compressed underlying file format may be stored, and/or one or more formats may be accessed via decompression and/or accelerated genomic data processing.

Similarly, in the transmission application, only a single compressed underlying file format need be transmitted, e.g., from a source network node to a destination network node, such as where the underlying format may be chosen primarily for smallest compressed file size, and/or where all desired file formats may be generated at the destination node by or for genomic data processing, such as on the fly. In this manner, only one compressed data file format need be used for storage and/or transfer, from which file format the other various file formats may be derived.

Accordingly, m view of A, hardware and/or quantum accelerated genomic data processing, as herein bed, may be utilized in (or by) both the source network node, to generate and/or compress the underlying format for ission, and the destination network node, to decompress and/or generate other desired file formats by accelerated genomic data processing. heless, JIT or other dynamic translation analysis continues to be useful in the transmission application even if only one of the source node or the destination node utilizes hardware and/or quantum accelerated genomic data processing.

For example, a data server that sends large amounts of genomic data may utilize re and/or quantum accelerated genomic data processing so as to generate the compressed underlying format for ission to s destinations. In such instances, each destination may use slower software genomic data processing to generate other desired data formats.

Hence, although the speed advantage of JIT analysis is lessened at the destination node, transmission time, and network utilization are still usefully reduced, and the source node is able to service many such transmissions efficiently due to its ponding hardware and/or quantum accelerated genomic data processing apparatus.

Further, in another example, a data server that receives uploads of large amounts of genomic data, e.g., from various sources, may utilize hardware and/or quantum accelerated genomic data processing and/or storage, while the various source nodes may use slower software run on a CPU/GPU to generate the compressed underlying file format for transmission. atively, hardware and/or quantum accelerated genomic data processing may be utilized by one or more ediate k nodes, such as a gateway server, between the source and destination nodes, to transmit and/or receive genomic data in a compressed underlying file format, ing to the JIT or other dynamic translation is methods, thus gaining the benefits of d transmission time and network utilization without overburdening the said intermediate network nodes with excessive software processmg.

Hence, as can be seen with respect to A, in certain instances, the local computing resource 100 may include a compiler 130, such as a JIT er, and may r include a compressor unit 150 that is configured for compressing data, such as generated and/or ed primary and/or secondary sed data (or tertiary data), which data may be compressed, such as prior to transfer over a local 10 and/or cloud 30 and/or hybrid cloud based 50 network, such as in a JIT analysis procedure, and which may be ressed subsequent to transfer and/or prior to use.

As described above, in various instances, the system may include a first integrated and/or quantum circuit 100 such as for performing a mapping, aligning, sorting, and/or variant calling operation, so as to generate one or more ed, d, sorted, icated , and/or t called results data. Additionally, the system may include a further integrated and/or quantum circuit 300 such as for employing the results data in the performance of one or more genomics and/or ormatics pipeline analyses, such as for tertiary processing. For instance, the result data generated by the first integrated and/or quantum circuit 100 may be used, e.g., by the first or a second integrated and/or quantum circuit 300, in the performance of a further genomics and/or bioinformatics pipeline processing procedure. Specifically, secondary processing of genomics data may be performed by a first hardware and/or m accelerated processor 100 so as to produce results data, and tertiary processing may be performed on that results data, such as where the further processing is performed by a CPU and/or GPU and/or QPU 300 that is operatively coupled to the first integrated circuit. In such an instance, the second circuit 300 may be configured for ming tertiary processing of the genomics ion data produced by the first t 100. Accordingly, the results data derived from the first integrated server acts as an is engine driving the further processing steps described herein with nce to tertiary processing, such as by the second integrated and/or quantum processing circuit 300.

However, the data generated in each of these primary and/or secondary and/or tertiary process steps may be immense, requiring very high resource and/or memory costs such as for storage, either locally 200 or remotely 400. For instance, in a first primary processing step, generated nucleic acid sequence data 110, such as in a BCL and/or FASTQ file format, may be received 120, such as from an NGS 110. Regardless of the file format of this sequence data, the data may be employed in a secondary sing protocol as described herein. The ability to receive and process primary sequence data directly from an NGS, such as in a BCL and/or FASTQ file format, is very useful. Particularly, instead of converting the sequence data file from the NGS, e.g., BCL, to a FASTQ file, the file may be directly received from the NGS, e.g., as a BCL file, and may be processed, such as by being received and ted by the JIT , e.g., on the fly, into a FASTQ file that may then be processed, as described herein, such as to produce a mapped, aligned, sorted, d, and/or variant called results data that may then be ssed, such as into a SAM, BAM, and/or CRAM file, and/or may be subjected to further processing, such as by one or more of the disclosed genomics tertiary processing pipelines.

Accordingly, such data once produced needs to be stored in some manner.

However, such storage is not only resource intensive, it is also costly. Specifically, in a typical cs protocol, the sequenced data once generated is stored as a large FASTQ file.

Then, once processed such as by being subjected to a mapping and/or aligning protocol, a BAM file is created, which file is also typically stored, increasing the expense of genomic data storage, such as by having to store both a FASTQ and a BAM file. Further, once the BAM file is processed, such as by being subjected to variant calling protocol, a VCF file is produced, which VCF also typically needs to be stored. In such an instance, in order to tely provide and make use of the generated genetic data, all three of the FASTQ, BAM, and VCF files may need to be stored, either locally 200 or remotely 400. Additionally, the original BCL file may also be stored. Such storage is inefficient as well as being memory resource intensive and expensive.

However, the computational power of the hardware and/or quantum processing architectures implemented herein, along with the JIT compilation, compression, and storage, greatly ameliorates these inefficiencies, resource costs, and expenses. For instance, in view of the methods implemented and the sing speeds achieved by the present rated integrated circuits, such as for the conversion of a BCL file to a FASTQ file, and then the conversion of a FASTQ file to a SAM or BAM file, and then the conversion of a BAM file to a CRAM and/or VCF file, and back again, the present system greatly reduces the number of computing resources and/or file sizes needed for the efficient processing and/or storage of such data. The benefits ofthese systems and methods are further enhanced by the fact that only one file format, e.g., a BCL, FASTQ, SAM, BAM, CRAM, and/or VCF, need be stored, from which all the other file formats may be derived and processed. Particularly, only one file format needs to be saved and from such file any of the other file formats may be generated rapidly, e.g., on the fly, in accordance with the methods sed herein, such as in a just in time, or JIT, compiling .

For example, in accordance with typical prior methods, a large amount of computing resources, e.g., server farms and large memory banks, is needed for the sing and storage of FASTQ files being generated by a NGS sequencer. Particularly, in a typical instance, once the NGS produces the large FASTQ file, the server farm would then be employed to receive and convert the FASTQ file to a BAM and/or CRAM file, which processing may take up to a day or more. r, once produced, the BAM file itself must then be stored, requiring further time and resources. Likewise, the BAM or CRAM file may be processed in such a manner to te a VCF, which may also take up another day or more, and which file will also need to be stored, thereby ing r resource costs and expenses. More particularly, in a typical instance, the FASTQ file for a human genome consumes about 90 GB ofstorage, per file. Likewise, a typical human genome BAM file may e about 160 GB. The VCF file may also need to be stored, albeit such files are quite smaller than the FASTQ and/or BAM files. SAM and CRAM files may also be generated throughout the secondary processing procedures, and these too may need to be stored.

Prior to the technologies provided herein, it has been computationally ive to go from one step to another, e.g., from one file format to r, and hence, all of the data for these file formats would typically have to be stored. This is in part due to the fact that if a user ever wanted to go back and regenerate one or more of the files, it would require a large amount of computing resources and time to re-do the processes involved to regenerate the various files thereby incurring a high monetary expense. Further, where these files are compressed before storage, such compression may take from about 2 to about 5 to about 10 or more hours, with about the same amount of time required for decompression, prior to reuse. Because of these high expenses, l users would not compress such files prior to storage, and would also typically store all two, three or more file formats, e.g., BCL, FASTQ, BAM, VCF, incurring increased costs over increased time.

Accordingly, the JIT ols employed herein make use of the accelerated processing speeds achieved by the present re and/or quantum accelerators, so as to realize enhanced efficiency, at reduced time and costs both for sing as well as for storage. Instead of storing 2, 3, or more copies of the same general data in different file formats, only one file format needs to be , and on the fly, any ofthe other file types can be regenerated, such as using the accelerated processing platforms discussed herein.

Particularly, from storing a FASTQ file, the present devices and systems make it easy to go backwards to a BCL file, or forwards to a BAM file, and then further to a VCF, such as in under 30 minutes, such as within 20 minutes, or about within 15 or 10 minutes, or less.

Hence, using the pipelines and the speed of processing offered by the hardwired/quantum processing engines herein disclosed, only a single file format need be stored, while the other file formats may easily and rapidly be generated therefrom. So instead of needing to store all three file formats, a single file format need be stored from which any other file format may be regenerated such as on the fly, just in time for the further sing steps desired by the user. Consequently, the system may be configured for ease of use such that if a user simply interacts with a graphical user ace, such as presented at an associated y of the device, e.g., the user clicks on the FASTQ, BAM, VCF, etc. button ted in the GUI, the desired file format may be presented, while in the background, one or more of the processing engines of the system may be performing the accelerated processing steps ary for regenerating the requested file in the requested file format from the stored file.

Typically, one or more of a compressed version of a BCL, FASTQ, SAM, BAM, CRAM, and/or VCF file will be saved, along with a small metafile that includes all of the configurations of how the system was run to create the compressed and/or stored file.

Such metafile data s how the particular file format, e.g., FASTQ and/or BAM file, was generated and/or what steps would be necessary for going backwards or forwards so as to generate any ofthe other file formats. This process is described in greater detail herein below.

In a manner such as this the process can proceed forwards or be reversed going rds using the configuration stored in the metafile. This can be about an 80% or more reduction in e and economic cost ifthe computing on is bundled with the storage functions.

Accordingly, in view of the above and as can be seen with respect to A, a cloud based server system for data analytics and storage is provided. For instance, using a cloud accessible server system, as disclosed herein, a user may connect with a storage device, such as for the storage of input data. For example, a remote user may access the system so as to input genomics and/or bioinformatics data into the system, such as for storage and/or the processing thereof. Particularly, a remote user of the system, e.g., using local computing resource 100, may access the system 1 so as to upload genomic data, e.g., such as one or more sequenced genomes of one or more individuals. As described in detail below, the system may include a user interface, e.g., accessing a suitably configured API, which will allow a user to access the BioIT platform so as to upload data to be processed, control the parameters ofthe processing, and/or ad output, e.g., results data, from the platform.

Specifically, the system may include an API, e.g., an S3 or "S3-like" object that allows access to one or more memories of the system, for the e 400 and/or receipt of stored files. For instance, a cloud accessible API object may be present, such as where the API is configurable so as to store data files in the cloud 50, such as into one or more storage buckets 500, e.g., an S3 bucket. Accordingly, the system may be configured so as to allow a user to have access to ly stored files, e.g., via an S3 or S3-like API, such as by ing the API via a cloud based ace on a personal computing device.

WO 14320 PCT/0S2017/036424 Such an API therefore may be configured for allowing access to the cloud 50 to thereby connect the user with one or more ofthe cloud based servers 300 disclosed herein, such as to upload and/or download a given stored file, e.g., so as to make files accessible between the cloud server 300 and the local hard drive 100. This may be useful, for instance, to allow a remote user to provide, access data, and/or download data, on or from the server 300, and further to run one or more applications and/or calculations on that data, either locally 100 or on the server 300, and then to call the API to send the transformed data back to or from the cloud 50, e.g., for storage 200 and/or further processing. This is specifically useful for the retrieval, analyses, and storage ofgenomics data.

However, typical cloud based e of data, e.g., "S3" storage, is expensive.

This expense is increased when storing the large amounts of data associated with the fields of genomics and bioinformatics, where such costs often become prohibitive. Additionally, the time required to record, upload, and/or download the data for use, e.g., either locally 100 or remotely 300, and/or for storage 400 also makes such expensive cloud based storage solutions less tive. The t ons disclosed herein overcome these and other such needs.

Particularly, instead of going through a typical "S3" or other typical cloud based object API, presented herein, is an alternative patible API, which may be implemented so as to reduce the speed of transmission and/or the cost of storage of data. In such an instance, when a user wants to store a file, d of going through a typical cloud based, e.g., S3, API, the alternative service API system, e.g., the proprietary S3 compatible API disclosed herein, will launch a compute instance, e.g., a CPU and/or FPGA instance of the system, which will function to compress the file, will generate a metadata index with respect to indicating what the data is and/or how the file was generated, etc., and will then store the compressed file via an S3 Compatible storage-like bucket 400. Accordingly, presented herein is a cloud-based 50 e that employs a compute instance 300, which may be launched by an alternative API, so as to sses data before storage 400, and/or decompress data upon retrieval. In such an instance, what is stored, therefore, is not the actual file, but rather what is stored is a compressed n ofthe original file.

] Specifically, in such ce, the initial file may be in a first format, which may be loaded into the system via the proprietary S3 compatible API, which receives the file, e.g., an FI file, and may then perform a compute function on the file, and/or then compresses the file, such as via a suitably configured CPU/GPU/QPU/FPGA processing engine 300, which then prepares the ssed file for storage, as a compressed, e.g., a compressed FI file. However, when the compressed and stored file needs to be retrieved, it may then be decompressed, which decompressed file may then be returned to the user. The advantage of this accelerated compression and decompression system is that the storage 400 of the compressed file means an incredible savings in storage costs, which advantage is made possible by the computing and/or compressing functionalities achieved by the systems disclosed herein.

Hence, because of the rapid and efficient computing and/or compressmg functionalities ed by the present systems, the user need not even know that the file is being compressed before storage, and subsequently decompressed post storage and ted at the user's interface. Particularly, the system functions so rapidly and ently that the user need not be aware of the multiplicity of compression, computation, and/or decompression steps that take place when storing and/or retrieving the requested data, to the user, this all appears seamless and timely. However, the fact that the present storage system will cost less and be more efficient than previous storage systems will be apparent.

Accordingly, in view of the above, object-based storage services are provided herein, wherein the storage services can be offered at lower costs, by combining a compute and/or compress instance along with a storage functionality. In such an instance, the typical storage costs can be substituted for computing costs, which are offered at a much lower level, because, as set forth herein, the computing costs may be implemented in an rated fashion such as by an FPGA and/or quantum computing platform 300, as described herein.

Hence, the accelerated platforms disclosed herein can be configured as a rapid and efficient e and retrieval system that allows for the rapid ssed storage of data that may be both compressed and stored as well as rapidly decompressed and retrieved at much lower costs and with greater efficiency and speed. This is particularly useful with respect to cs data storage 400, and is compatible with the Just In Time sing onalities disclosed herein, above. Therefore, in accordance with the devices, systems, and methods disclosed herein is an object storage service that may be provided, wherein the storage service implements a rapid compression functionality, such as genomics specific compression so as to store cs sing results data.

More particularly, as can be seen with t to A, in one exemplary implementation, the BioIT systems ed herein may be ured such that a pipeline server system 300, e.g., a n thereof, receives the request at the API, e.g., S3 compatible API, which is operably connected to a database 400 that is adapted for associating the initial (FI) file with the compressed version of the (CPI) file, e.g., based on the coupled metadata.

Likewise, once the original CPI files are decompressed and processed, the resulting results data (F2) files may then be compressed and stored as a CF2 file. ingly, when retrieval of the file is desired from the database 400, the server 300 has an API that has already associated the original file with the compressed file via appropriately configured metadata, hence, when retrieval is requested, a work flow management controller (WMS) ofthe system will launch the compute instance 300, which will launch the appropriate compute instance so as to perform any necessary computations and/or decompress the file for further processing, transmission, and/or presentation to the requesting user 100.

Hence, in various embodiments, an exemplary method may include one or more steps, in any logical order: 1) The request comes in through the API, e.g., S3 compatible API, 2) API communicates with the WMS, 3) the WMS populates the database and initiates the e instance(s), 4) the compute instance(s) performs the requisite compression on the FI file, and tes the characteristic metadata and/or other relevant file associations (X), e.g., to e a CPI XI file, 4) thereby preparing the data for storage 400. This process may then be repeated for F2, F3, Fn files, e.g., other sed information, so that the WMS knows how the compressed file was generated, as well as where and how it was stored. It is to be noted that a unique e of this system is that several different users 100 may be allowed to access the stored data 400 substantially simultaneously. For instance, the compression systems and methods disclosed herein are useful in conjunction with the BioT platforms disclosed herein, whereby at any time during the processing process the results data may be compressed and stored in accordance with the methods herein, and accessible to , with the right permissions.

] With respect to performing genomic is, a user 100 may access the system 300 herein, e.g., via a genomic analysis API such as an S3 or S3 compatible API, upload genomic data, such as in a BCL and/or FASTQ file or other file , and thereby request the performance of one or genomics operations, such as a mapping, aligning, sorting, de-duplicating, variant calling, and/or other operations. The system 300 receives the request at a ow r API, the workflow manager system then assesses the incoming requests, indexes the jobs, forms a queue, allocates the ces, e.g., instance tion, and generates the pipeline flow. Accordingly, when a request comes in and is preprocessed and queued, an instance allocator, e.g., API, will then spin up the various job specific instances, bed in greater detail herein below, in accordance with the work projects.

Hence, once the jobs are indexed, queued, and/or stored in an riate database 400, the ow manager will then pull the data from storage 400, e.g., S3 or S3 ible storage, cycle up an appropriate ce, which retrieves the file, and runs the appropriate processes on the data to perform one or more ofthe requested jobs.

Additionally, where a plurality of jobs are requested to be performed on the data, requiring the performance of a plurality of instances, then once the first instance has performed its operations, the results data may be ssed and , such as in an appropriate memory instance, e.g., a first data base, such as an elastic or le storage device, so as to wait while the further pipeline instance(s) is spun up and retrieves the results data for r sing, such as in accordance with the systems and methods disclosed herein above. Further, as new requests come in and/or current jobs are being run, the workflow management system will constantly be updating the queue so as to allocate jobs to the appropriate instances, via an instance allocator API, so as to keep the data flowing through the system and the processes ofthe system running efficiently.

Likewise, the system 300 may constantly be taking the results data and storing the data 200/400, e.g., in a first or a second database, prior to further processing and/or transmission, such as transmission back to the original requestor 100 or a designated party. In certain instances, the results data may be compressed, as disclosed herein, prior to storage 400 and/or transmission. Further, as indicated above, the generated results data files when compressed may include appropriate meta data and/or other associated data, where in the results data may designated differently as it flows through the system, such as going from an FI file to an FIC file to an F2 file, to an F2C, file, and so on, as the data is processed and moves through the platform pipeline e.g., as directed by a file associations APL Accordingly, because of the proprietary dedicated APis, as sed herein, the system may have a common ne to which other services may be coupled and/or additional resources, e.g., instances, may be brought online so as to make sure all of the pipeline operations run smoothly and efficiently. se, when desired the compressed and stored results data files may be called, whereby the workflow manager will spin up the appropriate compute and/or decompress database instance to decompress the results data for tation to the requester. It is noted that in various instances, the specified compute and ss instance, as well as the specified e and decompress instance, may be a single or multiple instances, and may be implemented as a CPU, FPGA, or a tightly coupled CPU/FPGA, tightly coupled CPU/CPU, or tightly coupled FPGA/FPGA. In certain instances, one or more of these and the other instances disclosed herein may be implemented as a quantum processing unit.

Accordingly, in view of the disclosures , in one , a device for performing one or more of a multiplicity of functions in performing cs sequence analysis operations is ed. For instance, once the data has been received, e.g., by a remote user 100, and/or stored 400 within the cloud based system, the input data may be accessed by the WMS, and may be prepared for further processing, e.g., for ary analysis, the s thereof may then be transmitted back to the local user 100, e.g., after being compressed, stored 400, and/or subjected to additional processing, e.g., tertiary sing by the system server 300.

In certain instances, the secondary processmg steps disclosed herein, in particular implementations, may be performed by a local computing resource 100, and may be implemented by software and/or hardware, such as by being executed by a box-top computing resource 200, where the computing resource 200 includes a core of CPUs, such as from about 4 to about 14 to about 24 or more CPU cores, and may r include one or more FPGAs. The local p computing resource 100 may be configured to access a large storage block 200, such as 120 GBs ofRAM memory, which access may be directly, such as by being directly coupled therewith, or indirectly, such as by being communicably coupled therewith over a local cloud based network 30.

Specifically, within a local system, data may be transmitted to or from the memory 200 via suitably configured SSD drives that are adapted for writing processing jobs data to, e.g., genomics jobs to be processed, and reading sed results data from the memory 200. In various embodiments, the local computing resource 100 may be communicably coupled to a sequencer 110 from where a BCL and/or FASTQ file may be obtained e.g., from the cer, and written to the SSD drivers, directly such as through a suitably configured interconnect. The local computing resource 100 may then perform one or more secondary processing ions on the data. For instance, in one embodiment, the local computing resource is a LINUX® server having 24 CPUs, which CPUs may be coupled to a suitably configurable FPGA that is adapted for performing one or more of the secondary processing operations disclosed herein.

] Hence, in particular instances, the local computing device 100 may be a "work bench" computing on having a BioIT chip set that is configured for performing one or more of secondary and/or tertiary sing on genetics data. For instance, as disclosed , the computing resource 100 may be ated with a PCie card that is inserted into the computing device so as to thereby be associated with the one or more internal CPUs, GPUs, QPU cores and/or associated memories. ularly, the components of the computing device 100 including the sing units, associated memories, and/or ated PCie card(s), having one or more FPGA/ASIC chipsets therein, may be in communication with one another, all ofwhich may be ed within a housing, such as in a box set manner that is typical within the art. More ularly, the box set may be configured for work-bench use, or in various instances, it may be configured and provided and/or usable within a remotely accessible server rack. In other embodiments, the CPU/FPGA/Memory chip sets and/or associated interconnect express card(s) can be associated within a Next Gen sequencing device so as to form one unit there with.

Accordingly, in one particular instance, a desktop box set may include a plurality of CPUs/GPUs/QPUs coupled to one or more FPGAs, such as 4 CPUs/GPUs, or 8, or 12, 16, 20, 22, or 24 CPUs, or more, which may be coupled to 1, or 2, or 3, or more FPGAs, such as within a single housing. Specifically, in one particular instance, a box set computing resource is provided wherein the ing resource includes 24 CPU cores, a reconfigurable FPGA, a database, e.g., 128x8 RAM, one or more SSDs, such as where the FPGA is adapted to be at least partially reconfigurable between operations, such as between performing mapping and aligning. Hence, in such an instance, BCL and/or FASTQ files generated by the sequencing apparatus 110 may be read into the CPU and/or transferred into the FPGA, for processing, and the results data thereof may be read back to the associated CPU via the SSD drives. Consequently, in this embodiment, the local computing system 100 may be ured to offload various high-compute functionalities to an associated FPGA, thereby enhancing speed, accuracy, and efficiency of bioinformatics processing. However, although a desktop box set solution 100 is useful, e.g., at a local facility, it may not be suitable for being accessed by a ity ofusers that may be located remotely from the box Particularly, in various instances, a cloud-based server solution 50 may be ed, such as where the server 300 may be accessible remotely. Accordingly, in particular instances, one or more of the integrated circuits (CPU, FPGA, QPU) disclosed herein may be provided and configured for being accessed via a cloud 50 based ace.

Hence, in particular instances, a work bench box set computing resource, as described above, may be provided where the box set configuration is adapted so as to be le to the cloud and accessible remotely. However, such a configuration may not be ient for ng a large of amount of traffic from remote users. ingly, in other cases, one or more of the integrated ts disclosed herein may be configured as a server based solution 300 configurable as part ofa server rack, such as where the server accessible system is configured specifically for being accessed remotely, such as via the cloud 50.

For ce, in one embodiment, a computing resource, or local server 100, having one or more, e.g., a multiplicity, of CPU and/or GPU and/or QPU cores, and associated memories, may be provided in ction with one or more of the FPGAs/ASICs disclosed herein. Particularly, as indicated above, in one implementation, a desktop box set may be provided, wherein the box set includes an 18 to 20 to 24 or more CPU /GPU core box set having SSDs, 128 x 8 RAM, and one or more BioIT FPGA/ASIC circuits, and further includes a suitably configured communications module having transmitters, receivers, antennae, as well as WIFI, Bluetooth, and/or cellular communications capabilities that are adapted in a manner so as to allow the box set to be accessible remotely. In this implementation, such as where a single FPGA is provided, the FPGA(s) may be adapted for being reconfigured, such as partially reconfigured, between one or more of the various steps ofthe cs analysis pipeline.

However, in other ces, a server system is ed and may include up to about 20 to 24 to 30 to 34 to 36 or more CPU/GPU cores and about 972 GB of RAM, or more, which may be associated with one or more, such as about two or four or about six or about eight or more FPGAs, which FPGAs may be configurable as herein described. For instance, in one implementation, the one or more FPGAs may be adapted for being reconfigured, such as partially reconfigured, between one or more of the various steps of the genomics analysis pipeline. However, in various other implementations, a set of dedicated FPGAs may be provided, such as where each FPGA is dedicated for performing a specific BioIT operation, such as mapping, aligning, variant calling, etc., thereby obviating the iguration step.

Accordingly, in various instances, one or more FPGAs may be provided, such as where the FPGA(s) are adapted so as to be reconfigurable between various pipeline ions. However, in other instances, one or more of the FPGAs may be configured so as WO 14320 PCT/0S2017/036424 to be dedicated to performing one or more functions without the need to be partially or fully configured. For instance, the FPGAs provided herein may be configured so as to be dedicated to performing one or more computationally intensive operations in the BioIT pipeline, such as where one FPGA is provided and dedicated to performing a mapping operation, and another FPGA is provided and configured for performing an alignment operation, although, in some instances, a single FPGA may be provided and configured for being at least partially reconfigured between performing both a mapping and an alignment operation. onally, other operations in the pipeline that may also be performed by reconfigurable or dedicated FPGAs may include performing a BCL sion/transposition operation, a Smith-Waterman ion, an HMM operation, a local realignment operation, and/or various other variant g operations. Likewise, s of the pipeline operations may be configured for being med by one or more of the associated CPUs/GPUs/QPUs of the system. Such operations may be one or more less computationally intensive ions of the pipeline, such as for preforming a sorting, deduplication, and other variant g operations. Hence, the overarching system may be ured for performing a combination of operations part by CPU/GPU/QPU, and part by hardware, such as by an FPGA/ASIC of the system.

Accordingly, as can be seen with respect to B, in vanous implementations of the cloud based system 50, the system may include a plurality of computing resources, including a plurality of instances, and/or levels of instances, such as where the instances and/or layers of instances are configured for performing one or more of the BioIT pipeline of operations discloed herein. For instance, various CPU/GPU/QPU and/or red integrated circuit instances may be provided for performing dedicated functions of the genomic pipeline analysis provided herein. For example, various FPGA instances may be provided for performing dedicated genomic analysis operations, such as an FPGA instance for ming mapping, another for performing aligning, another for ming local realignment and/or other Smith-Waterman operations, another for performing HMM operations, and the like.

Likewise, various CPU/GPU/QPU instances may be provided for performing ted genomic analysis operations, such as CPU/GPU/QPU instance for performing signal processing, sorting, de-duplication, compression, various variant g operations, and the like. In such instances, an associated memory or memories may be provided, such as between the various computation steps of the pipeline, for receiving results data as it is computed, compiled, and sed throughout the system, such as between the various CPU and/or FPGA instances and/or layers thereof. Further, it is to be noted that the size of the various CPU and/or FPGA instances may vary ent on the ational needs of the cloud based , and may range from small to medium to large to very large, and the number ofCPU/GPU/QPU and FPGA/ASIC instances may vary likewise.

Additionally, as can be seen with respect to B, the system may further include a workflow manager that is ured for scheduling and directing the movement of data throughout the system and from one instance to another and/or from one memory to another. In some cases, the memory may be a plurality of memories that are dedicated memories that are instance specific, and in other cases the memory may be one or more memories that are configured to be elestic and therefore capable ofbeing ed from one instance to another, such as a switchable elastic block e memory. In yet other instances, the memory may be instance non-specific and therefore capable of being communicably coupled to a plurality ofinstances, such as for elastic file storage.

Further, the workflow manager may be a dedicated instance itslef such as a CPU/GPU/QPU core that is dedicated and/or configured for determining what jobs need to be performed, and when and what ces will be utilized in the performance ofthose jobs, as well as for queuing up the jobs and directing them from resource to resource, e.g., instance to instance. The workflow manager may include or may otherwise be configured as a load estimator and/or form an elastic control node that is a dedicated ce that may be run by a processor, e.g. a CPU/GPU/QPU core. In s instances, the ow manager may have a database connected to it, which may be configured for managing all the jobs that need to be, are being, or have been processed. Hence, the WMS manager may be configured for detecting and ng how data flows hout the system, determining how to allocate system resources, and when to bring more resources online.

As indicated above, in certain instances, both a work bench and/or server based solution may be provided where the computing device includes a plurality of X CPU core servers having a size Y that may be configured to feed into one or more FPGAs with a size of Z, where X, Y, and Z are numbers that may vary depending on the processing needs of the system, but should be selected and/or otherwise configured for being optimized, e.g., , 14, 18, 20, 24, 30, etc. For instance, typical system configurations are optimized for ming the BioIT operations of the system herein described. Specifically, certain system urations have been optimized so as to maximize the flow of data from various CPU/GPU/QPU instances to various integrated circuits, such as FPGAs, of the system, where the size ofthe CPU and/or FPGA may vary in relation to one another based on the processing needs ofthe system. For example, one or more ofthe CPU and/or FPGA may have a size that is relatively small, medium, large, extra-large, or extra-extra-large. More specifically, the system architecture may be ured in such a manner that the CPU/FPGA hardware are sized and configured to run in an optimally efficient manner so as to keep both instance platforms busy during all run times, such as where the CPUs outnumber the ) 4 to 1, 8 to 1, 16 to 1, 32 to 1, 64 to 2, etc.

Hence, although it 1s generally good to have large FPGA capabilities, however, it may not be efficient to have a high capacity FPGA to process data, if there is not enough data needing to be sed being fed into the system. In such an instance, only a single or a partial FPGA may be implemented. Particularly, in an ideal arrangement, the workflow management system s the flow of data to identified CPUs and/or FPGAs that are configured in such a manner as to keep the system and its components computing full time. For instance, in one exemplary configuration, one or more, e.g., 2, 3, or 4 or more CPU/GPU/QPU cores may be configured to feed data into a small, medium, large, extra-large FPGA, or a portion thereof. Specifically, in one embodiment, a CPU specific instance may be provided, such as for ming one or more of the BioIT processing operations disclosed herein, such as where the CPU instance is cloud accessible and includes up to 4, 8, 16, 24, 30, 36 CPU cores, which cores may or may not be configured for being operably coupled to a portion of one or more FPGAs.

For example, a cloud accessible server rack 300 may be provided wherein the server includes a CPU core instance having about 4 CPU cores to about 16 to about 24 CPU cores that are ly connectable to an FPGA instance. For instance, an FPGA instance may be provided, such as where an average size of an FPGA is X, and the included FPGA may be of a size of about 1/8X, X, 2.5X up to 8X, or even about 16X, or more. In s instances, additional CPU/GPU/QPU cores and/or FPGAs may be included, and/or provided as a combined instance, such as where there is a large amount of data to process, and where the number of CPU cores is ed so as to keep the FPGA(s) full time busy. Hence, the ratio of the CPUs to FPGA(s) may be proportioned by being combined in a manner to ze data flow, and thus, the system may be configured so as to be cally scaled up or down as needs be, e.g., to minimize e while optimizing utilization based on workflow.

However, where the CPU(s) do not generate enough work to keep the FPGA busy and/or fully utilized, the configuration will be less than ideal. Provided herein, therefore, is a le architecture of one or more instances, which may be directly coupled together, or capable of being coupled together, in a manner that is adapted such that the CPU/FPGA software/hardware are run ently so as to ensure the present CPUs/GPUs/QPUs optimally feed the ble FPGA(s), and/or a portion thereof, in such a manner to keep both instance platforms busy during all run times. Pursuantly, allowing such a system to be accessible from the cloud will ensure a plurality ofdata being provided to the system so as to be queued up by the workflow manager and directed to the specific CPU/FPGA resources that are configured and capable ofreceiving and processing the data in an optimally efficient manner.

For instance, in some configurations, cloud accessible instances may e a plurality of numbers and sizes of CPUs/GPUs/QPUs, and onally, there may be cloud accessible instances that include a plurality of numbers and sizes of FPGAs (or ASICs) and/or QPUs. There may even be instances that have a combination of these ces.

However, in various iterations, the provided CPU/GPU/QPU and/or FPGA/QPU and/or mixed instances, may have too many of one instance and/or to less of the other instance for efficiently running the present BioIT pipeline processing platforms disclosed herein. ingly, herein presented, are systems and ectures, flexible combinations of the same, and/or methods for implementing them for the efficient formation and use of a ormatics and/or genomics processing platform of pipelines, such as is made accessible via the cloud 50.

In such systems, the number and configurations of the selected CPU(s)/GPUs/QPUs may be selected and configured to process the less computationally intensive ions, and the number and configurations of FPGA(s) and/or QPUs may be adapted for handling the computationally intensive tasks, such as where the data is seamlessly passed back and forth between the CPU/GPU/QPU and FPGA/QPU instances. Additionally, one or more memories may be provided for the g of data, e.g., s data, between the various steps of the procedures and/or between the various different ce types, thereby ng substantial period of instance latency. Specifically, during mapping and aligning, very little of the CPU/GPU is utilized, because of the intensive nature of the computations, these tasks are configured for being performed by the hardware implementations. Likewise, during variant calling, the tasks may be split in such a way as to be roughly fairly distributed n the CPU/FPGA instances in their tasks, such as where Smith-Waterman and HMM operations may be performed by the hardware, and vanous other operations may be performed by software run on one or more CPU/GPU/QPU instances.

] Accordingly, the architectural parameters set forth herein are not necessarily limited to one-set architecture, but rather the system is configured so as to have more flexibility for organizing its implementations, and relying on the workflow r to determine what ces are active when, how, and for how long, and directing which computations are performed on which instances. For instance, the number of CPUs and/or FPGAs to be brought online, and operationally coupled together, should be selected and configured in such a manner that the activated CPUs and FPGAs, as well as their attendant software/hardware, are kept optimally busy. Particularly, the number of CPUs, and their functioning, should be configured so as to keep the number of FPGAs, or a portion thereof, full time busy, such that the CPUs are optimally and efficiently feeding the FPGA(s) so as to keep both instances and their ent parts g proficiently.

Hence, in this manner, the work flow management controller of the system may be configured for accessing the workflow and organizing and dividing it in such a manner that the tasks that may be more optimally performed by the CPUs/GPUs/QPUs are ed to the number of CPUs necessary so as to optimally perform those operations, and that the tasks that may be more optimally performed by the FPGA(s)/ASICs/QPUs are directed to the number of FPGAs necessary so as to optimally perform those operations. An elastic and/or an efficient memory may further be ed for efficiently itting the results data ofthese operations from one ce to another. In this , a combination of machines and memories may be configured and combined so as to be optimally scaled based on the extent of the work to be performed, and the l configuration and usage of the instances so as to best perform that work efficiently and more cost effectively.

] Specifically, the cloud based architectures set forth herein shows that various known deficiencies in previous architectural ngs may cause ciencies that can be overcome by flexibly allowing more CPU/GPU/QPU core instances to access various different hardware instances, e.g., of FPGAs, or portions thereof, that have been organized in a more intentional manner so to be able to dedicate the right instance to performing the appropriate functions so as to be optimized by being implemented in that format. For instance, the system may be configured such that there is a greater proportion of available CPU/GPU instances that may be accessible remotely so as to be full time busy producing results data that can be optimally fed into the available FPGA/QPU instance(s) so as to keep the ed FPGA ce(s) full time busy. Therefore, it is desirable to provide a structured architecture that is as efficient as possible and is full time busy. It is to be noted that configurations where too few CPUs feed into too many FPGAs such that one or more of the FPGAs are being underutilized is not efficient and should be avoided.

In one implementation, as can be seen with respect to B, the architecture can be configured so as to virtually include several different layers or levels, such as a first level having a first number of X CPU cores, e.g., from 4 to about 30 CPU cores, and a second level having from 1 to 12 or more FPGA instances, where the size ofthe FPGAs may range from small to medium to large, etc. A third level of CPU cores and/or a fourth level of further FPGAs, and so on, may also be included. Hence, there are many available instances in the cloud based server 300, such as instances that simply include CPUs or GPUs and/or instances that include FPGAs and/or combinations ofthem, such as in one or more levels described herein. Accordingly, in a manner such as this, the architecture may be ly or cally organized so that the most intensive, specific ing functions are performed by the hardware instances or QPUs, and those functions that can be run h the CPUs, are directed to the appropriate CPU/GPU at the appropriate level for general processing purposes, and where necessary the number of CPU/FPGA instances may be increased or decreased within the system as needs be.

For example, the architecture can be elastically sized to both minimize system expense while at the same time maximizing optimal ation. ically, the architecture may be configured to maximize efficiency and reduce latency by combining the various instances on various different virtual levels. Particularly, a plurality, e.g., a significant and/or all, of the Level 1 CPU/GPU instances can be configured to feed into the various Level 2 FPGA ces that have been specifically configured to perform specific functions, such as a mapping FPGA and an aligning FPGA. In a further level, one or more onal (or the same as Level I) CPUs may be provided, such as for performing a sorting and/or deduplicating operations and/or various variant calling operations. Further still, one or more additional layers of FPGAs may be configured for performing a Needleman-Wunsch, Smith- an, an HMM, t calling ion, and the like. Hence, the first level CPUs can be engaged to form an initial level of a genomics analysis, such as for performing general processing steps, including the queuing up and preparing of data for further pipeline analysis, which data once processed by one or a multiplicity of CPUs, can be fed into one or more further levels of dedicated FPGA instances, such as where the FPGA instance is configured for ming ive computing functions.

In this manner, in a particular implementation, the CPU/GPU instances in the pipeline route their data, once prepared, to the one or two g and aligning Level 2 FPGA instances. Once the mapping has been performed the result data may be stored in a memory and/or then fed into an aligning instance, where aligning may be performed, e.g., by at least one dedicated Level 2 FPGA instance. Likewise, the processed mapped and aligned data may then be stored in a memory and/or directed to a Level 3 CPU instance for further processing, which may be the same Level 1 or a different instance, such as for performing a less processing intense cs analysis function, such as for performing a sorting function.

Additionally, once the Level 3 CPUs have med their processing, the resultant data may then be ded either back up to other Level 2 instances of the FPGAs, or to a Level 4 FPGA instance, such as for further genomics processing intense functions, such as for performing a Needleman-Wunsch (NW), Smith-Waterman (SW) processing function, e.g., at a NW or SW dedicated FPGA ce. Likewise, once the SW analysis has been performed, such as by an SW dedicated FPGA, then the processed data may be sent to one or more associated es and/or further down the processing pipeline, such as to another, e.g., Level 4 or 5, or back up to Level I or 3, CPU and/or FPGA instance, such as for performing HMM and/or Variant Calling analysis, such as in a dedicated FPGA and/or further layer of CPU processing core.

In a manner such as this latency and efficiency issues can be overcome by combining the various different instances, on one or more different levels, so as to provide a pipeline platform for cs processing. Such a configuration may involve more than a scaling and/or ing instances, the instances may be configured so that they specialize in performing dedicated functions. In such an instance, the Mapping FPGA instance only ms mapping, and likewise the aligning FPGA instance only performs ng, and so on, rather than a single instance performing end-to-end processing of the pipeline. Albeit, in other configurations, one or more of the FPGAs may be at least partially reconfigured, such as between performing pipeline tasks. For instance, in certain embodiments, as the genomics analyses to be performed herein is a multi-step process, the code of on FPGA may be configured so as to be changed halfway through processing s, such as when the FPGA completes the mapping operation, it may be reconfigured so as to perform one or more of aligning, t calling, Smith-Waterman, HMM, and the like.

] Hence, the pipeline manager, e.g., workflow management system, may function to manage the queue of genomic processing requests being formulated by the Level I CPU instances so as to be broken down into discrete jobs, aggregated, and be routed to the appropriate job specific CPU and then to the job specific FPGA ces for further processing, such as for mapping and/or ng, e.g., at Level 2, which mapped and aligned data once processed can be sent backwards or forwards to the next level of CPU/FPGA processing of the results data, such as for the performance of various steps in the variant calling module.

For instance, the variant calling function may be divided into a plurality of operations, which can be performed in software, then forwarded to Smith-Waterman and/or HMM processing in one or more FPGA hardware instances, and then may be sent to a CPU for continued variant calling operations, such as where the entire platform is elastically and/or efficiently sized and implemented to minimize cost of the expensive FPGA instances, while max1m1zmg utilization, minimizing latency, and ore optimizing operations.

Accordingly, in this manner, less hardware instances are needed because of their pure processing capabilities and hardwired specificity, and therefore, the number of FPGAs to the number of CPUs may be minimized, and their use, e.g., of the FPGAs, may be maximized, and therefore, the system optimized so as to keep all instances full time busy. Such a configuration is optimally designed for genomics processing analysis, especially for mapping, aligning, and variant calling.

An additional structural t that may be included, e.g., as an attachment, to the pipeline architecture, disclosed herein, is one or more elastic and/or ent memory modules, which may be configured to function for providing block storage of the data, e.g., results data, as it is transitioned throughout the pipeline. Accordingly, one or more Elastic Block Data Storage (EBDS) and/or one or more efficient (flexible) block data e s may be inserted between one or more of the sing levels, e.g., between the different instances and/or instance levels. In such an instance, the e device may be configured such that as data gets processed and results obtained, the processed results may be directed to the storage device for storage prior to being routed to the next level ofprocessing, such as by a dedicated FPGA processing module. The same storage device may be ed between all instances, or instance , or a multiplicity of storage devices may be employed between the various instances and/or instance levels, such as for storing and/or ing and/or for g of results data. ingly, one or more memories may be ed in such a manner that the various instances of the system may be coupled to and/or have access to the same memory so as to be able to see and access the same or similar files.

Hence, one or more elastic es ies capable of being coupled to a plurality of instances sequentially) and/or efficient memories (memories e of being coupled to a plurality of instances simultaneously) may be present whereby the various instances of the system are configured to read and write to the same or similar memory.

For ce, in one exemplary embodiment with respect to urations employing such elastic memories, prior to sending data directly from one instance and/or one level of processing to another, the data may be routed to an EBDS, or other memory device or structure, e.g., an efficient memory block, for storage and thereafter routed to the appropriate hardwired-processing module. ically, a block storage module may be attached to a node for memory storage where data can be written to the BSD for storage at one level, and the BSD may be flipped to another node for routing the stored data to the next processing level. In this manner, one or more, e.g., multiple, BDS modules may be included in the pipeline and ured for being flipped from one node to another so as to participate in the transitioning ofdata throughout the ne.

Further, as indicated above, a more flexible File Storage Device may be employed, such as a device that is capable of being coupled to one or more instances concurrently, such as without having to be switched from one to the other. In a manner such as this, the system may be elastically scaled at each level ofthe system, such as where at each level there may be a different number of nodes for processing the data at that level, and once processed the results data can be written to one or more associated EBDS devices that may then be switched to the next level of the system so as to make the stored data available to the next level ofprocessors for the performance r specific tasks at that level.

Accordingly, there are many steps in the processing ne, e.g., at its attendant nodes, as data is prepared for processing, e.g., preprocessing, which data once it is prepared is directed to an appropriate processing instance at one level where results data may be generated, then the result data may be stored, e.g., within an EDS device, queued and prepared for the next stage of processing by being flipped to the next node of instances and routed to the next instance for processing by the next order of FPGA and/or CPU processing instances, where further results data may be generated, and again once generated the results data may be directed either back to the same or forward to the next level of EDS for storage prior to being advanced to the next stage ofprocessing.

Particularly, in one specific implementation, flow through the pipeline may look like the following: CPU (e.g., a 4 CPU core, or C4 instance): data prepared (queued and/or stored); FPGA (e.g. a 2XL FPGA - 1/8 of a full server, or an FI instance): g, temporary storage; FPGA (e.g. a 2XL FPGA - 1/8 of a full server, or an FI instance): aligning, temporary storage; CPU: sorting, temporary storage; CPU: de-duplication, temporary storage; CPU: variant calling 1, temporary storage; FPGA (e.g., an FI or a 16XL, or F2 instance): Smith-Waterman, temporary storage; FPGA (e.g. FI or F2 instance): HMM, temporary storage; CPU: variant calling 2, temporary storage; CPU: VCGF, temporary storage, and so on. Additionally, a work flow management system may be included to control and/or direct the flow of data through the system, such as where the WMS may be implemented in a CPU core, such as a 4 core CPU, or C4 instance. It is noted, one or more of these steps may be performed in any logical order and may be ented by any suitably configured resource such as implemented in software and/or hardware, in various different combinations. And it is to be noted that any of these operations may be performed on one or more CPU instances and one or more FPGA instances on one or more theoretical levels of processing, such as to form the BioIT processing described herein.

] As indicated, a work flow manager may be included, such as where the WMS is implemented in one or more CPU cores. Hence, in s instances, the WMS may have a database operationally coupled to it. In such an instance, the database includes the various operations or jobs to be , g jobs, as well as the history of all jobs previously or currently to be performed. As such, the WMS monitors the system and database to identify any new jobs to be performed. uently, when a g job is fied, the WMS initiates a new analysis protocol on the data and farms it out to the appropriate instance ). Accordingly, the workflow manager keeps track of and knows where all the input files are, either stored, being processed, or to be stored, and therefore, directs and instructs the instances of the various sing nodes to access respective files at a given location, to begin reading files, to begin implementing processing instructions, and where to write results data. And, hence, the WMS directs the systems as to the g results data to down line processing nodes. The WMS also determines when new instance needs to be fired up and brought online so as to allow for the dynamic scaling of each step or level of processing.

Hence, the WMS identifies, organizes, and directs discrete jobs that have to be performed at each level, and further directs the results data being n to the memory to be stored, and once one job is completed, another node fires up, reads the next job, and performs the next ive operation.

In a manner such as this, the input jobs may be spread across a lot of different instances, which instances can be scaled, e.g., independently or collectively, by including less or more and more ces. These instances may be employed to build nodes so as to more efficiently balance the use of ces, where such instances may comprise a partial or full instance. The workflow manager may also direct and/or control the use of one or more memories, such as in between the processing steps disclosed herein. The various ces may also e mentary programing so as to allow them to communicate with each other and/or the various memories, so as to virtualize the server. The WMS may also include a load estimator so as to elastically control the usage ofthe nodes.

] Further, with respect to the use of es, one or more EBDS, or other suitably configured data and/or file storage devices, may be attached to one or more of the various nodes, e.g., between the various levels of instances, such as for temporary storage between the various different processing steps. Hence, the storage device may be a single storage device configured for being coupled to all of the various instances, e.g., an efficient memory block, such as elastic file storage, or may be multiple e s, such as one storage device per instance or instance type that is switchable between instances, e.g., elastic block e device. Accordingly, in a manner such as this, each level of processing instances and/or memory may be elastically scaled on an as needed basis, such as between each ofthe different nodes or levels ofnodes, such as for processing one or several genomes.

In view of the architecture herein, one or a multiplicity of genomes may be introduced into the system for processing, such as from one or more lanes of a flow cell of a Next Gen Sequencer, as indicated in Specifically, providing a cloud based server system 300, as herein described, will allow a multiplicity ofjobs to be piled up and/or queued for processing, which jobs may be processed by the various different instances of the system simultaneously or sequentially. Hence, the pipeline may be configured to support a multiplicity of jobs being processed by a virtual matrix of processors that are coupled to ly configured memory devices so as to facilitate the efficient processing and data from one instance to another. Further, as indicated, a single memory device may be ed, where the memory device is configured for being coupled to a ity of different instance, e.g., at the same time. In other instances, the memory device may be an elastic type memory device that may be configured for being coupled to a first instance, e.g., at a single time, and then being reconfigured and/or otherwise decoupled from the first instance, and switched to a second instance.

As such, in one implementation, one or more elastic block storage devices may be included and the system may be configured so as to include a switching control mechanism. For instance, a switch controller may be included and configured so as to control the functioning of such memory devices as they switch from one ce to another. This configuration may be arranged so as to allow the transfer of data through the pipeline of dedicated processors, thereby increasing the efficiency of the system, e.g., among all of the instances, such as by flowing the data h the system, allowing each level to be scaled independently and to bring processors online as needed to efficiently scale.

Additionally, the workflow management system thm may be configured so as to determine the number of jobs, the number of resources to process those jobs, the order of processing, and directs the flow ofthe data from one node to another by the flipping or switching of one or more flexible switching devices, and where needed can bring additional resources online to handle an increase in ow. It is to be noted that this configuration may be adapted so as to avoid the copying ofdata from one instance to the next to the next, which is inefficient and takes up too much time. Rather, by flipping the elastic storage from one set of instances to another, e.g., pulling it from one node and attaching to a second node, can y enhance the efficiency of the system. Further, in various instances, instead of employing EBSD, one or more elastic file storage devices, e.g., single memory devices e of being coupled to a multiplicity of instances without needing to be flipped from one to another, may be employed, so as to further e the transmission of data between ces, making the system even more ency. Additionally, it is to be noted, as indicated earlier herein, in r configuration the CPUs of the architecture can be directly to one another. Likewise, the various FPGAs may be directly d together. And, as indicated above, the CPUs can be directly coupled to the FPGAs, such as where such coupling is via a tight coupling interface as described above.

Accordingly, with respect to user storage and accessmg of the generated results data, from a system wide ctive, all of the generated results data need not be stored. For instance, the generated results data will typically be in a particular file , e.g., a BCL, FASTQ, SAM, BAM, CRAM, VCF file. However, each one of these files is extensive and the storage of all of them would e a lot of memory thereby incurring a lot of expense. Nevertheless, an advantage of the present s, systems, and methods herein, all of these files need not be stored. , given the rapid sing speeds and/or the rapid compression and decompression rates achievable by the components and methods ofthe system, only a single file format, e.g., a compressed file format, need be stored, such as in the cloud based database 400. Specifically, only a single data file format need be stored, from which file format, implementing the s and methods of the system, all other file formats may be derived. And, because of the rapid compression and decompression rates ed by the system, it is typically a compressed file, e.g., a CRAM file.

Particularly, as can be seen with respect to A, in one implementation, a user of a local computing resource 100 may upload data, such as genomics data, e.g., a BCL and/or FASTQ file, into the system via the cloud 50 for receipt by the cloud based computing resource, e.g., server 300. The server 300 will then either temporarily store the data 400, or will begin processing the data in accordance with the jobs request by the user 100. When processing the input data, the computing ce 300 will thereby generate results data, such as in a SAM or BAM and/or VCF file. The system may then store one or more of these files, or it may compress one or more ofthese files and store those. However, in order to lower cost and more efficiently make use of the resources, the system may store a singe, e.g., compressed, file, from which file all other file formats may be generated, such as by using the devices and methods herein disclosed. Accordingly, the system is configured for generating data files, e.g., results data, which may be stored on a server 300 associated database 400 that is accessible via the cloud 50, in a manner that is cost effective.

Accordingly, using a local computing resource 100, a user of the system may log on and access the cloud 50 based server 300, may upload data to the server 300 or database 400, and may request one or more jobs be med on that data. The system 300 will then m the requested jobs and store the results data in database 400. As noted, in particular instances, the system 300 will store the generated results data in a single file format, such as a CRAM file. r, with the click of a button, the user can access the stored file, and with another click of a button, all of the other file formats may then be made accessible. For instance, in accordance with the methods disclosed herein, given the systems rapid sing capabilities, which would then be processed and ted behind the scene, e.g., on the fly, thus g down on both processing time and burden as well as storage costs, such as where the computing and the storage functions are bundled together.

Particularly, there are two parts of this efficient and rapid storage process that are enabled by the speed of performing the accelerated operations herein disclosed. More particularly, e the various processing operations of g, aligning, sorting, deduplicating , and/or variant calling, may be implemented in a hardwired and/or quantum processing configuration, the production of s data, in one or more file formats, may be achieved rapidly. Additionally, because ofthe close coupling ectures disclosed herein, a seamless ssion and storing ofthe results data, e.g., in a FASTQ, SAM, BAM, CRAM, VCF file , is further achieved.

Further still, because of the accelerated processing provided by the devices of the system, and because of their seamless integration with the associated e devices, the data that results from the processing operations ofthe system, which data is to be stored, may be both efficiently compressed prior to storage and decompressed subsequent to storage.

Such encies thereby lower storage costs and/or the ies related to decompression of files before use. Accordingly, e of these advantages, the system may be configured so as to enable seamless compression and storing of only a single file type, with on-the-fly ration of any of the other file types, as needed or requested by the user. For instance, a BAM file, or a compressed SAM or CRAM file associated therewith, may be be stored, and from that file the others may be generated, e.g., in a forward or a e direction, such as to reproduce a VCF or FASTQ or BCL file, respectively.

For instance, in one embodiment, a FASTQ file may originally be input into the system, or otherwise generated, and stored. In such an instance, when going in the d direction, a checksum of the file may be taken. Likewise, once result data is produced, when going backward, another checksum may be ted. These checksums may then be used to ensure that any further file formats to be generated and/or recreated by the system, in the forward or reverse direction, match identically to one another and/or their compressed file formats. In a manner such as this it may be ensured that all of the necessary data is stored, in as ent as manner as possible, and the WMS knows exactly where the data is stored, in what file format it is stored in, what the original file format was in, and from this data the system can regenerate any file format in an identical manner going forwards or backwards between file formats (once the template is originally generated).

Hence, the speed advantage of the "just in time" compiling is enabled in part by the hardware and/or quantum implemented generation of the relevant files, such as in generating a BAM file from a previously generated FASTQ file. Particularly, compressed BAM files, including SAM and CRAM files, are not typically stored within a database because of the increased time it takes prior to processing to decompress the compressed stored file. However, the JIT system allows this to be done without substantial penalties.

More particularly, implementing the devices and ses disclosed herein, not only can generated sequence data be compressed and decompressed rapidly, e.g., almost instantaneously, it may also be stored efficiently. Additionally, from the stored file, in whatever file format it is stored, any of the other file formats may be regenerated in mere moments.

Hence, as can be seen with reference to C, when the accelerated hardware and/or m processing performs various secondary processing procedures, such as mapping and aligning, sorting, de-duplicating, and variant calling, a further step of compression may also be performed, such as in an all in one process, prior to storage in the compressed form. Then when the user desires to analyze or otherwise use the compressed data, the file may be ved, decompressed, and/or ted from one file format to another, and/or be analyzed, such as by the JIT (s) being loaded into the hardwired processor, or ured within the quantum processor, and subjecting the compressed file to one or more procedures ofthe JIT pipeline.

Accordingly, in various instances, where the system includes an associated FPGA, the FPGA can be fully or partially reconfigured, and/or a quantum processing engine may be organized, so as to perform a JIT procedure. Particularly, the JIT module can be loaded into the system and/or configured as one or more engines, which engines may include one or more compression engines 150 that are configured for g in the background.

Hence, when a given file format is called, the JIT-like system may perform the ary ions on the requested data so as to produce a file in the requested format. These ions may include compression and/or decompression as well as conversion so as to derive the requested data in the identified file format.

For instance, when genetic data is generated, it is usually ed in a raw data format, such as a BCL file, which then may get converted into a FASTQ file, e.g., by the NGS that generates the data. However, with the present system, the raw data files, such as in BCL or other raw file format, may be streamed or ise transmitted into the JIT module, which can then convert the data into a FASTQ file and/or into another file format. For example, once a FASTQ file is generated, the FASTQ file may then be processed, as disclosed herein, and a ponding BAM file may be generated. And likewise, from the BAM file a ponding VCF may be generated. Additionally, SAM and CRAM files may also be generated during appropriate steps. Each one of these steps may be performed very rapidly, especially once the riate file format has once been generated. Hence, once the BCL file is received, e.g., straight from the sequencer, the BCL can be converted into a FASTQ file or be directly converted into a SAM, BAM, CRAM, and/or VCF file, such as by a hardware and/or quantum implemented mapping/aligning/sorting/variant calling procedure.

For example, in one use model, on a typical sequencing instrument, a large number of different subject's genomes may be loaded into individual lanes of a single sequencing instrument to be run in parallel. Consequently, at the end of the run, a large number of diverse BCL files, derived from all the different lanes and enting the whole genomes ofeach ofthe different subjects, are generated in a multiplex x. Accordingly, these multiplexed BCL files may then be de-multiplexed, and respective FASTQ files may be ted representing the c code for each individual subject. For instance, if in one sequencing run N BCL files are ted, these files will need to be de-multiplexed, layered, and stitched together for each subject. This stitching is a complex process where each subject's genetic material is converted to BCL files, which may then be converted to a FASTQ file or used directly for mapping, aligning, and/or sorting, variant calling, and the like. This process may be ted so as to greatly speed up the various steps of the process.

Further, as can be seen with respect to A, once this data has been generated 110, and ore needs to be stored, e.g., in which ever file format is selected, the data may be stored in a password protected and/or encrypted memory cache, such as in a dedicated genomics dropbox-like memory 400. Accordingly, as the generated and/or processed genetic data comes off of the cer, the data may be processed and/or stored and made available to other users on other s, such as in a dropbox-like cache 400. In such an ce, the automated bioinformatics analysis pipeline system may then access the data in the cache and automatically begin processing it. For example, the system may include a management system, e.g., a workflow management system 151, having a controller, such as a microprocessor or other intelligence, e.g., artificial intelligence, that manages the retrieving ofthe BCL and/or FASTQ files, e.g., from the memory cache, and then directs the processing of that information, so as to te a BAM, CRAM, SAM, and/or VCF, thereby automatically generating and outputting the various processing results and/or storing the same in the dropbox memory 400.

A unique benefit of JIT sing, as implemented within this use model, is that JIT allows the various genetic files produced to be compressed, e.g., prior to data storage, and to be decompressed rapidly prior to usage. Hence, JIT processing can compile and/or compress and/or store the data as it is coming off the cer, where such storage is in a secure c dropbox memory cache. This genomic dropbox cache 400 may be a cloud 50 ible memory cache that is configured for the storing of genomics data ed from one or more automated sequencers 110, such as where the sequencer(s) are located remotely from the memory cache 400.

Particularly, once the sequence data has been generated 110, e.g., by a remote NGS, it may be compressed 150 for transmission and/or storage 400, so as to reduce the amount of data that is being uploaded to and stored in the cloud 50. Such uploading, transmission, and storage may be performed rapidly because ofthe data compression 150 that takes place in the system, such as prior to transmission. Additionally, once uploaded and stored in the cloud based memory cache 400, the data may then be retrieved, locally 100 or remotely 300, so as to be processed in accordance with the devices, systems, and methods of the BioIT pipeline disclosed herein, so as to te a mapping, aligning, sorting, and/or variant call file, such as a SAM, BAM, and/or CRAM file, which may then be stored, along with a metafile that sets forth the information as to how the generated file, e.g., SAM, BAM, CRAM, etc. file, was produced.

Hence, when taken together with the metadata, the compressed SAM, BAM, and/or CRAM file may then be sed to produce any of the other file formats, such as FASTQ and/or VCF files. Accordingly, as discussed above, on the fly, JIT can be used to rate the FASTQ file or VCF from the compressed BAM file and vice versa. The BCL file can also be regenerated in like manner. It is to be noted that SAM and CRAM files can likewise be compressed and/or stored and can be used to produce one or more of the other file s. For instance, a CRAM file, which can be un-CRAMed, can be used to produce a variant call file, and likewise for the SAM file. Hence, only the SAM, BAM and/or CRAM file need be saved and from these files, the other file s, e.g., VCF, FASTQ, BCL files, can be reproduced.

Accordingly, as can be seen with respect to A, a mapping and/or aligning and/or sorting and/or variant calling instrument 110, e.g., a work bench computer, may be on-site 100 and/or another second corresponding instrument 300 may be located ly and made accessible in the cloud 50. This configuration, along with the devices and methods disclosed herein, is adapted to enable a user to rapidly perform a BioIT analysis "in the cloud", as herein disclosed, so as to produce results data. The results data may then be processed so as to be compressed, and once compressed, the data may be configured for transmittal, e.g., back to the local computing resource 100, or may be stored in the cloud 400, and made accessible via a cloud based interface by the local computing resource 100. In such an instance, the compressed data may be a SAM, BAM, CRAM, and/or VCF file.

Specifically, the second computing ce 300 may be another work-bench on, or it may be a server configured ce, such as where the computing resource is accessible via the cloud 50, and is configured for ming mapping and/or aligning and/or g and/or variant calling instrument. In such an instance, a user may requests the cloudbased server 300 perform one or more BioIT jobs on uploaded data, e.g., BCL and/or FASTQ data. In this instance, the server 300 will then access the stored and/or compressed file(s) and may process the data so as to rapidly process that data and generate one or more results data, which data may then be compressed and/or stored. Additionally, from the results data file one or more BCL, FASTQ, SAM, BAM, VCF, or other file formats may be ted, e.g., on the fly, using JIT processing. This configuration thereby alleviates the l transfer speed bottleneck.

Hence, in various embodiments, the system 1 may include, a first mapping and/or aligning and/or sorting and/or variant calling instrument 100, which may be positioned locally 100, such as for local data production, compression 150, and/or storage 200; and a second instrument 300 may be positioned remotely and associated in the cloud 50, whereby the second instrument 300 is configured for receiving the generated and compressed data and storing it, e.g., via an associated storage device 400. Once stored, the data may be accessed decompression and conversion ofthe stored files into one or more ofthe other file formats.

Therefore, in one implementation of the system, data e.g., raw sequence data such as in a BCL or FASTQ file format, which is generated by a data generating tus, e.g., a sequencer 110, may be uploaded and stored in the cloud 50, such as in an associated genomics dropbox-like memory cache 400. This data may then be accessed directly by the first mapping and/or aligning and/or sorting and/or variant calling instrument 100, as described , or may be accessed ctly by the server resource 300, which may then process the ce data to produce mapped, aligned, sorted, and/or variant results data.

] Accordingly, in various embodiments, one or more of the storage devices herein disclosed may be configured so as to be ible, with the riate permissions, via the cloud. For instance, various of the results data of the system may be compressed and/or stored in a memory, or other suitably ured se, where the database is configured as a cs dropbox cache 400, such as where various results data may be stored in a SAM, BAM, CRAM and/or VCF file, which may be accessible ly.

Specifically, it is to be noted that, with respect to FIG 40A, a local instrument 100 may be provided, where the local instrument may be associated with the sequencing instrument 110 itself, or it may be remote therefrom but and associated with the sequencing instrument 110 via a local cloud 30, and the local instrument 100 may further be associated with a local storage facility 200 or remote memory cache 400, such as where the remote memory cache is configured as the genomics dropbox. r, in various instance, a second mapping and/or aligning and/or sorting and/or variant calling instrument 300, e.g., a cloud based instrument, with the proper authorities, may also be connected with the genomics dropbox 400, so as to access the files, e.g., compressed files, stored thereby the local computing resource 100, and may then decompress those files to make the results available for further, e.g., secondary or tertiary, processing.

Accordingly, in various instances, the system may be streamlined such that as data is generated and comes off of the sequencer 110, such as in raw data format, it may either be ately uploaded into the cloud 50 and stored in a cs dropbox 400, or it may be transmitted to a BioIT processing system 300 for further processing and/or ssion prior to being uploaded and stored 400. Once stored within the memory cache 400, the system may then immediately queue up the data for retrieval, compression, decompression, and/or for further processing such as by another associated BioIT processing apparatus 300, which when processed into results data may then be compressed and/or stored 400 for r use later. At this point, a tertiary processing pipeline may be initiated whereby the stored results data from secondary processing may be decompressed and used such as for ry analysis, in accordance with the methods disclosed herein.

Hence, in various embodiments, the system may be pipelined such that all of the data that comes off of the sequencer 110 may either be ssed, e.g., by a local computing resource 100, prior to er and/or storage 200, or the data may be erred directly into the genomics dropbox folder for storage 400. Once ed thereby, the stored data may then substantially immediately be queued for retrieval and compression and/or decompression, such as by a remote computing resource 300. After being decompressed the data may substantially immediately be available for processing such as for mapping, aligning, sorting, and/or variant calling to produce secondarily processed results data that may then be re-compressed for storage. ard, the compressed secondary results data may then be accessed, e.g., in the genomics dropbox 400, be decompressed, and/or be used in one or more tertiary sing procedures. As the data may be compressed when stored and substantially immediately ressed when retrieved, it is available for use by many different systems and in many different bioanalytical protocols at different times, simply by accessing the dropbox storage cache 400.

Therefore, in such manners as these, the BioIT platform pipelines presented herein may be configured so as to offer ible flexibility of data tion and/or analysis, and are adapted to handle the input of ular forms of genetic data in multiple formats so as to process the data and produce output formats that are compatible for various downstream analysis. Accordingly, as can be seen with respect to C, presented herein are devices, systems, and s for performing genetic sequencing analysis, which may e one or more of the following steps: First, a file input is received, the input may be in one or more of a FASTQ or BCL or other form of c sequence file format, such as in a compressed file format, which file may then be decompressed, and/or processed through a number of steps disclosed herein so as to generate a VCF/gVCF, which file may then be compressed and/or stored and/or transmitted. Such compression and/or decompression may occur at any suitable stage throughout the process.

For instance, once a BCL file is received, it may be subjected to a pipeline of analyses, such as in a sequential manner as disclosed herein. For example, once ed, the BCL file may be converted and/or de-multiplexed such as into a FASTQ and/or FASTQgz file format, which file may be sent to a mapping and/or aligning module, e.g., of a sever 300, so as to be mapped and/or aligned in accordance with the apparatuses and their methods of use described herein. Additionally, in various instances, the mapped and aligned data, such as in a SAM or BAM file format, may be position sorted and/or any duplications can be marked and removed. The files may then be compressed, such as to produce a CRAM file, e.g., for transmission and/or storage, or may be forwarded to a variant calling, e.g., HMM, module, to be processed so as to produce a variant call file, VCF or gVCF.

More specifically, as can be seen with respect to FIGS. 40C and 40D, in n instances, the file to be received by the system may be streamed or ise transferred to the system directly from the sequencing apparatus, e.g., NGS 110, and as such the transferred file may be in a BCL file . Where the received file is in a BCL file format it may be converted, and/or otherwise de-multiplexed, into a FASTQ file for processing by the system, or the BCL file may be processed directly. For instance, the platform pipeline processors can be configured to e BCL data that is streamed directly from the cer, as described with respect to or it may receive data in a FASTQ file format. r, receiving the sequence data directly as it is streamed off of the cer is useful because it enables the data to go directly from raw sequencing data to being directly processed, e.g., into one or more of a SAM, BAM, and/or VCF/gVCF for output.

] Accordingly, once the BCL and/or the FASTQ file is ed, e.g., by a computing resource 100 and/or 300, it may be mapped and/or aligned by the ing resource, which mapping and/or ng may be performed on single end or paired end reads. For instance, once received, the sequence data may be compiled into reads, for analysis, such as with read lengths that may range from about 10 or about 20, such as 26, or 50, or 100, or 150 bp or less up to about lK, or about 2.5K, or about 5K, even about lOK bp or more. Likewise, once mapped and/or aligned the sequence may then be sorted, such as on sorted, such as through binning by reference range and/or sorting of the bins by reference position. Further, the ce data may be processed via duplicate marking, such as based on the starting position and CIGAR string, so as to generate a high quality duplicate , and any marked duplicates may be removed at this point. Consequently, a mapped and aligned SAM file may be generated, which may be compressed so as to form a AM file, such as for storage and/or further processing. Furthermore, once the BAM/CRAM file has been retrieved, the mapped and/or aligned sequence data may be forwarded to a variant calling module of the system, such as a haplotype variant caller with reassembly, which in some instances, may employ one or more of a Smith-Waterman Alignment and/or Hidden Markov Model that may be implemented in a combination ofsoftware and/or hardware, so as to generate a VCF.

Hence, as seen in D, the system and/or one or more of its components may be configured so as to be able to convert BCL data to FASTQ or SAM/BAM/CRAM data formats, which may then be sent throughout the system for r processing and/or data reconstruction. For instance, once the BCL data is received and/or converted into a FASTQ file and de-multiplexed and/or deduped, the data may then be forwarded to one or more of the pipeline modules disclosed herein, such as for mapping and/or aligning, which dependent on the number of samples being processed will result in the production of one or more, e.g., several, SAM/BAM files. These files may then be sorted, de-duped, and forwarded to a variant calling module, so as to produce one or more VCF files. These steps may be ed for greater context and accuracy. For example, once the sequence data is mapped or d, e.g., to produce a SAM file, the SAM file may then be compressed into one or more BAM files, which may then be transmitted to a VCF engine so as to be converted throughout the processing of the system to a VCF/gVCF, which may then be compressed into a CRAM file. uently, the files to be output along the system may be a Gzip and/or CRAM file.

Particularly, as can be seen with respect to FIGS. 40C and 40D, one or more of the files, once ted may be compressed and/or transferred from one system component to another, e.g., from a local 100 to a remote resource 300, and once received may then be decompressed, e.g., if previously compressed, or converted/de-multiplexed. More particularly, once a BCL file is received, either by a local 100 or remote 300 resource, it may be converted into a FASTQ file that may then be processed by the integrated circuit(s) ofthe system, so as to be mapped and/or aligned, or may be transmitted to a remote resource 300 for such processing. Once mapped and/or aligned, the resulting sequence data, e.g., in a SAM file format, may be processed further such as by being ssed one or more times, e.g., into a BAM/CRAM file, which data may then be processed by position sorting, duplicate marking, and/or variant calling, the s of which, e.g., in a VCF format, may then be compressed once more and/or stored and/or transmitted, such as from a remote resource 300 to local 100 resource.

More particularly, the system may be d so as to process BCL data directly, thereby eliminating a FASTQ file sion step. Likewise, the BCL data may be fed directly to the pipeline to produce a unique output VCF file per sample. Intermediate M/CRAM files can also be generated on . The system, therefore, may be configured for receiving and/or transmitting one or more data files, such as a BCL or FASTQ data file containing sequence information, and processing the same so as to e a data file that has been compressed, such as a SAM/BAM/CRAM data file.

Accordingly, as can be seen with respect to A, a user may want to access the compressed file and convert it to an al version of the generated BCL 111 c and/or FASTQ file 11 ld, such as for subjecting the data to further, e.g., more advanced, signal processing 111 b, such as for error correction. Alternatively, the user may access the raw sequence data, e.g., in a BCL or FASTQ file format 111, and subject that data to further processing, such as for mapping 112 and/or aligning 113 and/or other related functions 114/115. For instance, the results data from these ures may then be compressed and/or stored and/or subjected to further processing 114, such as for sorting 114a, de-duplication 114b, recalibration 114c, local realignment 114d, and/or compression/decompression 114e.

The same or r user may then want to access the compressed form ofthe mapped and/or aligned results data and then run another analysis on the data, such as to produce one or more variant calls 115, e.g., via HMM, Smith-Waterman, Conversion, etc., which may then be ssed and/or stored. An additional user of the system may then access the compressed VCF file 116, decompress it, and subject the data to one or more tertiary processing protocols.

Further, a user may want to do a pipeline compare. The mapping/aligning/sorting/variant calling is useful for preforming various genomic analysis.

For ce, if a further DNA or RNA analysis, or some other kind of analysis, is ard desired, a user may want to run the data h another pipeline, and hence having access to the regenerated original data file is very useful. se, this process may be useful such as where a different SAM/BAM/CRAM file may be desired to be created, or recreated, such as where there is a new or different reference genome generated, and hence it may be desired to re-do the mapping and aligning to the new reference genome.

] Storing the compressed SAM/BAM/CRAM files is further useful because it allows a user of the system 1 to take advantage ofthe fact that a reference genome forms the backbone of the results data. In such an instance, it is not the data that agrees with the reference that is ant, but rather how the data disagrees with the reference. Hence, only that data that disagrees with the reference is essential for storage. Consequently, the system 1 can take advantage ofthis fact by storing only what is important and/or useful to the users of the system. Thus, the entire genomic file (showing agreement and disagreement with the reference), or a sub-portion of it (showing only agreement or disagreement with the reference), may be configured for being compressed and stored. It may be seen, ore, that as only the differences and/or variations between the reference and the genome being examined are the most useful to examine, in various embodiments, only these differences need be stored, as anything that is the same as the nce need not be reviewed again. ingly, since any given genome differs only slightly from a reference, e.g., 99% of human genomes are typically identical, after the BAM file is created, it is only the variations between the reference genome that need be reviewed and/or saved.

Additionally, as can be seen with respect to B, another useful component of a cloud ible system 1, provided herein, is a workflow management controller 151, which may be used to te the system flow. Such system animation may e utilizing the various system componentry to access data, either locally 100 or remotely 300, as and/or where it becomes available and then substantially automatically subjecting the data to further processing steps, such with respect to the BioIT nes disclosed herein. Accordingly, the workflow management controller 151 is a core automation technology for directing the various pipelines of the system, e.g., 111, 112, 113, 114, and/or 115, and in various instances may employ an artificial intelligence component 121a.

For instance, the system 1 may include an artificial intelligence (A/I) module that is configured to analyze the various data of the system, and in response thereto to icate its findings with the workflow management system 151. Particular, in various instances, the A/I module may be configured for analyzing the various genomic data presented to the system, as well as the results data that is ted by the processing of that data, so as to identify and determine various onships between that data and/or with any other data that may be entered into the system. More particularly, the A/I module may be configured for ing various genomic data in pondence with a plurality of other s, so as to determine any relationship, e.g., effect based relationships, between the various factors, e.g., data points, which may be informative as to the effects ofthe considered factors on the determined genomic data, e.g., variance data, and vice-versa.

Specifically, as described in r detail below, the A/I module may be configured to correlate the genomics data of a subject generated by the system with any electronic l records, for that t or others, so as to determine any relationships between them and/or any other relevant factors and/or data. Accordingly, such other data that may be used by the system in determining any relevant effects and/or onships that these s may have on a subject and/or their genomic data and/or health include: NIPT data, NICU data, Cancer d data, LDT data, Environmental and/or Ag Bio data, and/or other such data. For instance, further data to be analyzed may be derived by such other factors as environmental data, clad data, microbiom data, methylation data, structural data, e.g., chimeric or mate read data, germline variants data, allele data, RNA data, and other such data related to a subject's genetic material. Hence, the A/I module may be used to link various related data flowing through the system to the variants determined in the genome of one or more subjects along with one or more other possible related effect based factors.

Particularly, the A/I engine may be configured to be run on a CPU/GPU/QPU, and/or it may be configured to be run as an accelerated AI engine, which may be ented in an FPGA and/or Quantum Processing Unit. Specifically, the AI engine may be associated with one or more, e.g., all, ofthe various databases ofthe system, so as to allow the AI engine to explore and s the various data flowing through the .

Additionally, where a subject whose genome is being processed gives the appropriate authorization to access both genomic and patient record data, the system is then configured for correlating the various data sets one with the other, and may further mine the data to determine various significant correspondences, associations, and or relationships.

] More ically, the A/I module may be configured so as to implement a machine learning protocol with t to the input data. For instance, the genomics data of a plurality ofsubjects that is generated from the analyses being performed herein may be stored in a database. Likewise, with the appropriate authorizations and authentications, the Electronic Medical/Health Records (EMR), for the t's whose c DNA has been processed, may be obtained, and may likewise be stored in the database. As described in greater detail below, the processing engine(s) may be configured to analyze the subjects genomic data, as well as their EMR data, so as to determine any correlations n the two. These correlations will then be explored, observed relationships thened, and the results thereof may be used to more effectively and more efficiently perform the various ons ofthe system.

For example, the AI processing engine may access the genomic data of the subject, in correlation with the known diseases or conditions of those subjects, and from this analysis, the AI module may learn to perform tive ations based on that data, so as to become more and more capable of predicting the presence of disease and/or other similar conditions in other individuals. ularly, by determining such correlations between the genomes of others with their EMR, e.g., with respect to the presence of disease markers, the A/I module may learn to identify such correlations, e.g., system determined disease markers, in the genomes of others, thereby being able to predict the possibility of a disease or other identifiable conditions. More particularly, by analyzing a subject's genome in comparison to known or determined genetic disease markers, and/or by determining variance in the subject's genome, and/or further, by determining a potential relationship between the genomic data and the subject'shealth condition, e.g., EMR, the A/I module may be able draw conclusions not only for the subject being sampled, but for others who may be sampled in the future. This can be done, e.g., in a systematic manner, on a subject by subject basis, or may be done within populations and/or within geographically distinct locations.

More particularly, with t to the present s, a pileup of reads is produced. The pileup may overlap regions known to have a higher probability of a significant variance. ingly, the system on one hand will analyze the pileup to determine the ce of variance, while at the same time, based on its us findings, will already know the likelihood that a variance should or should not be there, e.g., it will have an initial prediction as to what the answer should be. Whether or not the expected variance is or is not there will be informative when analyzing that region of the genomes of others. For instance, this may be one data point in a sum of data points being used by the system to make better variant calls, and/or better associating those variants with one or more disease states or other health conditions.

For e, in an ary learning protocol, the A/I analysis may include taking an electronic image of a pileup of one or more regions in a genome, such as for those regions suspected of coding for one or more health conditions, and associating that image with the known variance calls form other pileups, such as where those variance may be known or not known to be related to disease states. This may be done again and again with the system learning to process the ation, make the appropriate associations, and make the correct calls quicker and quicker, and with greater cy. Once this has been performed for various, e.g., all, of the known regions of the genome suspected of causing disease, the same may be repeated for the rest of the genome, e.g., until the whole genome has been reviewed. Likewise, this may be repeated again and again for a plurality of sample genomes, over and over, so as to train the system, e.g., the variant caller, so as to make more accurate calls, sooner, and with greater ency, and/or to allow the tertiary sing module to better identify unhealthy conditions.

Accordingly, the system receives many inputs with known s, performs the analysis and computes the answer, and thereby learns from the process, e.g., renders an image of a pileup, with t to one genome, and then learns to make a call based on another genome, sooner and sooner, as it is more readily determined that future pileups resemble the previously captured images that are known to be related to unhealthy conditions.

Thus, the system may be configured so as to learn to make predictions as to the presence of ts, e.g., based on pattern recognitions, ad/or predicting the relationship between the presence ofthose variance with one or more medical conditions.

More specifically, the more the system performs partial or whole genome analyses, and determines the relationship between variations and various conditions, e.g., in a ity of samples, the better at making predictions, e.g., based on partial or whole genome images of pileups, the system becomes. This is useful when predicting diseased states based on images ofpileups and/or other read analysis, and may include the ng of a correlation between one or more of the EMR (including ypic data), the pileup image, and/or known variants (genotypic data) and/or disease states or ions, e.g., from which the predictions may be made. In various instances, the system may include a ription function, so as to be able to transcribe any of the physical notes that may be a part of the subject'smedical record, so as to include that data within the associations.

In one use model, a subject may have a mobile tracker and/or sensor, such as mobile phone or other computing device, which may be configured for both tracking the location of the t as well as for sensing the environmental and/or physiological conditions of the user at that location. Other sensed data may also be collected. For instance, the mobile computing device may include a GPS r, and/or its location may be determined by triangulation by cellular towers, and may further be configured for transmitting its collected data, e.g., via cellular, WIFI, Bluetooth, or other suitably configured communications protocol. Hence, the mobile device may track and categorize environmental data pertaining to the geographical locations, environmental conditions, physiological status, and other sensed data of the subject owner of the mobile computer encounters in their daily life. The collected location, environmental, physiological, health data, and/or other associated data, e.g., ZNA data, may then be transmitted, e.g., regularly and periodically, to one or more of the system databases herein, wherein the collected ZNA data may be ated with the subject's patient history, e.g., EMR records, and/or their genomic data, as ined by the system .

Likewise, in various instances, one or more of these data may be forwarded from the ZNA tion and analysis rm, to a central repository, e.g., at a government facility, so as to be analyzed on a greater, e.g., nationwide, scale, such as in accordance with the Artificial Intelligence disclosed herein. For instance, the database, e.g., mental controlled database, may have recorded environmental data to which the environmental data of the subject may be compared. For example, in one exemplary instance, a NICU test may be performed on a mother, a father, and their child, and then throughout the lives ofthe three, their environmental and c and medical record data may be continually collected and correlated with one another and/or on or more models, such as over the lifespan of the individuals, especially with respect to the onset of mutations, such as due to environmentally impactful factors. This data collection may be performed over the life of the individual, and may be performed on a family as whole basis, so as to better build a data collection database and to better predict the effects of such factors on genetic variation, and vice versa.

] Accordingly, the workflow management controller 151 allows the system 1 to receive inputs from one or more sources, such as one or multiple sequencing instruments, e.g., 110a, 110b, 110c, etc., and multiple inputs from a single sequencing instrument 110, where the data being received represents the genomes ofmultiple subjects. In such ces, the workflow management controller 151 not only keeps track of all ofthe incoming data, but it also efficiently organizes and facilitates the secondary and/or tertiary processing of the received data. Accordingly, the workflow management controller 151 allows the system 1 to seamlessly connect to both small and large sequencing centers, where all kinds of c material may be coming through one or more cing instruments 110 at the same time, all ofwhich may be erred into the system 1, such as over the cloud 50.

More specifically, as can be seen with respect to A, m vanous instances, one or a multiplicity of samples may be received within the system 1, and hence the system 1 may be configured for receiving and efficiently processing the s, either sequentially or in parallel, such as in a multi sample processing regime. Accordingly, to line and/or automate multi sample processing, the system may be lled by a comprehensive Workflow Management System (WMS) or LIMS atory information management system) 151. The WMS 151 s users to easily schedule multiple ow runs for any pipeline, as well as to adjust or accelerate NGS analysis algorithms, platform pipelines, and their attendant applications.

In such an ce, each run sequence may have a bar code on it indicating the type of sequence it is, the file format, and/or what processing steps have been performed, and what sing steps need to be performed. For instance, the bar code may include a manifest indicating "this is a genome run, ofsubject X, in file format Y, so this data has to go through pipeline Z," or likewise may indicate "this is A's result data that needs to go in this reporting system." Accordingly, as the data is received, processed, and transmitted through the system, the bar codes and results will get loaded into the ow management system 151, such as LIMS (laboratory information management system). LIMS, in this instance, may be a standard tool that is employed for the management of laboratories, or it may be a ically designed tool used for managing process flow.

In any instance, the workflow ment controller 151 tracks a ded sample from when it arrives in a given site, e.g., for storage and/or processing, until the s are sent out to the user. Particularly, the workflow management controller 151 is configured to track all data as it flows through the system end-to-end. More particularly, as the sample comes in, the bar code associated with the sample is read, and based on that reading the system determines what the requested work flows are, and prepares the sample for processing. Such processing may be simple, such as being run through a single genome pipeline, or it may be more complex, such as by being run through multiple, e.g., five pipelines, that need to be stitched er. In one particular model the generated or received data may be run through the system to produce processed data, the processed data may then be run through a GATK equivalent module, the s may be ed, and then the sample may be transmitted to another pipeline for further, e.g., tertiary processing 700. See B.

Hence, the system as a whole can be run in accordance with several different processing pipelines. In fact, many of the system processes can be interconnected, where the workflow manager 151 is notified or otherwise determines that a new job is pending, quantifies the job matrices, identifies available resources for performing the required analyses, loads the job into the system, receives the data coming in, e.g., off the sequencer 110, loads it in, and then processes it. Particularly, once the ow is set up, it can be saved, and then a modified bar code gets ed to that workflow, and the automated process takes place in accordance with the directives ofthe workflow.

Prior to the present automated workflow management system 151, it would take a number of Bioinformaticians a long period of time to configure and set up the system, and its component parts, and it would then require further time for actually running the is. To make matters more complicated, the system would have to be reconfigured prior to receiving the next sample to analyze, requiring even more time to igure the system for analyzing the new sample set. With the technology disclosed herein the system can be entirely automated. The present system, particularly, is configured so as to automatically receive multiple samples, map them to multiple ent workflows and pipelines, and run them on the same or le different system cards.

Accordingly, the workflow management system 151 reads the job requirements of the bar codes, allocates resources for ming the jobs, e.g., less of location, updates the sample barcode, and directs the samples to the ted resources, e.g., sing units, for processing. Hence, it is the workflow manager 151 that ines the secondary 600 and/or tertiary 700 analyses protocols that will be run on the received samples.

These processing units are resources that are ble for ating and performing the operations allocated to each data set. ularly, the work flow controller 151 ls the various ions ated with receiving and reading the sample, ining jobs, allocating resources for the performance ofthose jobs, e.g., ary processing, connecting all system components, and advancing the sample set through the system from component to component. The controller 151, therefore, acts to manage the overall system from start to finish, e.g., from sample receipt to VCF generation, and/or through to tertiary processing, see B.

In onal instances, as can be seen with respect to C, the system 1 may include a further tier of processing modules 800, such as configured for rendering additional processing, e.g., of the secondary and/or tertiary processing results data, such as for diagnosis, disease and/or therapeutic discovery, and/or prophylaxis thereof. For instance, in various instances, an additional layer of processing 800 may be provided, such as for disease diagnostics, therapeutic treatment, and/or prophylactic prevention 70, such as including NIPT 123a, NICU 123b, Cancer 123c, LDT 123d, AgBio 123e, and other such disease diagnostics, prophylaxis, and/or treatments employing the data generated by one or more ofthe present primary and/or secondary and/or tertiary pipelines.

Accordingly, herein presented is a system 1 for producing and using a local 30 and/or global hybrid 50 cloud network. For ce, presently, the local cloud 30 is used primarily for private storage, such as at a remote storage location 400. In such an instance, the computing of data is performed locally 100 by a local computing resource 140, and where storage needs are extensive, the local cloud 30 may be accessed so as to store the data generated by the local computing resource 140, such as by use of a remote private storage resource 400. Hence, generated data is typically managed wholly on site locally 100. In other embodiments, data may be generated, computed, and managed completely offsite by securely connecting to a remote computing resource 300 via a private cloud interface 30.

Particularly, in a general implementation of a bioinformatics analysis platform, the local computing 140 and/or storage 200 functions are maintained locally on site WO 14320 PCT/0S2017/036424 100. However, where storage needs exceed local storage capacity, the data may be uploaded via a local cloud access 30 so as to be stored privately off site 400. Further, where there is a need for stored data 400 to be made available to other remote users, such data may be transferred and made ble via a global cloud 50 interface for remote storage 400 thereby, but for global access. In such an instance, where the computing ces 140 required for performance of the computing functions are minimal, but the storage requirements extensive, the computing function 140 may be maintained y 100, while the storage function 400 may be maintained remotely, e.g., for either private or global access, with the fully processed data being erred back and forth between the local processing function 140, such as for local processing only, and the storage function 400, such as for the remote storage 400 ofthe sed data, such as by employing the JIT protocols sed herein above.

] For instance, this may be exemplified with respect to the sequencing function 110, such as with a typical NGS, where the data generation and/or computing resource 100 is configured for performing the functions required for the sequencing ofthe genetic material so as to produce genetic sequenced data, e.g., reads, which data is produced onsite 100 and/or transferred onsite locally 30. These reads, once generated, such as by the onsite NGS, may then be transferred, e.g., as a BCL or FASTQ file, over the cloud network 30, such as for storage 400 at a remote location 300 in a manner so as to be recalled from the cloud 30 when necessary, such as for further processing. For example, once the sequence data has been ted and stored, e.g., 400, the data may then be ed, e.g. for local usage, such as for the performance of one or more of secondary 600 and/or tertiary 700 processing functions, that is at a location remote from the storage facility 400, e.g., y 100. In such an instance, the local storage resource 200 serves merely as a storage cache where data is placed while waiting transfer to or from the cloud 30/50, such as to or from the remote storage facility 400.

Likewise, where the computing function is extensive, such as requiring one or more remote computing servers or computing cluster cores 300 for processing the data, and where the storage demands for g the processed data 200 are relatively minimal, as compared to the computing resources 300 required to process the data, the data to be sed may be sent, such as over the cloud 30, so as to be processed by a remote computing resource 300, which resource may include one or more cores or clusters of computing resources, e.g., one or more super computing ces. In such an instance, once the data has been processed by the cloud based computer core 300, the processed data may then be transferred over the cloud network 30 so as to be stored locally 200 and made readily available for use by the local computing resource 140, such as for local is and/or diagnostics. Ofcourse, the remotely generated data 300 may also be stored remotely 400.

This may further be exemplified with respect to a typical ary processing function 600, such as where the pre-processed sequenced data, e.g., read data, is stored locally 200, and is accessed, such as by the local computing resource 100, and transmitted over the cloud internet 30 to a remote computing facility 300 so as to be r sed thereby, e.g., in a secondary 600 or tertiary 700 processing on, to obtain processed s data that may then be sent back to the local facility 100 for storage 200 thereby. This may be the case where a local practitioner generates sequenced read data using a local data ting resource 110, e.g., automated sequencer, so as to produce a BCL or FASTQ file, and then sends that data over the network 50 to a remote computing facility 300, which then runs one or more ons on that data, such as a Burrows-Wheeler transform or Needlemen- Wunsch and/or Smith-Waterman ent function on that sequence data, so as to generate results data, e.g., in a SAM file format, that may then be compressed and transmitted over the internet 30/50, e.g., as a BAM file, to the local ing resource 100 so as to be examined thereby in one or more local administered processing protocols, such as for producing a VCF, which may then be stored locally 200. In various instances the data may also be stored remotely 400.

What is needed, however, is a seamless integration between the engagement between local 100 and remote 300 computer processing as well as between local 200 and remote 400 storage, such as in the hybrid cloud 50 based system ted herein. In such an instance, the system can be configured such that local 100 and remote 300 computing resources are configured so as to run seamlessly together, such that data to be processed thereby can be allocated real time to either the local 200 or the remote 300 computing resource without paying an extensive penalty due to transfer rate and/or in operational efficiency. This may be the case, for instance, where the software and/or hardware and/or quantum processing to be deployed or otherwise run by the computing resources 100 and 300 are configured so as to correspond to one r and/or are the same or functionally similar, e.g., the hardware and/or software is configured in the same manner so as to run the same algorithms in the same manner on the generated and/or received data.

For instance, as can be seen with respect to A a local computing resource 100 may be configured for ting or for receiving generated data, and therefore may include a data generating mechanism 110, such as for primary data generation and/or analysis 500, e.g., so as to produce a BCL and/or a FASTQ sequence file. This data generating mechanism 110 may be or may be associated with a local computer 100, as described herein throughout, having a processor 140 that may be configured to run one or more software applications and/or may be hardwired so as to perform one or more algorithms such as in a wired configuration on the generated and/or acquired data. For example, the data generating mechanism 110 may be ured for one or more of ting data, such as sequencing data 111. In various ments, the generated data may be sensed data 111 a, such as data that is detectable as a change in voltage, ion concentration, electromagnetic radiation, and the like; and/or the data ting mechanism 110 may be configured for generating and/or processing signal, e.g., analog or digital signal data, such as data representing one or more nucleotide identities in a sequence or chain of ated nucleotides. In such an instance, the data generating mechanism 110, e.g., sequencer 111, may r be configured for performing preliminarily processing on the generated data so as for signal processing 111 b or to perform one or more base call operations 111 c, such as on the data so as to produce sequence identity data, e.g., a BCL and/or FASTQ file 11 ld.

It is to be noted that in this instance, the produced data 111 may be generated locally and directly, such as by a local data generating 110 and/or computing ce 140, e.g., an NGS or sequencer on a chip. Alternatively, the data may be produced locally and indirectly, e.g., by a remote computing and/or generating resource, such as a remote NGS.

The data 111, e.g., in BCL and/or FASTQ file format, once produced may then be transferred indirectly over the local cloud 30 to the local ing resource 100 such as for secondary processing 140 and/or storage thereby in a local storage resource 200, such as while awaiting further local processing 140. In such an instance, where the data tion resource is remote from the local processing 100 and/or e 200 resources, the corresponding resources may be configured such that the remote and/or local storage, remote and local processing, and/or icating protocols ed by each resource may be adapted to smoothly and/or seamlessly integrate with one another, e.g., by running the same, similar, and/or equivalent software and/or by having the same, similar, and/or equivalent hardware configurations, and/or employing the same communications and/or er protocols, which, in some instances, may have been implemented at the time ofmanufacture or later thereto. ically, in one implementation, these functions may be implemented in a hardwired configuration such as where the sequencing function and the secondary processing function are maintained upon the same or associated chip or chipset, e.g., such as where the sequencer and secondary processor are directly interconnected on a chip, as herein described.

In other implementations, these functions may be implemented on two or more te devices via software, e.g., on a quantum processor, CPU, or GPU that has been optimized to allow the two remote devices to communicate seamlessly with one another. In other implementations, a combination of optimized hardware and software implementations for performing the recited ons may also be employed.

More specifically, the same configurations may be implemented with respect to the performance of the mapping, aligning, g, variant calling, and/or other functions that may be deployed by the local 100 and/or remote 300 computing resources. For example, the local computing 100 and/or remote 300 resources may e software and/or hardware configured for performing one or more secondary 600 tiers ofprocessing functions 112-115, and/or or ry tiers 700/800 of processing functions, on y and/or remotely ted data, such as genetic sequence data, in a manner that the processing and s thereof may be seamlessly shared with one another and/or stored thereby. Particularly, the local computing function 100 and/or the remote computing function 300 may be ured for generating and/or receiving primary data, such as genetic sequence data, e.g., in a BCL and/or a FASTQ file , and running one or more secondary 600 and/or tertiary 700 processing protocols on that ted and/or acquired data. In such an instance, one or more of these protocols may be implemented in a software, hardware, or combinational format, such as run on a quantum processor, a CPU, and/or a GPU. For ce, the data generating 110 and/or the local 100 and/or the remote 300 processing resource may be configured for performing one or more of a mapping operation 112, an alignment ion 113, variant calling 115, or other related function 114 on the acquired or generated data in software and/or in hardware.

] Accordingly, in various embodiments, the data generating resource, such as the sequencer 111, e.g., NGS or sequencer on a chip, whether implemented in software and/or in hardware, or a combination of the same, may further be configured to include an initial tier of processors 500 such as a scheduler, various analytics, comparers, graphers, releasers, and the like, so as to assist the data generator 111, e.g., sequencer, in converting biological information into raw read data, such as in a BCL or FASTQ file format 111 d.

Further, the local computing 100 resource, whether implemented in software and/or in hardware, or a combination ofthe same, may further be ured to include a further tier of processors 600 such as may include a mappmg engine 112, or may otherwise include programming for running a mapping algorithm on the genetic sequence data, such as for performing a Burrows-Wheeler transform and/or other algorithms for building a hash table and/or running a hash function 112a on said data, such as for hash seed mapping, so as to generate mapped ce data. Further still, the local computing 100 resource whether implemented in software and/or in hardware, or a combination of the same, may further be configured to include an l tier of processors 600 such as may also include an ent engine 113, as herein described, or may otherwise include programming for running an alignment algorithm on the genetic sequence data, e.g., mapped sequenced data, such as for performing a gapped and/or gapless Smith-Waterman alignment, and/or Needleman-Wunsch, or other like scoring algorithm 113a on said data, so as to generate aligned sequence data.

The local computing 100 and/or data generating ce 110 may also be configured to include one or more other modules 114, whether implemented in software and/or in hardware, or a combination of the same, which may be adapted to perform one or more other processing functions on the genetic sequence data, such as on the mapped and/or aligned sequence data. Thus, the one or more other s may include a suitably configured engine 114, or ise include programming, for running the one or more other sing functions such as a sorting 114a, de-duplication 114b, bration 114c, local realignment 114d, duplicate marking 114f, Base Quality Score Recalibration 114g function(s) and/or a compression function (such as to produce a SAM, Reduced BAM, and/or a CRAM ssion and/or decompression file) 114e, in accordance with the methods herein described. In various instances, one or more of these processing functions may be configured as one or more nes ofthe system 1.

Likewise, the system 1 may be configured to include a module 115, whether implemented in software and/or in hardware, or a combination of the same, which may be adapted for processing the data, e.g., the sequenced, mapped, aligned, and/or sorted data in a manner such as to e a variant call file 116. Particularly, the system 1 may include a variant call module 115 for running one or more variant call functions, such as a Hidden Markov Model (HMM) and/or GATK function 115a such as in a wired configuration and/or via one or more software applications, e.g., either locally or remotely, and/or a converter 115b for the same. In various instances, this module may be configured as one or more pipelines ofthe system 1.

] In particular embodiments, as set forth in B, the system 1 may include a local computing function 100 that may be configured for ing a computer processing resource 150 for performing one or more further processing ons on data, e.g., BCL and/or FASTQ data, generated by the system data generator 110 or acquired by the system acquisition mechanism 120 (as described herein), such as by being transferred thereto, for instance, by a third party 121, such as via a cloud 30 or hybrid cloud network 50. For example, a third-party analyzer 121 may deploy a remote ing ce 300 so as to generate relevant data in need offurther processing, such as genetic sequence data or the like, which data may be communicated to the system 1 over the network 30/50 so as to be r processed. This may be , for instance, where the remote computing resource 300 is a NGS, ured for taking raw biological data and converting it to a digital representation thereof, such as in the form of one or more FASTQ files containing reads ofgenetic ce data; and where further sing is desired, such as to determine how the generated sequence of an individual differs from that of one or more reference sequences, as herein described, and/or it is desired to subject the results thereof to furthered, e.g., ry, processmg.

] In such an instance, the system 1 may be adapted so as to allow one or more parties, e.g., a primary and/or secondary and/or third party user, to access the associated local processing resources 100, and/or a suitably configured remote processing resource 300 associated therewith, in a manner so as to allow the user to perform one or more quantitative and/or qualitative processing functions 152 on the generated and/or acquired data. For instance, in one configuration, the system 1 may include, e.g., in addition to primary 500 and/or secondary 600 processing pipelines, a third tier of processing modules 700/800, which processing modules may be configured for performing one or more processing functions on the ted and/or acquired primary and/or secondary processed data.

Particularly, in one embodiment, the system 1 may be configured for generating and/or receiving processed genetic sequence data 111 that has been either remotely or locally mapped 112, d 113, sorted 114a, and/or r processed 114 so as to generate a variant call file 116, which variant call file may then be subjected to further processing such as within the system 1, such as in response to a second and/or third party analytics requests 121. More particularly, the system 1 may be configured to receive processing requests from a third party 121, and further be configured for performing such requested secondary 600 and/or tertiary processing 700/800 on the generated and/or acquired data. Specifically, the system 1 may be configured for producing and/or acquiring genetic sequence data 111, may be configured for taking that genetic sequence data and mapping 112, aligning 113, and/or g 114a it and processing it to produce one or more variant call files (VCFs) 116, and additionally the system 1 may be configured for performing a tertiary sing function 700/800 on the data, e.g., with respect to the one or more VCFs generated or received by the system 1.

Particularly, the system 1 may be configured so as to perform any form of tertiary processing 700 on the generated and/or acquired data, such as by subjecting it to one or more ne sing functions 700 such as to generate genome, e.g., whole genome, data 122a, epigenome data 122b, metagenome data 122c, and the like, including genotyping, e.g., joint genotyping, data 122d, variants analyses data, including GATK 122e and/or MuTect2 122f analysis data, among other potential data analytic pipelines, such as a microarray analysis pipeline, exome analysis pipeline, microbiome analysis pipeline, RNA sequencing pipelines, and other genetic analyses pipelines. r, the system 1 may be configured for ming an additional tier of processing 800 on the ted and/or processed data, such as including one or more of non-invasive prenatal testing (NIPT) 123a, NIP ICU 123b, cancer related diagnostics and/or therapeutic modalities123c, various laboratory developed tests (LDT) 123d, agricultural biological (Ag Bio) applications 123e, or other such health care d 123fprocessing function. See C.

] Hence, in various embodiments, where a primary user may access and/or ure the system 1 and its various components directly, such as through direct access therewith, such as through the local computing resource 100, as presented herein, the system 1 may also be d for being accessed by a secondary party, such as is connected to the system 1 via a local network or intranet connection 10 so as to configure and run the system 1 within the local environment. onally, in certain embodiments, the system may be adapted for being accessed and/or configured by a third party 121, such as over an associated hybrid-cloud network 50 connecting the third party 121 to the system 1, such as through an application program interface (API), accessible as through one or more graphical user interface (GUI) components. Such a GUI may be ured to allow the party user to access the system 1, and using the API to configure the various components of the system, the modules, associated pipelines, and other associated data generating and/or processing functionalities so as to run only those system ents necessary and/or useful to the third party and/or requested or desired to be run thereby.

Accordingly, in various instances, the system 1 as herein presented may be adapted so as to be configurable by a primary, secondary, or tertiary user of the system. In such an instance, the system 1 may be d to allow the user to configure the system 1 and thereby to arrange its components in such a manner as to deploy one, all, or a ion ofthe analytical system resources, e.g., 152, to be run on data that is either generated, acquired, or otherwise transferred to the system, e.g., by the primary, secondary, or third party user, such that the system 1 runs only those portions of the system necessary or useful for running the analytics requested by the user to obtain the desired results thereof. For example, for these and other such purposes, an API may be included within the system 1 wherein the API is configured so as to include or otherwise be operably associated with a cal user interface (GUI) including an operable menu and/or a related list of system function calls from which the user can select and/or otherwise make so as to configure and operate the system and its components as desired.

In such an instance, the GUI menu and/or system function calls may direct the user selectable operations of one or more of a first tier of operations 600 including: sequencing 111, g 112, aligning 113, sorting 114a, variant calling 115, and/or other associated functions 114 in accordance with the teachings herein, such as with relation to the primary and/or secondary processing functions herein described. Further, where d the GUI menu and/or system function calls may direct the operations of one or more of a second tier of operations 700 including: a genome, e.g., whole genome, analysis pipeline 122a, epigenome pipeline 122b, metagenome pipeline 122c, a genotyping, e.g., joint, genotyping pipeline 122d, variants pipelines, e.g., GATK 122e and/or MuTect2 122f analysis pipelines, including structural variants pipelines, as well as other ry analyses pipelines, such as a micro-array analysis pipeline, exome analysis ne, iome analysis pipeline, RNA sequencing pipelines, and other genetic analyses pipelines. Furthermore, where desired the GUI menu and system function calls may direct the user selectable ions of one or more of a third tier of operations 800 including: non-invasive prenatal testing (NIPT) 123a, NIP ICU 123b, cancer related diagnostics and/or therapeutic modalities 123c, various laboratory developed tests (LDT) 123d, agricultural biological (Ag Bio) applications 123e, or other such health care related 123fprocessing functions.

Accordingly, the menu and system function calls may e one or more primary, secondary, and/or tertiary sing functions, so as to allow the system and/or its ent parts to be configured such as with respect to performing one or more data analysis pipelines as selected and ured by the user. In such an instance, the local computing resource 100 may be ured to correspond to and/or mirror the remote ing resource 300, and/or se the local storage resource 200 may be configured to correspond and/or mirror the remote storage resource 400 so that the various components of the system may be run and/or the data generated thereby may be stored either locally or remotely in a seamless distributed manner as chosen by the use ofthe system 1. Additionally, in particular embodiments, the system 1 may be made accessible to third parties, for running proprietary analysis protocols 121a on the generated and/or processed data, such as by running through an artificial intelligence interface designed to find ations there The system 1 may be ured so as to perform any form of tertiary processing on the generated and/or acquired data. Hence, in various embodiments, a primary, secondary, or tertiary user may access and/or configure any level of the system 1 and its various components either directly, such as through direct access with the computing resource 100, indirectly, such as via a local network connection 30, or over an associated hybrid-cloud network 50 connecting the party to the system 1, such as through an riately configured API having the appropriate permissions. In such an instance, the system components may be presented as a menu, such as a GUI selectable menu, where the user can select from all the various processing and storage options desired to be run on the user presented data. r, in various instances, the user may upload their own system protocols so as to be adopted and run by the system so as to process various data in a manner designed and ed for by the user. In such an instance, the GUI and associated API will allow the user to access the system 1 and using the API add to and configure the various components of the system, the modules, associated pipelines, and other associated data generating and/or sing functionalities so as to run only those system components necessary and/or useful to the party and/or requested or desired to be run thereby.

With respect to C, one or more ofthe above ated modules, and their respective functions and/or ated resources, may be configured for being performed remotely, such as by a remote computing resource 300, and further be adapted to be transmitted to the system 1, such as in a ss transfer protocol over a global cloud based internet connection 50, such as via a ly configured data acquisition mechanism 120.

Accordingly, in such an instance, a local computing resource 100 may include a data acquisition mechanism 120, such as ured for transmitting and/or receiving such acquired data and/or associated information.

For instance, the system 1 may include a data acquisition mechanism 120 that is configured in a manner so as to allow the continued processing and/or storage of data to take place in a seamless and steady manner, such as over a cloud based network 50 where the processing functions are distributed both locally 100 and/or remotely 300. Likewise, where one or more ofthe results ofsuch processing may be stored locally 200 and/or remotely 400, such that the system ssly allocates to which local or remote resource a given job is to be sent for processing and/or storage regardless of where the ce is physically positioned. Such distributed processing, transferring, and ition may include one or more of cing 111, mapping 112, aligning 113, sorting 114a, duplicate marking 114c, deduplication, recalibration 114d, local realignment 114e, Base y Score Recalibration 114f function(s) and/or a ssion function 114g, as well as a variant call function 116, as herein described. Where stored locally 200 or remotely 400, the processed data, in whatever state it is in in the process may be made available to either the local 100 or remote processing 300 resources, such as for further sing prior to re-transmission and/or re-storage.

Specifically, the system 1 may be configured for ing and/or acquiring genetic sequence data 111, may be configured for taking that genetic sequence data and processing it locally 140, or transferring the data over a suitably configured cloud 30 or hybrid cloud 50 network such as to a remote processing facility for remote sing 300.

Further, once processed the system 1 may be configured for storing the processed data remotely 400 or transferring it back for local e 200. Accordingly, the system 1 may be configured for either local or remote generation and/or sing of data, such as where the generation and/or processing steps may be from a first tier of primary and/or secondary processing functions 600, which tier may include one or more of: cing 111, mapping 112, aligning 113, and/or g 114a so as to produce one or more t call files (VCFs) Further, the system 1 may be configured for either local or remote generation and/or processing of data, such as where the generation and/or processing steps may be from a second tier of tertiary processing functions 700, which tier may include one or more of generating and/or acquiring data pursuant to a genome pipeline 122a, epigenome pipeline 122b, metagenome pipeline 122c, a genotyping pipeline 122d, variants, e.g., GATK 122e and/or MuTect2, analysis 122fpipeline, as well as other tertiary analyses nes, such as a micro-array analysis pipeline, a microbiome analysis pipeline, an exome analysis pipeline, as well as RNA sequencing pipelines and other genetic analyses pipelines. Additionally, the system 1 may be configured for either local or remote generation and/or processing of data, such as where the generation and/or processing steps may be from a third tier of tertiary processing functions 800, which tier may include one or more of generating and/or acquiring data related to and including: non-invasive prenatal testing (NIPT) 123a, NIP ICU 123b, cancer related diagnostics and/or therapeutic modalities 123c, s laboratory developed tests (LDT) 123d, agricultural biological (Ag Bio) applications 123e, or other such health care related ocessing functions.

In particular embodiments, as set forth in C, the system 1 may further be configured for allowing one or more parties to access the system and transfer ation to or from the associated local processing 100 and/or remote 300 processing resources as well as to store ation either locally 200 or remotely 400 in a manner that allows the user to choose what ation get processed and/or stored where on the system 1. In such an instance, a user can not only decide what primary, secondary, and/or tertiary processing functions get performed on generated and/or acquired data, but also how those resources get deployed, and/or where the results of such sing gets stored. For instance, in one configuration, the user may select whether data is generated either locally or remotely, or a combination thereof, whether it is subjected to secondary processing, and if so, which modules ofsecondary processing it is subjected to, and/or which resource runs which ofthose processes, and further may determine whether the then generated or acquired data is r subjected to tertiary processing, and if so, which modules and/or which tiers of tertiary processing it is ted to, and/or which resource runs which of those processes, and se, where the s ofthose ses are stored for each step ofthe ions.

Particularly, in one embodiment, the user may configure the system 1 of A so that the generating of genetic ce data 111 takes place remotely, such as by an NGS, but the secondary processing 600 of the data occurs locally 100. In such an instance, the user can then determine which of the secondary processing functions occur locally 100, such as by selecting the processing ons, such as mapping 112, aligning 113, sorting 111, and/or producing a VCF 116, from a menu of available processing options. The user may then select whether the locally processed data is subjected to tertiary processing, and if so which modules are activated so as to further process the data, and whether such tertiary processing occurs locally 100 or remotely 300. Likewise, the user can select various options for the various tiers of tertiary sing options, and where any generated and/or acquired data is to be stored, either locally 200 or remotely 400, at any given step or time ofoperation.

More particularly, a pnmary user may configure the system to receive processing requests from a third party, where the third party may configure the system for performing such requested primary, secondary, and/or tertiary processing on generated and/or acquired data. Specifically, the user or second and/or third party may configure the system 1 for producing and/or ing genetic sequence data, either locally 100 or remotely 200.

Additionally, the user may ure the system 1 for taking that genetic sequence data and mapping, aligning, and/or sorting it, either locally or remotely, so as to produce one or more variant call files (VCFs). Additionally, the user may configure the system for performing a ry processing function on the data, e.g., with respect to the one or more VCFs, either locally or remotely.

More particular still, the user or other party may configure the system 1 so as to perform any form of tertiary processing on the generated and/or acquired data, and where that processing is to occur in the system. Hence, in various embodiments, the first, second, and/or third party 121 user may access and/or configure the system 1 and its various components ly such as by directly accessing the local computing on 100, via a local network connection 30, or over an associated hybrid-cloud network 50 connecting the party 121 to the system 1, such as through an application program interface (API), accessible as through one or more graphical user interface (GUI) components. In such an instance, the third party user may access the system 1 and use the API to configure the various components of the system, the modules, associated pipelines, and other associated data generating and/or processing functionalities so as to run only those system components necessary and/or useful to the third party and/or requested or desired to be run thereby, and further allocate which computing resources will e the ted processing, and where the results data will be stored.

Accordingly, in various instances, the system 1 may be configurable by a y, secondary, or ry user of the system who can configure the system 1 so as to arrange its components in such a manner as to deploy one, all, or a selection of the analytical system resources to be run on data that the user either ly generates, causes to be ted by the system 1, or causes to be transferred to the system 1, such as over a network ated therewith, such as via the data acquisition mechanism 120. In such a , the system 1 is configurable so as to only run those portions ofthe system necessary or useful for the analytics desired and/or requested by the requesting party. For example, for these and other such purposes, an API may be included wherein the API is configured so as to include a GUI operable menu and/or a related list of system function calls that from which the user can select so as to configure and operate the system as desired.

Additionally, in particular embodiments, the system 1 may be made accessible to a primary user and/or third parties, such as governmental regulators, such as the Federal Drug stration (FDA) 70b, or allow primary users and/or third s to collate, compile, and/or access a data base of genetic ation derived or otherwise acquired and/or ed by the system 1 so as to form an electronic medical records (EMR) database 70a and/or to allow governmental access and/or oversight ofthe system, such as the FDA for Drug Development Evaluation. The system 1 may also be set up to conglomerate, compile, and/or annotate the data 70c and/or allow other high level users access thereto.

Accordingly, the system 1, and or its components, may be configured for being accessed by a remote user, such as a primary user or third party, and therefore, one or more of the computer ces 100 and/or 300 may include a user interface, and/or may further include a display device having a graphic user interface for allowing a potential user of the system to access the system so as to transmit sample data for entry into one or more of the BioIT pipelines disclosed herein, and/or for receiving s data rom. The GUI or other interface may be configured for allowing the user to manage the system components, e.g., via a suitably configured web portal, and to track sample sing progress, regardless of whether the computing resources to be engaged are available locally 100 or remotely 300.

Accordingly, the GUI may list a set of jobs that may be performed, e.g., mapping 112, ng 113, etc., and/or a set of resources for performing the jobs, and the user may selfselect which jobs they want to run and by which resources. Hence, in an instance such as this, each individual user may build thereon a unique, or may use a predetermined, analysis workflow, such as by clicking on, dragging, or otherwise selecting the particular work projects they desire to be run.

For instance, in one use model, a dashboard is presented with a GUI interface that may include a ity of icons representing the various processes that may be implemented and run on the system. In such an instance, a user can click on or drag the selected work process icons into a workflow interface, so as to build a desired workflow process, which once built may be saved and used to establish the control instructions for the sample set barcodes. Once the desired work projects have been ed, the work flow management controller 151 may ure the d workflow processes (e.g., ary analysis), and then identify and select the resources for performing the selected analysis.

Once the workflow analysis process begins, the dashboard may be viewed so as to track progress h the system. For example, the dashboard may indicate how much data is running through the system, what ses are being run on the data, how much has been accomplished, how much processing remains, what workflows have been completed, and which still need to be accessed, the latest projects to be run, and which runs have been completed. Essentially, full access to everything that's running on the system, or a tion thereof, may be provided to the desktop.

] Further, in various instances, the desktop may include various different user interfaces that may be accessible via one or more tabs. For ce, one tab for accessing the system controls may be a "local resources 100 tab," which when ed allows a user to select control functions that are capable of being implemented locally. Another tab may be configured for accessing "cloud resources 300," which when ed allows a user to select other control functions that are capable of being implemented remotely. Accordingly, in interacting with the dashboard, a user can select which ces to perform which tasks, and as such can increase or decrease resource usage as required so as to meet the project requirements.

Hence, as the computational xity increases, and/or increased speed is desired, the user (or the system itself, e.g., WMS 151) can bring more and more resources online, as needed, such as by the mere click of a button, cting the workflow manager to bring additional local 100 and/or cloud based 300 resources online, as needed to te the task within the desired timeframe. In this manner, although the system is automated and/or lled by the workflow manager controller 151, a user of the system can still set the control parameters, and when needed can bring cloud based resources 300 on line.

Accordingly, the controller 151 can expand to the cloud 50/300 as needed to bring on line additional processing and/or storage ces 400.

In various instances, the desktop interface may be configured as a mobile application or "app" that is accessible via a mobile device and/or desktop computer.

Consequently, in one aspect, a genomics market place, or cohort, may be provided so as to allow a plurality of users to collaborate in one or more ch projects, so as to form an electronic cohort market place that is accessible via the dashboard app, e.g., a web based browser interface. As such, the system may provide an online forum for performing collaborative research and/or a market place for developing various analytical tools for analyzing c data, which system may be accessible directly via the system interface, or via the app, to allow remote control ofthe system by a user.

Accordingly, in various embodiments, as can be seen with respect to A, a hybrid cloud 50 is provided wherein the hybrid cloud is configured for connecting a local computing 100 and/or storage resource 200 with a remote ing 300 and/or storage 400 resource, such as where the local and remote resources are separated one from the other distally, spatially, geographically, and the like. In such an instance, the local and distal resources may be configured for communicating with one r in a manner so as to share information, such as digital data, seamlessly between the two. Particularly, the local resources may be configured for performing one or more types ofprocessing on the data, such as prior to transmission across the hybrid network 50, and the remote resources may be configured for performing one or more types of further processing ofthe data.

For instance, in one particular configuration, the system 1 may be ured such that a generating and/or ing function 152 is configured for being performed locally 100 by a local ing resource, such as for the purpose of performing a primary and/or secondary processing function, so as to generate and/or process genetic sequence data, as herein described. Additionally, in various embodiments, the local resources may be configured for performing one or more tertiary processing functions on the data, such as one or more ofgenome, exome, and/or epigenome analysis, or a cancer, microbiome, and/or other DNA/RNA processing analysis. r, where such processed data is meant to be transferred, such as to a remote ing 300 and/or storage 400 resource, the data may be transformed such as by a suitably configured transformer, which transformer may be configured for indexing, converting, compressing, and/or encrypting the data, such as prior to transfer over the hybrid network 50.

In particular instances, such as where the generated and processed data is transferred to a remote computing resource, e.g., server 300, for further processing, such processing may be of a global nature and may include ing data from a plurality of local computing resources 100, ing such pluralities of data, ting the data, and comparing the same, such as to interpret the data, determine trends thereof, analyzing the same for s biomarkers, and aiding in the development of diagnostics, therapeutics, and/or prophylactics. Accordingly, in various instances, the remote ing resource 300 may be configured as a data sing hub, such as where data from a variety of sources may be transferred, processed, and/or stored while waiting to be transformed and/or transferred, such as by being accessed by the local ing resource 100. More particularly, the remote processing hub 300 may be configured for receiving data from a plurality of resources 100, processing the same, and distributing the processed data back to the variety oflocal resources 100 so as to allow for collaboration amongst chers and/or resources 100. Such collaboration may include various data sharing protocols, and may additionally include preparing the data to be transferred, such as by allowing a user of the system 1 to select amongst various ty protocols and/or privacy settings so as to control how the data will be prepared for transfer.

In one particular instance, as presented in B, a local computing 100 and/or storage 200 resource is provided, such as e at a user's location. The computing resource 100 and/or storage 200 resource may be coupled to a data ting resource 121, such as an NGS or sequencer on a chip, as herein described, such as over a direct or an intranet connection 10, where the sequencer 121 is configured for ting genetic sequencing data, such as BCL and/or FASTQ files. For instance, the sequencer 121 may be part of and/or housed in the same apparatus as that of the computing resource 100 and/or storage unit 200, so as to have a direct communicable and/or operable connection therewith, or the sequencer 121 and computing resource 100 and/or e resource 200 may be part of separate apparatuses from one r, but housed in the same facility, and thus connected over a cabled or intranet 10 connection. In some instances, the sequencer 121 may be housed in a separate facility than that of the computing 100 and/or storage 200 resource and thus may be connected over an internet 30 or hybrid cloud connection 50.

In such instances, the genetic sequence data may be processed 100 and stored locally 200, prior to being transformed, by a suitably ured transformer, or the generated sequence data may be itted ly to one or more of the transformer and/or analyzer 152, such as over a suitably configured local connection 10, intranet 30, or hybrid cloud connection 50, as described above such as prior to being sed locally. Particularly, like the data generating resource 121, the transformer 151 and/or analyzer 152 may be part of and/or housed in the same apparatus as that ofthe computing resource 100 and/or storage unit 200, so as to have a direct communicable and/or operable connection therewith, or the ormer and/or analyzer 152 and computing resource 100 and/or storage resource 200 may be part of separate apparatuses from one another, but housed in the same facility, and thus connected over a cabled or et 10 connection. In some instances, the transformer 151 and/or analyzer 152 may be housed in a separate facility than that of the computing 100 and/or storage 200 resource and thus may be ted over an internet 30 or hybrid cloud connection 50.

For instance, the transformer may be configured for preparing the data to be transmitted either prior to analysis or post analysis, such as by a suitably configured computing resource 100 and/or analyzer 152. For instance, the analyzer 152 may perform a secondary and/or tertiary processing function on the data, as herein described, such as for analyzing the generated sequence data with respect to determining its genomic and/or exomic characteristics 152a, its epigenomic features 152b, any various DNA and/or RNA markers of interests and/or indicators of cancer 152c, and its relationships to one or more microbiomes 152d, as well as one or more other secondary and/or tertiary processes as described herein.

As ted, the generated and/or processed data may be transformed, such as by a suitably configured transformer such as prior to transmission hout the system 1 from one component f to another, such as over a direct, local 10, internet 30, or hybrid cloud 50 connection. Such transformation may include one or more of conversion 151 d, such as where the data is converted from one form to another; comprehension 151 c, ing the coding, decoding, and/or otherwise taking data from an incomprehensible form and transforming it to a comprehensible form, or from one comprehensible form to r; indexing 151 b, such as including compiling and/or collating the generated data from one or more resources, and making it locatable and/or searchable, such as via a generated index; and/or encryption 151 a, such as ng a lockable and able, password protected dataset, such as prior to transmission over an internet 30 and/or hybrid cloud 50.

] Hence, as can be seen with t to C, in these and/other such instances, the hybrid cloud 50 may be configured for allowing seamless and protected transmission of data throughout the components of the system, such as where the hybrid cloud 50 is adapted to allow the various users ofthe system to configure its component parts and/or the system itself so as to meet the research, diagnostic, therapeutic and/or prophylactic discovery and/or development needs of the user. Particularly, the hybrid cloud 50 and/or the various ents of the system 1 may be operably connected with compatible and/or corresponding API aces that are adapted to allow a user to ly configure the various components of the system 1 so as to deploy the resources desired in the manner desired, and further to do so either locally, remotely, or a combination of the same, such as based on the demands of the system and the particulars of the analyses being performed, all the while being enabled to communicate in a secured, encryptable environment.

WO 14320 PCT/0S2017/036424 In particular instances, the system 1 may include a processing architecture 310, such as an interpreter, that is configured for performing an interpreting function 310.

The interpreter 310 may perform one or a series of ic functions on generated data, such as tion 311, interpretation 312, diagnostics 313, and/or a detection and/or an analysis function for ining the presence of one or more biomarkers, such as in the genetic data.

The interpreter 313 may be part ofor te from the local computing resource 100, such as where the interpreter 310 is coupled to the computing resource 100 via a cloud interface, such as a hybrid cloud 50.

Further an onal processing architecture 320 may be included, such as where the architecture 320 is configured as a collaborator. The collaborator 320 may be configured for performing one or more functions directed to ensuring the security and/or privacy of data to be transmitted. For instance, the collaborator may be configured for securing the data sharing process 321, for ensuring the privacy of transmission 322, setting control parameters 323, and/or for initiating a security protocol 324. The collaborator 313 is configured for ng for the sharing of data, such as for facilitating the collaboration of processing, as such the collaborator 320 may be part of or separate from the local computing resource 100, such as where the collaborator 320 is coupled to the computing ce 100 via a cloud interface, such as a hybrid cloud 50. The interpreter 310, collaborator 320, and/or the local computing resource 100 may further be coupled to a remote ing resource 300, such as for enhancing system efficiency by offloading computing 300 and/or storage 400 ons into the cloud 50. In various instance, the system 1 may be configured for allowing secure third party analysis 121 to take place, such as where the third party can connect with and engage the system such as through a ly configured APL As can be seen with respect to , the system 1 may be a multi-tiered and/or multiplexed bioanalytical processing platform that includes layers of data generating and/or data processing units each having one or more processing pipelines that may be deployed in a systematic and concurrent or sequential manner so as to process genetic information from its primary processing stage to a secondary and/or ry processing stage.

Particularly, presented herein are devices ured for performing bioanalysis in one or more of re and/or software and/or quantum processing implementations, as well as methods of their use, and systems including the same. For instance, in one embodiment, a genomics processing platform may be provided and configured as a multiplicity of integrated circuits, which integrated ts may be adapted as, or otherwise be included within, one or more of a central or cs processing unit, such as a general purpose CPU and/or GPU, a hardwired implementation, and/or a quantum processing unit. ularly, in various embodiments, one or more pipelines of the genomics processing platform may be configured by one or more integrated and/or quantum circuits of a quantum processing unit.

Accordingly, the platforms herein presented may be configured so as to harnesses the tremendous power of zed software and/or hardware and/or quantum processing implementations for the performance of the various genetic sequencing and/or secondary and/or tertiary processing functions, herein disclosed, which may be run on one or more integrated ts. Such integrated circuits may be ssly coupled together and may further be seamlessly coupled to various other integrated circuits, e.g., CPUs and/or GPUs and/or QPUs, ofthe system that are configured for running the various software and/or hardwired based applications oftertiary bioanlytical functions.

Particularly, in various embodiments, these processes may be performed by optimized re run on a CPU, GPU, and/or QPU, and/or may be implemented as a firmware configured integrated circuit, e.g., an FPGA, which may be part ofthe same device or separate devices that may be positioned on the same motherboard, different PCie cards within the same device, separate devices in the same ty, and/or located at different facilities. Accordingly, the one or more sing units and/or integrated circuits may be directly coupled together, e.g., tightly, such as by being physically incorporated into the same mother board, or separate mother boards oned within the same housing and/or otherwise coupled together, or they may be positioned on separate motherboards or pCIE cards that are capable of icating with one r remotely, such as wirelessly and/or via a networked interface, such as via a local cloud 30, and in various embodiments the one or more processing units and/or ated circuits may be positioned geographically remotely from one another but communicable via a hybrid cloud 50. In particular instances, the integrated circuit(s) forming or being a part of the CPU, GPU, and/or QPU, which ated circuit(s) may be arranged as and/or be a part of the secondary and/or tertiary analytics platform, may be configured so as to form one or more pipelines of analyses where the various data generated may be fed into and out of, back and forth n, the various processing units and/or integrated circuits, such as in a seamless and/or streaming fashion, so as to allow for the rapid transmission of data between the multiplicity of integrated circuit, and more particularly to expedite the analyses herein.

For instance, in some ces, the various devices for use in accordance with the methods disclosed herein may include, or otherwise be associated with, one or more sequencing devices, for performing a sequencing protocol, which sequencing protocol may be performed by software run on a remote sequencer, such as by a Next Gen sequencer, e.g., Illumina's HiSeq Ten, located in a core sequencing facility, such as made accessible via a cloud based interface. In other instances, the sequencing may be performed in a hardwired configuration run on a sequencing chip, such as implemented by Thermo Fisher's Ion Torrent, or other sequencer a chip technologies, where sequencing is performed by use of a semiconductor technology that delivers benchtop next gen sequencing, and/or by an integrated circuit configured as, or to otherwise e, a field effect transistor employing a graphene channel layer. In such instances, where the cing is performed by one or more integrated ts configured as, or to include, a semiconducting sequencing microchip, the chip(s) may be positioned remotely from the one or more other processing units and/or integrated circuits sed herein, which may be configured for performing secondary and/or tertiary analytics on the sequenced data. Alternatively, the chips and/or sing units may be positioned vely close to one another so as to be directly coupled together, or at least within the same general proximity of one another, such as within the same ty.

In this and other such instances, a sequencing and/or BioIT analytics pipeline may be formed such that the raw sequencing data generated by the sequencer may be rapidly communicated, e.g., streamed, to the other analytic components of the pipeline for direct analysis, such as in a streaming manner.

Further, once the raw sequencing data (e.g., BCL data) or read data (e.g., FASTQ data) is produced by the sequencing instrument, this data may be transmitted to, and be received by, an integrated circuit configured for performing s bioanalytic functions on genetic and/or protein sequences, such as with respect to analyzing the generated and/or received DNA, RNA, and/or protein sequence data. This ce analysis may e the comparing of a generated or received nucleic acid or protein sequence to one or more databases of known sequences, such as for performing secondary analysis on the received data, and/or in some ces, for performing disease diagnostics, such as where the database of known ces for performing the comparison may be a database containing morphologically distinct and/or abhorrent sequence data, that 1s data of genetic samples pertaining to or believed to pertain to one or more ed states.

Accordingly, in various instances, once isolated and sequenced, the genetic, e.g., DNA and/or RNA, data may be subjected to secondary analysis, which may be performed on the received data, such as for the performance of mapping, ng, sorting, variant calling, and/or the like, so as to generate mapped and/or aligned data that may then be used to derive one or more VCF detailing the ence between the mapped and/or aligned genetic sequence and a reference sequence. Particularly, once secondary processing has occurred, the genetic information may then be passed onto one or more tertiary processing modules of the system, such as for further processing thereby, such as to derive therapeutically and/or prophylactic results. More particularly, after variant calling, the mapper/aligner/variant caller may output a standard VCF file that is ready for and may be communicated to an additional ated circuit for performing tertiary analysis, such as analyses related to genome, e.g., whole genome, analysis, genotyping, e.g., joint genotyping, analysis, micro-array is, exome analysis, iome analysis, an epigenome analysis, a metagenome analysis, a joint genotyping analysis, a variance analysis, e.g., a GATK analysis, structural variants is, somatic variants analysis, and the like, as well as an RNA-sequencing or other genomics analysis.

Hence, the bioanalytic, e.g., the BioIT, platform herein presented may include highly zed algorithms for mapping, aligning, sorting, duplicate marking, haplotype variant calling, compression and/or ression, such as in a software, hardwired, and/or a quantum processing configuration. For example, although one or more ofthese functions may be configured to be performed entirely or lly in a hardwired configuration, in particular instances, the ary and/or tertiary processing platform may be configured for running one or more software and/or quantum processing applications, such as one or more programs directed at performing one or more bioanalytics functions, such as one or more of the ons disclosed herein below. Particularly, the sequenced and/or mapped and/or aligned and/or other processed data may then be r processed by one or more other highly optimized algorithms for one or more of whole genome analysis, genotyping analysis, microarray analysis, exome analysis, microbiome analysis, epigenome analysis, metagenome is, joint genotyping, and/or a t, e.g., GATK analysis, such as implemented by software being run on a general purpose CPU and/or GPU and/or QPU, albeit in n instances one or ore ofthese functions may at least partially implemented in hardware. ingly, as can be seen with reference to , in various embodiments, the multiplexed bioanalytical smg rms are configured for ming one or more of primary, secondary, and/or tertiary processing. For example, the primary processing stage produces genetic ce data, such as in one or more BCL and/or FASTQ files for er into the system 1. Once within the system 1 the ced genetic data, including any associated metadata, may be advanced to a secondary processing stage 600, so as to produce one or more variant call files. Hence, the system may also be configured to take the one or more variant call files along with any associated ta, and/or or other associated processed data, and in one or more tertiary processing stages, may perform one or more other operations thereon, such as for the purposes orming one or more diagnostics and/or prophylactic and/or therapeutic ures there with.

Particularly, an analysis ofthe data may be initiated, e.g., in response to a user request 120, e.g., made from a remote computing resource 100, and/or in response to data submitted by the third party 121, and/or data automatically retrieved from a local 200 and/or remote 400 storage facility. Such r processing may include a first tier of processing n various pipeline run protocols 700 are configured to perform analytics on the determined genetic, e.g., variation, data of one or more subjects. For instance, a first tier of ry processing units may include a genomics sing platform that is configured to m genome, epigenome, metagenome, ping, and/or various variant analysis, and/or other bioinformatics based analysis. Additionally, in a second tertiary processing tier, various e diagnostic, research, and/or analysis protocols 800 may be performed, which analysis may include one or more of NIPT, NICU, cancer, LDT, biological, AgBio applications and the like.

The system 1 may further be adapted so as to receive and/or transmit various data 900 related to the procedures and processes herein disclosed such as related to electronic medical records (EMR) data, Federal Drug Administration testing and/or structuring data, data relevant to annotation, and the like. Such data may be useful so as to allow a user to make and/or allow access to generated medical, diagnostic, therapeutic, and/or prophylactic modalities developed through use of the system 1 and/or made accessible thereby.

Accordingly, in various instances, the devices, s, and systems presented herein allow for the secure performance of genetic and bioanalytic analysis, as well as for the secure transfer of the results f, in a forum that may be easily usable for downstream processing. Additionally, in various instances, the devices, methods, and systems presented herein allow for the secure transmission of data into the system, such as from one or more health monitoring and/or data storage facilities and/or from a government agency, such as the FDA or NIH. For example, the system may be ured for securely receiving EMR/PHR data, such as may be transmitted from a health care and/or storage ty for use in accordance with the methods disclosed herein, such for the performance of genetic and bioanalytic analysis, as well as for the secure transfer of the s thereof, in a forum that may be easily usable for downstream processing. ularly, the first tertiary processing tier 700 may include one or more genomics processing platforms, such as for performing genetics analysis, such as on mapped and/or aligned data, e.g., in a SAM or BAM file format, and/or for processing variant data, such as in a VCF . For instance, the first tertiary processing rm may e one or more of a genome pipeline, epigenome pipeline, a metagenome pipeline, a joint genotyping pipeline, as well as one/or more variant analysis pipelines, including: a GATK pipeline, structural variant pipeline, somatic variant calling pipeline, and in some instances, may include an quencing analysis pipeline. One or more other genomic analysis pipelines may also be included.

More specifically, with reference to , in s instances, the multitiered and/or multiplexed bioanalytical processing platform includes a further layer of data generation and/or processing units. For instance, in n instances, the bioanalytical processmg platform incorporates one or more processing pipelines, in one or more of software and/or hardware entations, that are directed to ming one or more tertiary processing protocols. For example, in particular instances, a platform of ry processing pipelines 700 may include one or more of a genome pipeline, an epigenome pipeline, a metagenome pipeline, a joint genotyping ne, a variance pipeline, such as a GATK ne, and/or other pipelines, such as an RNA pipeline. Additionally, a second layer of the tertiary processing analyses platform may e a number of processing pipelines, such as one or more of a micro-array analysis pipeline, a , e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, epigenome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analysis pipeline, including joint genotyping, variants analyses pipeline, including structural variants pipelines, somatic variants pipelines, and GATK and/or MuTect2 pipelines, as well as RNA sequencing pipelines and other genetic analyses pipelines.

Accordingly, m one embodiment, the multi-tiered bioanalytical processing platform includes a metagenomics pipeline. For instance, a metagenomics pipeline may be included, such as for the performance of one or more environmental genomics processes.

Particularly, in s embodiments, the metagenomics analysis may be configured for determining if a group of organisms evolved from a common ancestor, such as a species or other clade. More particularly, in various embodiments, an environmental sample containing a multiplicity of living and/or dead organisms within it may be ed, from which the DNA/RNA present may be isolated, ced, and processed via, one or more of the processing platforms herein, so as to identify the particular species present and/or one or more other genomic factors relevant thereto. Such "environmental" samples may include a multiplicity of human microbiomes (e.g. related to the microorganisms that are found in association with both healthy and diseased humans, including microorganisms found in the skin, blood, sputum, stool samples) as well as external nmental agents.

There are a ity of methods for deriving the ced genetic s for performing metagenomic processing. A first method includes a targeted 16S ribosomal RNA cloning and/or gene sequencing ol. For instance, 16S ribosomal RNA is highly variable across species (or even strains of one species). Accordingly, this RNA may be isolated and sequenced to produce a genetic profile of bio-diversity that is derived from naturally ing biological samples, which may be used to inform the A/I or other databases of the system. r, a problem with such sequencing is that a large amount ofmicrobial biodiversity may be missed simply due to the manner by which it has been cultivated.

Accordingly, a second method includes a shotgun and/or PCR directed protocol that may be used to generate samples of a plurality, e.g., all, genes from all biological agents ofthe sampled communities, which once sequenced may reveal the genetic ity of microscopic life. Specifically, in the shotgun sequencing method, an ate reference sequence may be generated, e.g., from many (e.g., tens of thousands) of reference genomes of ent species. However, the aggregate size ofthis many nce s is huge. Hence, it is ageous to select one or more distinctive sub-sequences from each reference genome so as to build the aggregate reference sequence.

For instance, such a subsequence may range from l hundred bases to several thousand bases long, which ideally are unique sequences not occurring in other species (or strains). These subsequences may then be aggregated so as to construct the reference sequences. Accordingly, once isolated, sequenced, mapped and d, these metagenomic sequences can be compared against l or full reference genomes for many species, and genetic biodiversity can be determined.

Hence, metagenomics offers a powerful lens for viewing the microbial world that can revolutionize our understanding of the living world. Consequently, in either of these instances, when there is a significant presence of an organisms DNA present in a sample, that species can be identified as being within that environment. Ideally, in a manner such as this, s not common to other species generally present in that environment may be identified.

Specifically, when coverage of all species is normalized for the ed nmental samples, c diversity of all species present can be determined and can be compared against the entire coverage, such as by comparing a portion of a ular sm's DNA to that ofthe generated biologically diverse reference genetic sequence.

The significance of these analyses can be determined by Bayesian methods, such as by estimating the probability of ing the sequenced reads of a particular organism, assuming a given s is or is not present. Bayesian probability s are directed to describing the probability of an event, based on conditions that might be related to that event. For example, if one is interested in determining the presence of cancer in a subject, and if the subject's age is known, and if is determined that cancer is an age related disease, then, using Bayes' theorem, information about the subject's age can be used to more accurately assess the ility er.

Specifically, with the Bayesian probability retation the theorem expresses how a subjective degree ofbelief can rationally change to account for the observed evidence. Bayes' theorem is stated mathematically as the following equation: P (A/B) = P (B / A) P (A) I P (B) where A and Bare events and P(B) f. 0. P(A) and P(B) are the probabilities of observing A and B without regard to each other. P(A I B), a conditional probability, is the probability of observing event A given that B is true. P(B I A) is the probability ofobserving event B given that A is true.

Accordingly, one or more steps for performing a Bayesian Probability analyses in this context may include one or more of: Presence calls can be made for clades at various taxonomic levels: kingdom, phylum, class, order, family, genus, species, and/or strain. However, this is complicated by the fact that DNA tends to be singly similar between organisms sharing lower taxonomic levels. Additionally, often times a sample may match a nce genome from multiple species within a higher taxonomic level (or multiple strains of one species), and hence, in many instances, only a more general clade (such as a genus or ) can be called present unambiguously, rather than a specific species or strain.

Nevertheless, the devices, systems, and methods of using the same disclosed herein can be employed to overcome these and other such ulties.

Specifically, in one embodiment, a method for determining the presence of two or more species or clades nisms from a sample is provided. For instance, in a first step, reads of genomic sequence data may be obtained from a sample, such as where the reads may be in a FASTQ or BCL format. Mapping ofthe genomic sequence may be performed so to map the reads to multiple genomic reference sequences. In this instance, the genomic nce sequences may be a whole , or may be a partial genome in order to reduce the amount of data required for each s, strain, or clade. However, using larger ns of a genome will increase the sensitivity of detection, and each reference sequence used should be selected to represent each species, strain, or clade that will be distinct from one another.

For this purpose, all or a portion of the genomic sequence from the 16S ribosome of each s or clade may be used. In this manner, two or more genomic reference sequences of species, strains, or clades of organisms suspected to be in the sample, may be built so as to detect members of these groups in the sample. Once built, an index for each ofthe genomic reference sequences may also be built. The s may be a hash table or a tree index, such as a prefix or suffix tree index. Once the index has been built, the sample genomic ce reads may be compared with each ofthe two or more indexes. Then it may be determined ifthe sample genomic sequence reads map to each ofthe indexes.

Likewise, the reads of the c sequence may also be aligned to the genomic reference sequence(s) to which they are mapped. This will te an alignment score, in accordance with the methods herein, which may be used in analyzing the probability that a read indicates the presence or absence of a species or clade of organism in the sample.

Specifically, the mapping and/or aligning may be accomplished by the present software and/or hardware modules, as described . In some embodiments, the mapped and aligned data may then be communicated to the computing resource 100/300 for further analysis and processing.

For instance, the mapped and/or aligned genomic sequence reads may be analyzed to ine the likelihood that an organism having the genomic reference sequence is present in the sample. Likewise, a list of species, s, or clades that are determined to be present in the environmental sample may be reported. In certain embodiments, the list may be reported with a confidence metric (e.g. P-value) so as to indicate the statistical confidence ofthe evaluation. The entire list of species, strains, or clades of organisms analyzed may also be reported, along with an indication of which species, strains, or clades were t, and a confidence metric. It is to be noted that although described with respect to the analysis of microbiomes, various ofthe techniques and procedures disclosed herein may be employed in the is ofall other tertiary processing protocols, where appropriate.

] For Instance, B sets forth an ary entation of a method for performing environmental analysis, such as of microbiomes within an environmental . For example, in a first instance, an environmental sample may be obtained, and the various genetic al may be ed therefrom. The various genetic material may then be processed and sequenced, such as via a suitably configured NGS.

Consequently, in a first step 1000, once the various genetic material has been sequenced, e.g., by an NGS, it may be transmitted to the system 1 disclosed herein. In step 1010, one, two, or more genomic reference sequences of interest, e.g., to be detected within the sample, may be built. At step 1020, an index for each of the one, two, or more genomic reference sequences may be built. r, at step 1030, the obtained sequenced reads of the genomic sample may then be compared to the one, two, or more indexes, such as via a suitably configured mapping module. At step 1040, then it may be determined ifthe genomic sample ofsequenced reads map to each ofthe two or more s.

At this point, if desired, at step 1050, the mapped reads may be aligned with the genomic reference sequences to generate an alignment and/or an alignment score.

Accordingly, once the obtained genetic materials within the sample are mapped and/or aligned, at step 1060, the likelihood that a given organism having the reference sequence is present within the sample may be determined. And once processed a list of species, strains, and/or clades that are present in the sample may be identified and/or reported.

The ry processing platform disclosed herein may also include an epigenomic pipeline. Particularly, epigenetics studies the genetic effects not encoded in the DNA sequence of an organism. The term also refers to the s themselves: functionally relevant changes to the genome that do not involve a change in the tide sequence. heless, epigenetic changes are stably heritable phenotypes that result from changes in a chromosome that does not alter the DNA sequence. These alterations may or may not be heritable. Particularly, epigenetic changes modify the activation of certain genes, but not the genetic code sequence of DNA. It is the microstructure (not code) of DNA itself or the associated chromatin proteins may be modified, causing activation or silencing.

The epigenome is involved in regulating gene expression, development, tissue differentiation, and suppression oftransposable elements. Unlike the ying genome that is largely static within an individual, the epigenome can be dynamically altered by environmental conditions. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Additionally, epigenomics involves the study of the te set of epigenetic cations on the genetic material of a cell, known as the epigenome consisting of a record of the chemical changes to the DNA and histone proteins of an organism. These s can be passed down to an organism's offspring via transgenerational epigenetic inheritance. Changes to the epigenome can result in changes to the structure ofchromatin and changes to the function ofthe genome.

This epigenetic mechanism enables differentiated cells in a multicellular organism to express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Particularly, most etic changes only occur within the course of one individual sm's me. However, if gene inactivation occurs in a sperm or egg cell that results in fertilization, then some epigenetic changes can be transferred to the next generation. Several types of etic inheritance systems may play a role in what has become known as cell memory. For ce, various covalent modifications of either DNA (e.g., cytosine ation and hydroxymethylation) or of histone proteins (e.g. lysine acetylation, lysine and argmme methylation, senne and threonine phosphorylation, and lysine ubiquitination and sumoylation) may play central roles in many types of epigenetic inheritance. Because the ype of a cell or dual is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. Such effects on cellular and physiological phenotypic traits may result from external or environmental s that switch genes on and offand affect how cells express genes.

For instance, DNA damage can cause epigenetic s. DNA damage 1s very frequent. These damages are largely repaired, but at the site of a DNA repair, epigenetic changes can remain. In particular, a double strand break in DNA can initiate unprogrammed epigenetic gene silencing both by causmg DNA methylation as well as by promoting silencing types of histone modifications (chromatin ling). Other examples of mechanisms that produce such changes are DNA methylation and histone modification, each of which alters how genes are expressed without ng the underlying DNA sequence.

Nucleosome remodeling has also been found to cause epigenetic ing of DNA .

Further, DNA damaging chemicals, can also cause considerable hypomethylation of DNA, such as through the tion ative stress pathways. Additionally, gene expression can be controlled through the action ofrepressor proteins that attach to silencer regions of the These epigenetic changes may last through cell divisions for the duration of the cell's life, and may also last for multiple generations even though they do not involve changes in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. One example of an epigenetic change in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells become the various pluripotent cell lines ofthe embryo, which in tum become fully entiated cells. In other words, as a single fertilized egg cell - the zygote - continues to divide, the resulting er cells change into all the different cell types in an organism, including neurons, muscle cells, epithelium, elium of blood vessels, etc., by activating some genes while inhibiting the expression ofothers.

There are several layers of regulation ofgene expression. One way that genes are regulated is through the remodeling of chromatin. Chromatin is the x of DNA and the histone proteins with which it associates. If the way that DNA is wrapped around the histones changes, gene expression can change as well. A first way is post translational modification of the amino acids that make up histone proteins. Histone proteins are made up of long chains of amino acids. Ifthe amino acids that are in the chain are changed, the shape of the histone might be modified. DNA is not completely unwound during replication. It is possible, then, that the modified histones may be d into each new copy of the DNA.

Once there, these histones may act as templates, initiating the surrounding new histones to be shaped in the new manner. By altering the shape of the histones around them, these modified histones would ensure that a lineage-specific transcription program is maintained after cell division.

The second way is the addition of methyl groups to the DNA, mostly at CpG sites, to convert cytosine to 5-methylcytosine. ylcytosine ms much like a regular cytosine, pairing with a guanme m double-stranded DNA. However, some areas of the genome are methylated more y than others, and highly methylated areas tend to be less transcriptionally active, through a ism not fully understood. Methylation of cytosines can also persist from the germ line of one of the parents into the zygote, marking the chromosome as being inherited from one parent or the other (genetic ting). Although histone cations occur throughout the entire sequence, the unstructured N-termini of histones (called histone tails) are particularly highly modified. These cations include acetylation, methylation, ubiquitylation, phosphorylation, sumoylation, ribosylation and citrullination.

Accordingly, DNA methylation is the ce of methyl groups on some DNA nucleotides, especially 'C' bases followed by 'G's, or "CpG" dinucleotides.

Methylation in promotor regions tends to suppress gene expression. Methylation analysis is the process of detecting which 'C'bases are methylated in a given sample genome. Bisulfite sequencing (MethylC-seq) is the most common method of detecting ation using whole-genome sequencing, where hylated cytosine ('C') bases are ally converted to uracil ('U')bases, which become thymine ('T')bases after PCR amplification.

Methylated 'C'bases resist conversion.

Accordingly, in accordance with the devices and methods disclosed herein, detection of modifications of DNA molecules, where the modifications do not affect the DNA sequence, but do affect gene expression, are provided herein, such as by performing one or more mapping and/or aligning operations on epigenetic genetic material. In such methods, the obtained reads may be mapped and aligned to the reference genome in a manner allowing converted 'T' bases to align to reference 'C' positions, and 'C' bases may be replaced with 'T'sin the nce sequence, prior to mapping/alignment. This allows for accurate mapping and ent of the reads, which have bisulfite ted C's (now T's), thus revealing the non-bisulfite converted (methylated) C's in the genomic sequence reads.

For reverse-complemented ents, the complementary substitutions may be used, e.g., 'G'smay be replaced with 'A's.

Likewise, the reference index (e.g. hash table) builder and the /aligner may be modified to perform these substitutions automatically for MethylC-seq usage.

Alternatively, the mapper/aligner may be modified to allow the forward alignment of read 'T'sto reference 'C's, and the reverse-complemented ent of read 'A'sto reference 'G's. The methods disclosed herein improve accuracy, and prevent erroneous forward alignment of read 'C'sto reference 'T's,or erroneous reverse-complemented alignment of read 'G'sto reference 'A's.

Additionally, provided herein are methods for determining the ation state of cytosine bases in genomic ce reads. For instance, in a first step, reads of genomic sequence from bisulfite-treated nucleotide samples may be obtained. Particularly, one or more modified sequencing protocols may be employed so as to generate the reads for secondary processing, in these regards. Specifically, one or more of: whole genome bisulfate sequencmg; reduced representation bisulfate sequencmg; methylated DNA immunoprecipitation sequencing, and methylation-sensitive restriction enzyme sequencing may be used to identify DNA ation across ns ofthe genome, at g levels of resolution down to basepair level. Further, chromatin accessibility may be accessed, for instance, where DNase I hypersensitivity site sequencing may be performed, such as where the DNase I enzyme may be used to find open or accessible regions in the . Further, RNA-sequencing and expression arrays may be used to identify expression levels or protein coding genes. Particularly, sequencing may be used to identify expression of small noncoding RNA, primarily miRNAs.

Consequently, once sequenced to produce reads, a genomic reference sequence may be built for comparison with the reads. CpG locations in the genomic reference sequence may then be marked. r, the genomic nce ce may be preprocessed by replacing C's in genomic with T's. An index for the genomic reference sequence may be built. And once the index has been built the sample genomic sequence reads may be compared with the index, and it may be determined ifthe sample epi-genomic sequence reads map to the index.

Further, the mapped reads may be aligned with the genomic reference sequence so as to generate an alignment score. In certain embodiments, base substitutions may be made in the read sequence, and the read may be re-compared and re-aligned with the index. In some ments, an alignment orientation restriction may be utilized during mapping and/or alignment of a read, such that only forward alignments may be permitted with C to T replacements in the read and genomic sequence reference, and only reversecomplement alignments are ted with G to A replacements, in the read and genomic ce reference.

WO 14320 PCT/0S2017/036424 These mapping and aligning procedures may be accomplished by the various software and/or hardware modules described herein. In some embodiments, the mapped and aligned data may then be communicated to a CPU/GPU/QPU for further analysis and processing. For instance, the mapped and aligned reads may be sorted by their mapped reference position. In some embodiments, duplicate reads may be marked and removed.

Overlapping reads from a pileup of reads may be ed over each marked reference CpG location. In such an instance, A e (T) that has replaced a cytosine (C) indicates a nonmethylated cytosine and is marked as such. And a cytosine that remains in the read sequence may be marked as a methylated cytosine. Reverse-complemented alignments of CpG locations may also be marked as methylated or non-methylated. For example, a guanine (G) that has replaced an adenine (A) is marked as the reverse-complement of a non-methylated cytosine (C), while a guanine (G) that remains in the read sequence is marked as the reverse complement ofa methylated cytosine (C). The likely methylation status of each CpG location on each tide strand may be reported, and an associated confidence metric (e.g. p-value) in the methylation call may be made. In some embodiments, the methylation status of the marked CpG locations may also be indicated for each chromosome of a diploid pair of somes.

] With respect to histone modification, histone cation includes various naturally occurring chemical modifications of the histone ns that DNA wraps around, resulting in the DNA wrapping more or less tightly. Loosely wrapped DNA, for instance, is associated with higher rates of gene expression. Such histone modifications may be determined by Chromatin Immunoprecipitation Sequencing (ChIP-Seq), which may be used to identify genome wide patterns of histone modifications, such as by using antibodies against the modifications. Further, ChIP-seq is a method that may be employed so as to e and sequence DNA that is tightly bound to histones (or other selected proteins). After ChIP-seq has been med, the sample may be prepared, the DNA isolated and sequenced, and the sequenced DNA may then be /aligned to a nce genome as disclosed herein, and the mapped coverage may be used to infer the level ofhistone binding at various loci in the genome. Additionally ed herein are s of analyzing ChIP-derived nucleotide sequences, which is similar to the methods described below for analyzing ural variants.

Ofspecial note is that epigenetics is useful in cancer ch and diagnostics.

For instance, human tumors undergo a major disruption ofDNA methylation and histone modification patterns. In fact the nt epigenetic landscape of the cancer cell is characterized by a global c hypomethylation, CpG island promoter hypermethylation of tumor suppressor genes, an altered histone code for critical genes, and a global loss of monoacetylated and trimethylated histone H4. ingly, the methods disclosed herein may be used for the purposes ofcancer research and/or diagnostics.

Further, the methods herein disclosed may be useful for generating one or more epigenomic databases and/or reference genomes. For example, the methods herein disclosed, e.g., employing an A/I ng protocol of the system, may be useful for ting a human reference of epigenomes, such as from normal, healthy individuals across a large variety of cell lines, primary cells, and/or primary tissues. Such data produced may then be used to enhance the mapping and/or aligning protocols disclosed herein. Furthermore, once a database of epigenomic differences has been generated, the database may be mined, e.g., by the A/I module so as to better characterize and determine relevant s that occur in various e states, such as cancer, dementia, Alzheimer's disease, and other neurological conditions.

Accordingly, in s instances, an epigenomics analysis may be performed, such as to identify one or more or the entire set of epigenetic cations that have taken place on the genetic material of a cell. Particularly, employing the methods disclosed herein, the epigenome of an organism, and/or the cells thereof, may be determined, so as to catalog and/or record of the chemical changes to the DNA and histone proteins of the cells of the sm. For example, an exemplary omic analysis is set forth herein in C.

For instance, in a first step, a c sample may be obtained from an organism, and the genetic material ed therefrom and sequenced. Hence, once sequenced, at step 1000, the sequenced reads of the sample may be transmitted into and received by the system 1. In this instance, the reads may be derived from a bisulfate-treated tide sample. Likewise, at step 1010, a genomic reference ofsequences, e.g., for the organism, may be built such as for performing a comparison of the epigenomic sample reads. At step 1012, any various CpG locations in the genomic reference sequence(s) may be identified.

Once identified, at 1014, the "C's"ofthe CpG locations, in the reference, may be replaced with "Ts," and at step 1020, an index for the modified genomic reference sequence may be generated. Once the index for the ed reference is generated, at step 1030, the genomic sequence reads ofthe sample may be compared with the index, and at step 1040 it may be determined if the c sequence reads of the sample map to the index, such as by being mapped in accordance with the methods and apparatuses disclosed herein.

The mapped reads may then be aligned with the genomic reference sequence, and an alignment score may be generated, such as by performing one or more alignment operations, as discussed herein.

At this point, one of a couple of various analyses may be performed. For ce, at step 1051, if greater context is d, the base substitutions in the reads, as processed above, and/or the alignment orientation, and/or ter restrictions may be adjusted, and the comparison steps 1030 - 1050 may be repeated. This process itself may be repeated as desired until a sufficient level of context is achieved. Accordingly, once a ient level of context has been achieved, the mapped and/or aligned reads, at step 1080, may be sorted, such as in the processes disclosed herein, by the mapped/aligned reference position. And at step 1081, any duplicate reads may be marked and/or removed.

Further, at step 1082, the reads from the pileup of reads pping each marked reference CpG location may be analyzed. Where a "T" has been replaced with a "C", it may be marked as a non-methylated "C", at step 1083; and where a "C" remains in the sequence, at step 1084, the "C" may be marked as a methylated "C". Finally, at step 1086, a ination and/or report on the likely methylation status of each of the CpG location on each nucleotide strand, and a ence in the methylation call, may also be made.

Additionally, provided herein, are methods for analyzing genomic material where part ofthe genetic material may have, or may otherwise be associated with, a structural variant. ularly, a structural variation is a variation in the structure of an organism's chromosome. Structural variations involve many kinds of variations in the genome of a species, including microscopic and submicroscopic types, such as deletions, duplications, copy-number variants, insertions, inversions, and translocations. Many structural variants are associated with genetic diseases. In fact, about 13% of the human genome is defined as urally variant in the normal population, and there are at least 240 genes that exist as homozygous deletion polymorphisms in human populations. Such structural variations can comprise millions of nucleotides of geneity within every genome, and are likely to make an important contribution to human disease susceptibility.

Copy-number variation is a large category of structural variation, which es insertions, deletions, and ations. There are several inversions known that are related to human e. For instance, recurrent 400kb inversion in factor VIII gene is a common cause ofhaemophilia A, and smaller inversions affecting idunorate 2-sulphatase will cause Hunter syndrome. More examples include Angelman syndrome and Sotos syndrome.

The most common type of complex structural variation are non-tandem duplications, where sequence is duplicated and inserted in ed or direct orientation into another part of the genome. Other classes of complex structural variant include deletion-inversion-deletions, duplication-inversion-duplications, and tandem duplications with nested ons. There are also c translocations and segmental uniparental disomy (UPD).

However, the detection of abnormal DNA structures 1s problematic and beyond the scope of variant calling heretofore known. Such structural variants that are problematic to detect include those having: large insertions and deletions (e.g., beyond the 50-lO0bp indel size); duplications, and other copy-number ions ; inversions and translocations, and aneuploidy (abnormal chromosome copy counts: monosomy, disomy, y, etc.). In certain instances disclosed herein, identified copy-number variations may be tested on subjects who do not have genetic diseases, such as by using quantitative SNP genotyping.

Structural variation detection generally begins with performing a g and an aligning operation as using the s and methods disclosed . For instance, the reads of the c sample to be ed may be mapped and aligned to a reference genome, such as in a protocol that supports chimeric alignments. Specifically, some structural variants (e.g. CNVs and aneuploidy) can be detected by analysis of relative mapped coverage. However, other structural variants (e.g., large , inversions, translocations) can be detected by analysis ofclipped and chimeric alignments.

Specifically, each structural variant involves one or more "break" positions, where the read does not map to the reference genome, such as where the geometry changes between the sample and the reference. In such an instance, the pileup may be configured such that the reads therein that slightly overlap the structural variant breaks may be clipped at the break, and the reads substantially overlapping the structural variant breaks may be chimerically aligned, e.g., with two ns of a read mapped to different reference locations. However, read pairs overlapping structural variant breaks may be inconsistently aligned, with the two mate reads mapped to widely different reference locations, and/or with abnormal relative ation ofmate reads. Such obstacles may be overcome by the s disclosed herein.

For instance, in certain instances, data pertaining to known structural variants may be used to better determine the sequence of a structural variant. For example, a database having a list of the structural variations in human genome may be compiled, e.g., with an emphasis on CNVs, and such data may be used in determining the sequence of particular variants, such as in a suitably configured weighting protocol. Particularly, where a structural variant is known, its " and "outer" nates may be employed as a minimal and maximum range of sequence that may be ed by the structural variation. Additionally, known insertion, loss, gain, inversion, LOH, everted, transchr and UPD variations may be classified and fed into the knowledge base ofthe t system.

In various instances, the determination of a structural variant may be performed by a CPU/GPU/QPU running suitably configured software, such as employing previously determined sequencing data, and in other instances, ural variant analyses may be performed such as in the hardware disclosed . Accordingly, in particular instances, a method for ing genomic sequences for structural variants is provided. For ce, in a first step, genomic sequence reads may be received from a nucleotide sample.

In certain instances, the sequenced reads may have been derived from paired end or mate pair protocols for detecting structural variants. Next an index for the genomic reference sequence may be built, such as where the index may be a hash table or a tree, such as a prefix or suffix tree. Once the index has been built, the sample genomic sequence reads may be compared with the index so as to determine if the sample genomic sequence reads map to the index. If so, the sample genomic sequence reads may then be aligned to the genomic reference sequence to which they are , and an alignment score may be determined.

As indicated above, the g and aligning may be accomplished by the hardware module as described herein. In some embodiments, the mapped and aligned data may then be communicated to an associated CPU/GPU/QPU for further analysis and processing. The reads may be sorted by mapped reference position, and duplicate reads may be marked and deleted. Chimeric reads and/or unusual relative alignments of two mate reads may be determined, and le ural variants may be determined based on any ed ic reads and/or unusual relative alignments (e.g. large indel, an inversion, or a translocation). Likewise, posterior probabilities of each possible structural variant may be calculated. In some embodiments, structural t haplotypes may be determined, such as by using HMM analysis of the chimeric reads and/or the unusual relative alignments. For WO 14320 2017/036424 example, pair HMM may be used for such a determination. The pair HMM may be accomplished using the hardware .

Accordingly, in various instance, as can be seen with respect to D, a method for determining ions in the structure of an organism's chromosomes is ted. For instance, in accordance with the methods disclosed herein, at step 1000, reads of genomic sequence data may be ed. At step 1010 one or more genomic reference sequences may be built, so as to perform a comparison between the reads and the reference sequence(s). Specifically, at step 1010 a genomic nce sequence may be built so as to allow the ed reads to be compared against the generated nce. More specifically, for these purposes, at step 1020 an index for the genomic reference sequence may be generated, for example, at step 1020 a hash table or prefix/suffix tree may be generated.

Hence, at step 1030, the reads of the sample genomic ce may be compared with the generated index, such as in accordance with the software and/or hardware implementations disclosed herein.

If, at step 1040, it is determined that the reads ofthe sample c sequence map to the index, then at step 1050, the mapped reads may be aligned with the genomic reference sequence, and an alignment score may be generated. At step 1080, the sample reads may be sorted by their mapped reference positions. At this point, at step 1081, duplicate reads may be marked and removed. Further, at step 1090 chimeric reads and/or unusual relative alignments, e.g., of two mate reads, may be detected, and at 1092 possible structural variants may be determined, such as based on the detected chimeric reads and/or unusual ve alignments. Furthermore, ior probabilities of each possible structural variant may be calculated, and, optionally, at step 1096, structural variant haplotypes may be determined, such as by using HMM analysis, as described , of the chimeric reads and/or unusual relative alignments.

Further, the devices, systems, and methods disclosed herein may be employed for the processing of RNA sequences. Particularly, herein presented are methods for analyzing RNA-sequence reads, such as employing a spliced mapping and alignment protocol (e.g., with a suitably configured RNA mapper/aligner). For instance, in one embodiment, a transcriptome pipeline may be provided, such as for ultra-rapid RNA-sequence data analysis. ularly, this pipeline may be configured to perform secondary analysis on RNA transcripts, such as with respect to reference-only alignment as well as annotation-assisted alignment.

Accordingly, in a first method, raw read data, e.g., in a BCL and/or FASTQ file format, may produced by a sequencing instrument, and may be input into the system, where mapping, ng, and variant calling may be performed. However, in various instances, one or more gene annotations files (GTF) may be input into the system, such as to guide the spliced alignments, e.g., a splice junction LUT may be built and used. For instance, alignment accuracy and splice junction tables may be employed. Consequently, a 2-phase alignment may be performed, such as where in a first detection phase novel splice ons may employed, which may then be used to guide a second pass mapping/aligning phase.

After variant calling, the system will output a standard VCF file ready for tertiary analysis.

Particularly, once an input file is received, spliced mapping and aligning may be performed, such as on both single and paired read ends. As indicated, configurable junction filters may be employed to give a single junction output. Position sorting may be performed, which may include binning by the nce range, and then the sorting of the bins by reference position, and duplicate marking may take place, such as based on the starting position and CIGAR string so as to achieve a high quality duplicate report, whereby any duplicates may be d. Haplotype variant g may then be performed, e.g., using a SW and HMM processing engine, and ly may be med.

Additionally, the devices, systems, and methods disclosed herein may be ed for performing c variant calling. For instance, a somatic variant calling protocol may be employed so as to detect variants that may occur in cancer cells. Particularly, genomic samples for somatic calling may be obtained from single or multiple tumor biopsies, or from blood. Optionally, a "normal" (non-tumor) sample may also ed, such as for comparison during variant calling, e.g., where the somatic variants will occur in the tumor cells but not in the cells of the normal sample. The DNA/RNA form the sample(s) may be isolated and ced, such as by a Next Gen sequencer. The sequenced data, e.g., from each sample, may then be transmitted into the secondary processing platform, and the reads may be mapped and aligned. Further, the reads may be subjected to a plurality of variant calling procedures, including processing by one or both of SW and pair HMM engines.

However, the system should be ured so as to be able to detect low t allele frequencies, such as 3% to 10% (or higher). More particularly, a genotyping probability model may be employed, where the model is configured to allow arbitrary allele frequencies. One method for allowing this is to assign each variant pe allele frequencies corresponding to the observed allele ncies in the overlapping reads. For instance, if 10% of overlapping reads exhibit a certain variant, a genotype can be tested consisting of 90% reference allele and 10% alternate allele. For tumor/normal dual samples, the posterior ility that a variant is present in the tumor sample but not the normal sample can be estimated. ] r, the somatic variant caller ne may be configured to provide information on tumor heterogeneity, e.g., that a series of distinct on events occurred, such as where one or more sections ofa tumor with different genotypes (a subclone) has been identified. Such subclone information may be derived from a determination of variant allele frequencies and distributions thereof, and/or by explicitly calling variants entially among multiple tumor samples.

Accordingly, methods for detecting ce ts of cancer cells from a sample are provided. In a first step, genomic sequence reads from a nucleotide sample may be obtained from cancerous and/or normal cells. The sequence reads may be from paired end or mate pair protocols similar to that for detecting structural variants. An index for the genomic reference sequence may be built, such as where the index may be a hash table or a tree, such as a prefix or suffix tree. The sample genomic sequence reads, e.g., of the tumor and/or of the normal sample, may be compared with the index, and it may be determined if the sample genomic sequence reads map to the index.

The sample genomic sequence reads may then be aligned to the genomic reference sequence to which they are mapped, and an alignment score may be generated. The mapping and ng may be accomplished by a software and/or hardware , as described herein. In some embodiments, the mapped and aligned data may then be communicated to a CPU/GPU/QPU for r analysis and processing. The reads may be sorted by mapped reference position, and any duplicate reads may be marked and deleted.

Variants may be detected using a Bayesian analysis that is modified to expect arbitrary variant allele frequencies, and to detect and report possible low allele frequencies (e.g. 3% to %).

In some embodiments, germline variants may be ed in both noncancerous and cancerous samples, and c variants may be detected in only the cancerous samples. For example, the germline and somatic mutations may be distinguished by relative frequency. Posterior probabilities may be calculated of each possible cancer variant, and in some embodiments, structural variant haplotypes may be determined using HMM analysis of the chimeric reads and/or the unusual relative alignments. For example, pair HMM may be used for such a determination. The pair HMM may be accomplished using hardware modules as described herein.

Accordingly, in various embodiments, a somatic t calling procedure, as exemplified, in E, may be performed, such as to calculate the probability that a variant is a cancer variant. For ce, at step 1000 reads ofgenomic sequence samples may be generated, e.g., via sequencing ofan NGS, and/or be received, e.g., via transmission over a suitably ured cloud based network , such as from one or both of cancerous and non-cancerous genetic samples. At step 1010 a genomic reference sequence may be generated such as for comparison of the reads, at step 1020 an index may be built from the genomic reference sequence, and at step 1030 the sample genomic sequence may be compared with the index, such as employing the software and/or re implementations disclosed herein, so as to map the genomic sequence reads to the index, at step at 1040.

Further, at step 1050, the mapped reads may be aligned with the genomic reference sequence to generate an alignment score. The mapped and/or aligned reads may then be sorted with t to the reference position, at 1080, and optionally, at 1081 any duplicate reads may be marked and removed.

Additionally, once the reads have been mapped and/or aligned and/or sorted and/or ed, then at step 1100 variants may be detected, such as by employing a Bayesian analysis, and at 1101 germline variants in both non-cancerous and cancerous samples as well as somatic variants therein may optionally be detected. Likewise, at step 1094, posterior probabilities of each possible cancer variant may be ated. Further, at step 1096, cancer t haplotypes may optionally be determined, such as by implementing an HMM analysis in software and/or in hardware as disclosed herein.

Furthermore, the devices, systems, and s disclosed herein may be ured for performing a joint genotyping operation. Particularly, a joint genotyping operation may be ed so as to improve variant calling accuracy, such as by jointly considering reads from a cohort of multiple subjects. For instance, in various ces, genomic variations may be highly correlated in certain tions, e.g., where certain variants are common to a plurality of ts. In such ces, the sensitivity and specificity of variant calling can be improved by jointly considering the evidence for each variant from multiple DNA (or RNA) samples. Specifically, sensitivity may be improved because weak evidence for a variant in one subject can be enhanced by evidence for the same variant in other samples. More specifically, sensitivity may be ed because moderate evidence for a false-positive variant can be tempered by absence of evidence for the same variant in other samples. Generally, the more samples participating in joint genotyping, the more accurate the variant calls can be for any given subject.

Joint genotyping involves the estimation of posterior probabilities for s subsets of all the subjects having a given variant, using prior probabilities that express the observed correlations in genetic variation. In various instances, joint ping may be performed in a single variant-calling pass, where aligned reads from multiple samples are examined by the variant caller. This is usually only practical for small s of samples, because when dozens, hundreds, or thousands of samples are involved, the total data size becomes tical to rapidly access and manipulate.

Alternatively, joint ping can be done by first performing variant calling separately for each sample, then merging the results with a joint genotyping tool, which updates the variant probabilities for each subject using the joint information. This method uses additional output from each single-sample t g pass so as to better measure areas of weak ce for variants and/or in regions where no variant would be called without joint processing. Whereas the VCF format is commonly used to represent called variants from single-sample variant calling, a special gVCF format may be used to represent first-stage variant (and non-variant) calls in preparation for merging. The gVCF format includes records for locations, and/or blocks of multiple locations, where most likely no variant is present, so this information can be merged with other gVCF calls or non-calls at the same ons to yield improved joint genotype calls for each t. ingly, the joint genotyping ne may be configured to call variants from multiple samples faster and with r accuracy. Additionally, the joint genotyping ne may further be configured to supports ee as well as population variant calling from a cohort of samples. For instance, the ne may be configured to handle up to 10, 15, , 25, even 50 or more samples at one time. In various instances, a population calling configuration may be adapted to handle sample sizes of many thousands at once. Further, a combination of speed and hierarchical grouping of multiple samples provides a computationally efficient analysis solution for joint genotyping. Additionally, the sequencing of the samples for joint genotyping may be performed within the same flow cell of a Next Gen sequencer thereby allowing the system to simultaneously map/align multi-sample inputs thereby speeding up the overall process ofjoint calling, such as where the BCL data may be fed directly to the pipeline to produce unique gVCF files for each sample.

Therefore, provided herein is a method for improving variant calling accuracy by jointly considering reads from a cohort of multiple subjects. In a first step, reads of genomic sequence from two or more samples are received. A genomic reference sequence for comparison with the reads is built, and from the genomic reference sequence an index is generated. The genomic sequence reads of each sample are then compared with the index, and it is determined ifthe c sequence reads ofeach sample map to the index.

The mapped reads may then be aligned with the genomic reference sequence and an alignment score may be generated. The reads may be sorted by mapped reference position, and ate reads may be marked and/or removed. Additionally, overlapping reads from the pileup of reads may then be analyzed to determine if a majority ofreads agree with the reference genomic sequence. Posterior probabilities of each possible variant are calculated, and the variant call data from all samples may be merged so as to enhance the variant call accuracy for each individual sample. This can enhance the variant calling accuracy (e.g., the sensitivity and specificity) for each sample, and may be accomplished as a processing step after all of the samples have undergone variant calling analysis, or it may be accomplished cumulatively, after each of the samples undergoes variant calling analysis. The likelihood of non-reference alleles in regions where no variant is called may then be determined, and the ined likelihood of non-reference alleles in the regions where no variant is called may be ed.

Accordingly, in various embodiments, a somatic variant g procedure, as exemplified, in F, may be performed, such as to calculate the probability that a variant is a cancer t. For instance, at step 1000 reads ofgenomic sequence samples may be generated, e.g., via sequencing ofan NGS, and/or be received, e.g., via transmission over a ly configured cloud based network system, such as from one or both of cancerous and non-cancerous genetic samples. At step 1010 a genomic reference sequence may be generated such as for ison of the reads, at step 1020 an index may be built from the genomic reference sequence, and at step 1030 the sample genomic ce may be compared with the index, such as employing the software and/or hardware implementations disclosed , so as to map the genomic sequence reads to the index, at step at 1040.

Further, at step 1050, the mapped reads may be aligned with the genomic reference ce to te an alignment score. The mapped and/or aligned reads may then be sorted with WO 14320 PCT/0S2017/036424 respect to the nce position, at 1080, and optionally, at 1081 any duplicate reads may be marked and removed.

Likewise, at 1082, overlapping reads from a pileup of reads may be analyzed to determine if one or more, e.g., a majority of the reads, agree with the reference genomic sequence(s), and at step 1094, posterior ilities of each possible variant may be calculated. At this point, at step 1096, variant haplotypes may be determined, if desired, such as by performing an HMM analysis, and/or at step 1120, the variant call data, e.g., from all samples, may optionally be merged so as to e the variant call accuracy for each individual sample. Further, at step 1122, the likelihood of non-reference alleles, e.g., in regions where no variant is called may be determined and reported.

Additionally, as can be seen with reference to , in one aspect, an online app store is provided to allow users to develop, sell, and use genomics tools that can be incorporated into the system and be employed to analyze the genomic data transmitted to and entered into the system. Particularly, the genomic app store enables customers that desire to develop genetic tests, e.g., like a NICU test, and once developed may be uploaded on to the system, e.g., genetic marketplace, for purchase and running as a platform thereon, so that anyone g the newly developed system platform, can deploy the uploaded tests via the web portal. More particularly, a user can browse the web portal "app" store, find a desired test, e.g., the NICU test, download it, and/or configure the system to implement it, such as on their uploadable genetic data. The online "cohort" marketplace, ore, presents a rapid and efficient way to deploy new genetic analytic ations, which applications allow for identical results to be obtained from any of the present system platforms that runs the downloaded application. More particularly, the online market place es a mechanism for anyone to work with the system to develop genetic analysis applications that remote users can download and configure for use in ance with the present workflow models.

Another aspect of the cohort marketplace disclosed herein is that it allows for the secure sharing ofdata. For instance, the transmittal and storage ofgenomic data should be highly protected. However, often such genetic data is large and difficult to transfer in a secure and protected manner, such as where the subject's identity is restricted. ingly, the present genetics market place allows cohort participants to share genetic data without having to identify the subject. In such a market place, cohort participants can share questions and processes so as to e their ch in a protected and secure environment, t g the identity of their respective subject's genomes. Additionally, a user can enlist the WO 14320 PCT/0S2017/036424 help of other researchers in the analysis of their sample sets without identifying to whom those genomes belong.

For instance, a user can identify subjects having a specific genotype and/or phenotype, such as stage 3 breast cancer, and/or having been treated with a particular drug. A cohort can be formed to see how these drugs affect cancerous cell growth on a genetic level.

Therefore, these characteristics, t others, may form a cohort selection criteria that will allow other researchers, e.g., remotely located, to perform standard c analyses on the genetic data, using uniform analytic procedures, on subjects they have access to that fit within the cohort criteria. In this , a given researcher need not be responsible for fying and ng all members of a sample set, e.g., subjects fitting within the criteria, to substantiate his or her scientific inquiry.

Particularly, Researcher A may set up a research cohort within the marketplace, and fy the appropriate ion criteria for subjects, the genomic test(s) to be run, and the parameters by which the test is to be run. Researchers B and C, located remotely from Researcher A, may then sign up for the cohort, identify and select subjects matching the criteria, and then run the specified tests on their subjects, using the uniform procedures disclosed herein, so as to help Researcher A achieve or better accomplish his or her research goals in an expeditious manner. This is beneficial because only a portion of genetic data is being transmitted, subject identity is protected, and as the data is being ed using the same genetic analysis system employing the same ters, the results data will be the same regardless of where and on what machine the ) are run. uently, the cohort market place allows users to form and build cohorts simply by posting the selection criteria and run parameters on the dashboard. Compensation rates may also be posted and payments rendered by employing a ly configured commerce, e.g., monetary exchange, program.

Anyone that accepts participation in the cohort can then download the criteria and data file(s) and/or use c data of subjects they have already generated and/or stored in performing the requested analyses. For instance, each cohort participant will have, or be able to generate, a database of BCL and/or FASTQ files that are stored in their individual servers. These genetic files will have been derived from subjects who happen to meet the selection criteria. Specifically, this stored genetic and/or other data of the t may be scanned so as to determine suitability for inclusion within the cohort selection criteria. Such data may have been generated for a number ofpurposes, but regardless ofthe reasons for the generation, once generated it may be selected and subjected to the requested pipeline analyses and used for inclusion within the . ingly, in various embodiments, the cohort system may be a forum for connecting researchers, so as to allow them to pool their resources and data, e.g., genetic sequence data. For example, engaging a cohort would allow a first researcher to introduce a project requiring genetic data analyses requiring the mining and/or examination of a number of s from various subjects, such as with respect to mapping, aligning, t calling, and/or the like. Therefore, d of having to gather subjects and collect sample sets individually, the cohort initiator can advertise the need for a ibed analyses procedure to be run on sample sets previously or to be collected by others, and as such a collective approach to generating sample sets and analyzing the same is provided for by the cohort organization herein. Particularly, the cohort initiator can set up the cohort selection, create a configuration file to be shared with the potential cohort participants, create the workflow parameters, e.g., within a ow folder, and can thereby automate data generation and analyses, e.g., via the workflow management system. The system may also enable the commercial aspect of the transaction, e.g., the payment processing for compensating the cohort participants for their provision of genetic data sets that may be analyzed, such as with respect to mapping, aligning, variant calling, and/or with respect to tertiary es.

] In various embodiments, the cohort structured analyses may be directed to primary sing, e.g., of either DNA or RNA, such as with respect to image processing and/or base quality score recalibration, methylation analysis, and the like; and/or may be directed to the performance of secondary analysis, such as with respect to mapping, aligning, sorting, variant calling, and the like; and/or may be directed to tertiary analysis, such as with respect to array, genomic, epigenomic, metagenomic, ping, variants, and/or other forms of tertiary analyses. Additionally, it is to be understood that gh many of the pipelines and analyses performed thereby may involve primary and/or secondary processing, various analysis platforms herein may not be directed to primary or secondary processing.

For instance, in n instances, an analysis platform may be exclusively directed to performing tertiary analysis, such as on genetic data, or other forms of cs and/or bioinformatics analyses.

] For example, m particular embodiments, with respect to the particular analytical procedures to be run, the analyses to be performed may include one or more of mapping, aligning, sorting, t calling, and the like, so as to produce results data that may be subjected to one or more other secondary and/or tertiary analyses procedures, depending on the specific pipelines selected to be run. The workflow may be simple or it may be complex, e.g., it may require the performance of one pipeline module, e.g., mapping, or multiple modules, such as g, aligning, sorting, variant calling, and/or others, but an important parameter is that the ow should be identical for each person that takes part of the cohort. Particularly, a unique feature of the system is that the requester establishing the cohort sets forth the control parameters so as to ensure that the analysis to be performed are performed in the same manner, less of where those procedures are performed and on what machines.

] Consequently, when setting up the cohort the requester will upload both selection criteria along with a configuration file. Other cohort participants will then view the selection criteria to determine if they have data sets of genetic information falling within the set forth criteria, and if so will perform the requested is on the data, based on the gs of the configuration file. Researches may sign up to be selected as a cohort ipant, and if subscription is great a lottery or competition can be held to select the participants. In various instances, a bidding system could be initiated. The results data generated by the cohort ipants may be processed onsite or on the cloud, and as long as the configuration file is followed, the processing ofthe data will be the same. Particularly, the configuration file sets forth how the BioIT analytics device is to be configured, and once the device is set up in accordance with the prescribed uration, a device ated with the system will perform the ted genetic analyses in the same manner regardless of where located, e.g., locally or remotely. The results data may then be uploaded onto the cohort market place, and payment tendered and received in view ofthe received results data.

For instance, the analysis of the c data may be performed locally, and the results uploaded onto the cloud, or the genetic data itself may be uploaded and the analyses run on the cloud, e.g., a server or server network, such as quantum processing platform, associated with the cloud. In various instances, it may be useful to only upload the results data, so as to better protect the subjects' identities. Particularly, by uploading only results data, not only is security protected, but large amounts of data need not be transferred, thereby enhancing system ency.

More particularly, in various ces, a compressed file containing results data from one or more of the pipelines may be uploaded, and in some instances, only a file containing a description of variations need be uploaded. In some instances, only an answer need be given, such as a text answer, e.g., a "yes" or "no" answer. Such answers are preferable as they do not set forth the identity of the subject. However, if the analyses need to be performed , e.g., in the cloud, selected BCL and/or FASTQ files may be uploaded, the analyses performed, and the results data may then be pushed back to the initial submitter, who can then upload the results data at the cohort ace. The original raw data may then be deleted from the online . In this and other such manners, the cohort requester will not have access to the identities ofthe subjects.

Compression, such as that employed m "just in time analysis" (JIT), is particularly useful in enhancing cohort ency. For instance, using typical procedures, the movement of data into and out ofthe cohort system is very expensive. Accordingly, although in various configurations, raw and/or uncompressed data uploaded to the system may be stored there, in ular instances, the data can be compressed prior to being uploaded, the data may then be processed within the system, and the results can then be compressed prior to being transmitted out of the system, such as where the compression is effectuated in ance with a JIT protocol. In this instance, storage ofsuch data, such as in a ssed form is less expensive, and therefore the cohort system is very cost efficient.

Additionally, in various instances, a plurality of cohorts may be provided within an online marketplace, and given the compression processes herein described, data may be transmitted from one cohort to another, so as to allow researches ofvarious different cohorts to share data between them, which without the compression methods disclosed herein could be itively costly. Particularly, without the speed and efficiency of JIT compression data once transmitted into the cloud, would typically stay in the cloud, albeit it would be accessible therein for review and manipulation. However, JIT allows data to be quickly transmitted to and from the cloud for both local and/or cloud based processing.

Further, as can be seen with respect to B and 43, in particular ces, the system 1 may be configured for subjecting the generated and/or secondarily sed data to r processing, e.g., via a local 100 and/or a remote 300 computing resource, such as by g it through one or more tertiary processing pipelines, such as one or more of a micro-array analysis ne, a genome, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, epigenome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analysis pipeline, including joint genotyping, variants analyses pipeline, including structural variants pipelines, somatic ts pipelines, and GATK and/or MuTect2 pipelines, as well as RNA sequencing pipelines, and/or other ry processing pipeline. The results data from such processing may then be compressed and/or stored remotely 400 and/or be transferred so as to be stored locally 200.

Particularly, one or more, e.g., all, of these functions, may be performed y, e.g., on site 10, on a local cloud 30, or via controlled access through the hybrid cloud 50. In such an instance, a developer environment is created that allows a user to control the functionality of the system 1 to meet his or her individual needs and/or to allow access thereto for others seeking the same or similar results. Consequently, the various components, processes, procedures, tools, tiers, and hierarchies ofthe system may be configurable such as via a GUI interface that allows the user to select which components of the system to be run, on which data, at what time, and in what order in accordance with the user determined desires and ols, so as to generate relevant data and connections between data that may be securely communicated throughout the system whether locally or remotely. As indicated, these components can be made to communicate seamlessly er, e.g., regardless of location and/or how connected, such as by being in a tightly coupled configuration and/or a seamless cloud based coupling, and/or by being configurable, e.g., via a JIT protocol, so as to run the same or similar processes in the same or similar manner, such as by employing corresponding API aces dispersed throughout the system, the employment of which allows the s users to configure the various components to run the various procedures in like manner.

For instance, an API may be defined in a header file with respect to the processes to be run by each particular component of the system 1, wherein the header describes the functionality and determines how to call a function, such as the parameters that are , the inputs ed and s transmitted, and the manner in which this , what comes in and how, what goes out and how, and what gets returned, and in what manner.

For e, in various embodiments, one or more of the components and/or elements thereof, which may form one or more pipelines of one or more tiers of the system may be configurable such as by instructions entered by a user and/or one or more second and/or third party applications. These instructions may be communicated to the system via the ponding APis which communicate with one or more of the various drivers of the , instructing the driver(s) as to which parts of the system, e.g., which modules and/or which processes thereof are to be activated, when, and in what order, given a preselected parameter configuration, which may be determined by a user selectable interface, e.g., GUI.

Particularly, the one or more DMA drivers of the system 1 may be ured to run in corresponding fashion, such as at the kernel level ofeach component and the system 1 as a whole. In such an instance, one or more of the provided kernel's may have their own very low level, basic API that provides access to the hardware and functions of the various components of the system 1 so as to access able registers and modules so as to configure and direct the processes and the manners in which they are run on the system 1.

Specifically, on top of this layer, a virtual layer of service functions may be built so as to form the building blocks that are used for a multiplicity of functions that send files down to the kemel(s) and get results back, encodes, encrypts, and/or transmits the relevant data and r performs more higher level functions thereon. On top of that layer an additional layer may be built that uses those service functions, which may be an API level that a user may interface with, which may be adapted to function primarily for configuration of the system 1 as a whole or its component parts, downloading files, and ing results, which files and/or results may be transmitted throughout the system either y or globally. Additional APis may be ured and included as set forth in more detail above with respect to the secure storage ofdata.

Such configuring of the vanous APis, s, and/or firmware of the system may include communicating with registers and also ming function calls. For example, as bed herein above, one or more function calls necessary and/or useful to perform the steps, e.g., sequentially, to execute a mapping and/or aligning and/or sorting and/or variant call, or other secondary and/or tertiary functions as herein described may be implemented in accordance with the hardware operations and/or related algorithms so as to generate the necessary processes and perform the required steps.

Specifically, e in certain embodiments one or more of these operations may be based on one or more structures, the various structures needed for implementing these ions may need to be constructed. There will therefore be a function call that performs this function, which function call will cause the requisite structure to be built for the performance of the operation, and because of this a call will accept a file name ofwhere the structure parameter files are stored and will then generate one or more data files that n and/or configure the requisite structure. Another function call may be to load the structure that was generated via the respective algorithm and transfer that down to the memory on the chip and/or system 1, and/or put it at the right spot where the re is expecting them to be. Of course, various data will need to be downloaded onto the chip and/or otherwise be erred to the system generator, as well for the performance of the various other selected functions ofthe system 1, and the configuration manager can perform these functions, such as by loading everything that needs to be there in order for the modules ofpipelines of the tiers of the platforms of the chip and/or system as a whole to perform their functions, into a memory on, attached, or otherwise associated with the chip and/or .

Additionally, the system may be configured to allow various components of the system to icate with one another, such as to allow one or more chips of the system 1 to ace with the circuit board of the sequencer 121, the computing resource 100/300, transformer 151, analyzer 152, interpreter 310, collaborator 320, or other system component, when included therewith, so as to receive the FASTQ and/or other generated and/or sed genetic sequencing files directly from the sequencer or other processing component such as ately once they have been generated and/or sed and then transfers that information to the configuration manager which then directs that information to the appropriate memory banks in the hardware and/or re that makes that information available to the pertinent modules ofthe hardware, software, and/or system as a whole so that they can perform their designated functions on that information so as to call bases, map, align, sort, etc. the sample DNA/RNA with respect to the reference genome, and or to run associated secondary and/or tertiary processing operations thereon.

Accordingly, in various embodiments, a client level interface (CLI) may be included n the CLI may allow the user to call one or more of these functions ly.

In various embodiments, the CLI may be a software ation, e.g., having a GUI, which is adapted to configure the accessibility and/or use of the hardware and/or various other software applications of the system. The CLI, therefore, may be a program that s instructions, e.g., arguments, and makes functionality available simply by calling an application program. As indicated above, the CLI can be command line based or GUI (graphical user interface) based. The line based commands happen at a level below the GUI, where the GUI includes a windows based file r with click on function boxes that delineate which modules, which pipelines, which tiers, of which platforms will be used and the parameters of their use. For example, in ion, if instructed, the CLI will locate the reference, will determine if a hash table and/or index needs to be generated, or if already generated locate where it is stored, and direct the uploading ofthe generated hash table and/or index, etc. These types ofinstructions may appear as user options at the GUI that the user can select the associated chip(s)/system 1 to perform.

Furthermore, a library may be included wherein the library may include preexisting , editable, configuration files, such as files orientated to the typical user selected functioning of the hardware and/or associated software, such as with respect to a portion or whole genome and/or protein analysis, for instance, for various es, such as personal medical histories and ancestry is, or disease diagnostics, or drug discovery, therapeutics, and/or one or more ofthe other analytics, etc. These types ofparameters may be preset, such as for performing such analyses, and may be stored in the library. For example, if the platform herein described is employed such as for NIPT, NICU, Cancer, LDT, AgBio, and related research on a collective level, the preset ters may be configured differently than if the platform were ed simply to researching genomic and/or genealogy based research, such as on an individual level.

More particularly, for specific diagnosis of an individual, accuracy may be an important factor. Therefore, the parameters of the system may be set to ensure increased accuracy albeit in exchange for possibly a decrease in speed. However, for other genomics applications, speed may be the key determinant and therefore the parameters of the system may be set to ze speed, which however may sacrifice some accuracy. Accordingly, in various embodiments, often used parameter settings for performing different tasks can be preset into the library to facilitate ease of use. Such parameter settings may also include the necessary software applications and/or hardware configurations employed in running the system 1. For instance, the library may contain the code that es the API, and may further include sample files, scripts, and any other ary ation necessary for running the system 1. Hence, the library may be configured for compiling software for running the API as well as various ofthe executables.

Additionally, as can be seen with respect to C and 43, the system may be configured such that one or more of the system components may be med remotely, such as where the system component is d to run one or more ative functions on the data, such as an interpretive function 310 and/or collaborative function 320. For instance, where an interpretive protocol is employed on the data, the retive protocol 312 may be configured to analyze and draw sions about the data and/or determine vanous onships with respect thereto, one or more other analytical protocols may also be performed and include annotating the data 311, performing a diagnostic 313 on the data, and/or analyzes the data, so as to determine the presence or absence of one or more WO 14320 PCT/0S2017/036424 biomarkers 314. As indicated, one or more of these functions may be directed by the WMS, and/or performed by the A/I module disclosed herein.

Additionally, where a collaborative protocol is med, the system 1 may be configured for providing an electronic forum where data sharing 321 may occur, which data sharing protocol may e user selectable security 324 and/or privacy 322 settings that allow the data to be encrypted and/or password protected, so that the identity and sources of the data may be hidden from a user of the system 1. In particular instances, the system 1 may be configured so as to allow a 3rd party analyzer 121 to run virtual tions on the data. Further, one generated, the interpreted data and/or the data subjected to one or more collaborative analyses may be stored either ly 400 or locally 200 so as to be made available to the remote 300 or local 100 computing resources, such as for further processing and/or analysis.

In another aspect, as can be seen with respect to , a method for using the system to generate one or more data files upon which one or more ary and/or tertiary processing protocols may be run is provided. For instance, the method may include providing a genomic infrastructure such as for one or more of onsite, cloud-based, and/or hybrid c and/or bioinformatics generation and/or processing and/or analysis.

In such an instance, the genomic infrastructure may include a bioinformatics processing platform having one or more memories that are ured to store one or more configurable processing structures for configuring the system so as to be able to perform one or more analytical processing functions on data, such as data including a genomic sequence of interest or processed result data pertaining thereto. The memory may include the genomic sequence of interest to be processed, e.g., once generated and/or acquired, one or more genetic reference sequences, and/or may additionally include an index of the one or more c reference sequences and/or a list of splice junctions pertaining thereto. The system may also include an input having a platform application mming interface (API) for selecting from a list of options one or more ofthe urable processing ures, such as for configuring the system, such as by selecting which processing functions of the system will be run on the data, e.g., the pre- or processed genomic sequences of interest. A graphical user ace (GUI) may also be present, such as ly associated with the API, so as to present a menu by which a user can select which of the available options he or she desires to be run on the data.

Hence, in these and/other such instances, the hybrid cloud 50 may be configured for allowing seamless and protected transmission of data hout the components of the system, such as where the hybrid cloud 50 is adapted to allow the various users of the system to ure its component parts and/or the system itself, e.g., via the WMS, so as to meet the research, diagnostic, therapeutic and/or lactic discovery and/or development needs of the user. Particularly, the hybrid cloud 50 and/or the various ents of the system 1 may be operably connected with compatible and/or ponding API interfaces that are adapted to allow a user to remotely configure the s components of the system 1 so as to deploy the resources desired in the manner desired, and further to do so either locally, remotely, or a combination of the same, such as based on the demands of the system and the particulars of the analyses being performed, all the while being enabled to communicate in a secured, encryptable environment.

As bed above, the system may be ented on one or more ated circuits that may be formed ofone or more sets ofconfigurable, e.g., preconfigured and/or hardwired, digital logic ts that may be interconnected by a plurality of physical electrical interconnects. In such an instance, the integrated circuit may have an input, such as a memory interface, for receiving one or a ity of the configurable structure protocols, e.g., from the memory, and may further be adapted for implementing the one or more structures on the integrated circuit in ance with the configurable processing structure protocols. The memory interface of the input may also be configured for receiving the genomic sequence data, which may be in the form of a plurality of reads of genomic data.

The interface may also be adapted for accessing the one or more genetic reference sequences and the es).

In various instances, the digital logic circuits may be arranged as a set of sing engines that are each formed of a subset of the digital logic circuits. The digital logic circuits and/or processing engines may be configured so as to perform one or more preconfigurable steps of a primary, secondary, and/or tertiary processing protocol so as to generate the plurality of reads of genomic sequence data, and/or for processing the plurality of reads of genomic data, such as according to the genetic reference sequence(s) or other genetic sequence derived information. The ated circuit may further have an output so as to output result data from the primary, secondary, and/or tertiary processing, such as according to the platform application programming interface (API).

WO 14320 PCT/0S2017/036424 Particularly, in various embodiments, the digital logic circuits and/or the sets of processing engines may form a ity of genomic processing pipelines, such as where each pipeline may have an input that is d according to the platform application programming interface so as to receive the result data from the primary and/or secondary processing by the bioinformatics processing rm, and for performing one or more analytic processes thereon so as to produce result data. Additionally, the plurality of genomic processing pipelines may have a common ne API that defines a secondary and/or tertiary processing operation to be run on the result data from the primary and/or ary processed data, such as where each of the plurality of genomic processing pipelines is configured to perform a subset of the secondary and/or tertiary processing ions and to output result data ofthe secondary and/or tertiary processing according to the pipeline APL In such ces, a plurality of the genomic analysis applications may be stored in the memory and/or an associated searchable application repository, such as where each ofthe ity of c analysis applications are accessible via an electronic medium by a computer such as for execution by a computer processor, so as to perform a ed analysis of the genomic pre- or post processed data from the result data of the y, secondary, and/or tertiary processing, such as by one or more of the plurality of c processing pipelines. In particular instances, each of the plurality of genomic analysis applications may be defined by the API and may be configured for receiving the result data of the y, secondary, and/or tertiary processing, and/or for performing the target analysis of the pre- or post processed genomic data, and for outputting the result data from the ed analysis to one ofone or more genomic databases.

The method may additionally include, selecting, e.g., from the menu of the GUI, one or more genomic processing pipelines from a plurality of the available genomic processing pipelines of the system; selecting one or more genomic analysis applications from the plurality of genomic analysis applications that are stored in an application repository; and executing, using a computer processor, the one or more selected genomic analysis applications to perform a targeted analysis of genomic data from the result data of the primary, ary, and/or tertiary processing.

Additionally, in various embodiments, all of mapping, aligning, and sorting, and variant calling may take place on the chip, and local realignment, duplicate marking, base quality score bration may, and/or one or more ofthe tertiary processing protocols and/or pipelines, in various embodiments, also may take place on the chip or in software, and in various instances, various compression protocols, such as SAM and/or BAM and/or CRAM, may also take place on the chip. However, once the primary, secondary, and/or tertiary processed data has been produced, it may be compressed, such as prior to being transmitted, such as by being sent across the system, being sent up to the cloud, such as for the performance of the variant calling module, a secondary, ry, and/or other processing platform, such as including an interpretive and/or orative analysis protocol. This might be useful especially given the fact that variant calling, including the tertiary processing thereof, can be a moving target, e.g., there is not one standardized agreed upon algorithm that the industry uses.

Hence, different algorithms can be employed, such as by remote users, so as to achieve a different type ofresult, as desired, and as such having a cloud based module for the performance of this function may be useful for allowing the flexibility to select which algorithm is useful at any particular given moment, and also as for serial and/or parallel processing. Accordingly, any one of the s disclosed herein can be implemented as either hardware, e.g., on the chip, or software, e.g., on the cloud, but in certain embodiments, all of the s may be configured so that their function may be performed on the chip, or all of the modules may be configured so that their function may be med remotely, such as on the cloud, or there will be a mixture of modules wherein some are positioned on one or more chips and some are positioned on the cloud. Further, as indicated, in various embodiments, the ) itself may be configured so as to function in conjunction with, and in some ments, in immediate operation with a genetic sequencer, such as an NGS and/or sequencer on a chip.

More specifically, in various embodiments, an apparatus ofthe sure may be a chip, such as a chip that is ured for processing genomics data, such as by employing a pipeline of data analysis modules. ingly, as can be seen with respect to , a genomics pipeline processor chip 100 is provided along with ated hardware of a genomics pipeline processor system 10. The chip 100 has one or more connections to external memory 102 (at "DDR3 Mem Controller"), and a connection 104 (e.g., PCie or QPI Interface) to the outside world, such as a host computer 1000, for e. A crossbar 108 (e.g., ) provides access to the memory interfaces to various requestors. DMA engines 110 transfer data at high speeds between the host and the processor chip's 100 external memories 102 (via the crossbar 108), and/or between the host and a central controller 112.

The central controller 112 controls chip operations, especially coordinating the efforts of WO 14320 PCT/0S2017/036424 multiple sing engines 13. The processing engines are formed of a set of hardwired digital logic circuits that are interconnected by physical electrical interconnects, and are organized into engine clusters 11/114. In some implementations, the engines 13 in one r 11/114 share one crossbar port, via an arbiter 115. The central controller 112 has connections to each ofthe engine clusters. Each engine cluster 11/114 has a number ofprocessing engines 13 for processing genomic data, ing a mapper 120 (or mapping module), an aligner 122 (or aligning ), and a sorter 124 (or sorting module), one or more processing engines for the performance of other functions, such as t g, may also be provided. Hence, an engine cluster 11/114 can include other engines or s, such as a variant caller module, as well.

In accordance with one data flow model consistent with implementations bed herein, the host CPU 1000 sends commands and data via the DMA engines 110 to the l controller 112, which load-balances the data to the processing engines 13. The sing engines return processed data to the central controller 112, which streams it back to the host via the DMA engines 110. This data flow model is suited for mapping and alignment and variant calling. As indicated, in various instances, ication with the host CPU may be through a relatively loose or tight coupling, such as a low latency, high bandwidth interconnect, such as a QPI, such as to maintain cache coherency between associated memory elements ofthe two or more devices.

For instance, in various instances, due to vanous power and/or space constraints, such as when performing big data analytics, such as g/aligning/variant calling in a hybrid re/hardware accelerated environment, as described herein, where data needs to be moved both rapidly and seamlessly between system devices, a cache coherent tight coupling interface may be useful for performing such data transmissions throughout the system to and from the coupled devices, such as to and from the sequencer, DSP (digital signal processor), CPU and/or GPU or CPU/GPU hybrid, accelerated integrated circuit, e.g., FPGA, ASIC (on network card), as well as other Smart Network rators in a rapid, cache-coherent manner. In such instances, a suitable cache coherent, tight-coupling interconnect may be one or more of a single interconnect technology specification that is configured to ensure that processing, such as between a multiplicity of processing platforms, using different instruction set architectures (ISA), can coherently share data n the different platforms and/or with one or more associated accelerators, e.g., such as a hardwired FPGA implemented accelerator, so as to enable efficient heterogeneous computing, and thereby significantly improve the computing efficiency of the system, which in various instances may be configured as a based server system. Hence, in certain instances, a high bandwidth, low y, cache coherent onnect protocol, such as a QPI, Coherent Processor Accelerator Interface (CAPI), NVLink/GPU, or other suitable interconnect protocol may be employed so as to expedite various data transmissions between the various ents of the system, such as pertaining to the mapping, aligning, and/or variant calling compute functions that may involve the use of acceleration engines the functioning ofwhich requires the need to access, process, and move data seamlessly among various system components ective ofwhere the various data to be processed resides in the system. And, where such data is retained within an ated memory device, such as a RAM or DRAM, the ission activities may further involve expedited and coherent search and in-memory database processing.

Particularly, in ular embodiments, such heterogeneous computing may involve a multiplicity of sing and/or ration architectures that may be interconnected in a reduced instruct set computing . In such an instance, such an onnect device may be a coherent connect interconnect six (CCVI) device, which is configured to allow all computing entry within the system to address, read, and/or write to one or more associated memories in a single, consistent, and coherent manner. More particularly, a CCVI interconnect may be employed so as to connect various ofthe devices of the system, such as the CPU and/or GPU or CPU/GPU hybrid, FPGA, and/or associated memories, etc. one with the other, such as in a high bandwidth manner that is configured to increase transfer rates n the various components while evidencing extremely reduced latency rates. Specifically, a CCVI interconnect may be employed and configured so as to allow components of the system to access and process data irrespective of where the data resides, and without the need for complex programing environments that would otherwise need to be implemented to make the data coherent. Other such interconnects that may be employed so as to speed up, e.g., decrease, processing time and increase accuracy e QPI, CAPI, NVLink, or other interconnect that may be configured to interconnect the various components of the system and/or to ride on top of an associated PCI-express peripheral interconnect.

Hence, in accordance with an ative data flow model consistent with implementations described herein, the host CPU 1000 streams data into the external memory 1014, either directly via DMA engines 110 and the crossbar 108, or via the central controller 112. The host CPU 1000 sends commands to the central controller 112, which sends ds to the processing engines 13, which instruct the processing engines as to what data to process. Because of the tight ng, the processing engines 13 access input data directly from the al memory 1014 or a cache associated therewith, process it, and write results back to the al memory 1014, such as over the tightly coupled interconnect 3, reporting status to the central controller 112. The central controller 112 either streams the result data back to the host 1000 from the external memory 1014, or notifies the host to fetch the result data via the DMA engines 110. illustrates a genomics pipeline processor and system 20, g a full complement of processing engines 13 inside an engine cluster 11/214. The ne processor system 20 may include one or more engine clusters 11/214. In some implementations, the pipeline processor system 20 includes four or more engine clusters 11/214. The sing engines 13 or processing engine types can include, t limitation, a mapper, an aligner, a sorter, a local realigner, a base quality recalibrater, a duplicate marker, a t caller, a compressor and/or a decompressor. In some implementations, each engine cluster 11/214 has one of each processing engine type. Accordingly, all processing engines 13 of the same type can access the crossbar 208 simultaneously, through different crossbar ports, because they are each in a different engine cluster 11/214. Not every processing engine type needs to be formed in every engine cluster 11/214. Processing engine types that require massive parallel processing or memory bandwidth, such as the mapper (and attached aligner(s)) and sorter, may appear in every engine cluster ofthe pipeline processor system 20.

Other engine types may appear in only one or some of the engine clusters 214, as needed to y their performance requirements or the performance requirements of the pipeline processor system 20. illustrates a genomics pipeline sor system 30, showing, in addition to the engine clusters 11 described above, one or more embedded central processing units (CPUs) 302. Examples of such embedded CPUs include Snapdragons® or standard ARM® cores, or in other instances may be an FPGA. These CPUs execute fully programmable bio-IT algorithms, such as advanced t calling, such as the ng of a DBG or the performance ofan HMM. Such processing is rated by computing functions in the various engine clusters 11, which can be called by the CPU cores 302 as needed.

Furthermore, even engine-centric processing, such as mapping and alignment, can be managed by the CPU cores 302, giving them heightened programmability.

WO 14320 PCT/0S2017/036424 illustrates a processing flow for a genomics pipeline processor system and method. In some preferred implementations, there are three passes over the data. The first pass includes mapping 402 and ent 404, with the full set ofreads streamed through the engines 13. The second pass includes sorting 406, where one large block to be sorted (e.g., a substantial portion or all reads previously mapped to a single chromosome) is loaded into memory, sorted by the processing engines, and returned to the host. The third pass includes downstream stages (local realignment 408, duplicate marking 410, base quality score recalibration (BQSR) 412, SAM output 414, reduced BAM output 416, and/or CRAM compression 418). The steps and functions of the third pass may be done in any combination or bination, and in any order, in a single pass.

Hence, in this manner data is passed relatively ssly from the one or more processing engines, to the host CPU, such as in accordance with one or more of the methodologies described herein. Hence, a virtual pipeline architecture, such as described above, is used to stream reads from the host into circular buffers in memory, through one processing engine after r in sequence, and back out to the host. In some implementations, CRAM decompression can be a separate ing function. In some implementations, the SAM output 414, reduced BAM output 416, and/or CRAM compression 418 can be replaced with variant calling, compression and ression.

In various instances, a hardware implementation of a sequence is pipeline is described. This can be done in a number of different ways such as an FPGA or ASIC or structured ASIC implementation. The functional blocks that are implemented by the FPGA or ASIC or structured ASIC are set forth in . Accordingly, the system es a number ofblocks or modules to do ce analysis. The input to the hardware realization can be a FASTQ file, but is not limited to this format. In addition to the FASTQ file, the input to the FPGA or ASIC or structured ASIC consists of side information, such as Flow Space Information from technology such as from the NGS. The blocks or s may include the following blocks: Error Control, g, Alignment, Sorting, Local Realignment, Duplicate Marking, Base Quality Recalibration, BAM and Side Information reduction and/or variant calling.

These blocks or s can be present inside, or implemented by, the hardware, but some of these blocks may be omitted or other blocks added to achieve the purpose ofrealizing a sequence analysis pipeline. Blocks 2 and 3 describe two alternatives of the sequence analysis pipeline platform. The sequence analysis pipeline platform comprising an FPGA or ASIC or structured ASIC and software assisted by a host (e.g., PC, server, cluster or cloud computing) with cloud and/or cluster storage. Blocks 4-7 describe different interfaces that the sequence analysis pipeline can have. In Blocks 4 and 6 the interface can be a PCie and/or PI/CCVI/NVLink interface, but is not limited to a PCie, QPI, or other interface. In Blocks 5 and 7 the hardware (FPGA or ASIC or structured ASIC) can be directly integrated into a sequencing machine. Blocks 8 and 9 describe the integration of the hardware sequence analysis pipeline integrated into a host system such as a PC, server cluster or sequencer. Surrounding the hardware FPGA or ASIC or structured ASIC are a ity of DDR3 memory elements and a PCie/QPI/CAPI/CCVI/NVLink interface. The board with the FPGA/ASIC/sASIC connects to a host computer, consisting of a host CPU and/or GPU, that could be either a low power CPU such as an ARM®, Snapdragon®, or any other processor.

Block 10 illustrates a hardware ce is pipeline API that can be accessed by third party applications to perform tertiary analysis.

FIGS. 50A and 50B depict an expansion card 104 having a processing chip 100, e.g., an FPGA, of the disclosure, as well as one or more associated elements 105 for coupling the FPGA 100 with the host CPU/GPU, such as for the erring of data, such as data to be sed and result data, back and forth from the CPU/GPU to the FPGA 100.

B s the expansion card ofA having a plurality, e.g., 3, slots containing a plurality, e.g., 3, processing chips ofthe disclosure. ically, as ed in FIGS. 50A and 50B, in various embodiments, an apparatus of the sure may include a computing architecture, such as embedded in a silicon field gate programmable array (FPGA) or application specific integrated circuit (ASIC) 100. The FPGA 100 can be integrated into a printed circuit board (PCB) 104, such as a Peripheral Component Interface - Express (PCie) card, which can be plugged into a computing platform. In various instances, as shown in A, the PCie card 104 may include a single FPGA 100, which FPGA may be nded by local memories 105, however, in various embodiments, as depicted in B, the PCie card 104 may include a plurality of FPGAs 100A, 100B and 100C. In various instances, the PCI card may also include a PCie bus. This PCie card 104 can be added to a computing platform to execute algorithms on extremely large data sets. In an alternative embodiment, as noted above with respect to , in various embodiments, the FPGA may be adapted so as to be directly associated with the CPU/GPU, such as via an interloper, and tightly coupled therewith, such as via a QPI, CAPI, CCVI interface. Accordingly, in s instances, the overall work flow WO 14320 PCT/0S2017/036424 of c sequencing involving the FPGA may include the following: Sample preparation, ent (including mapping and alignment), Variant analysis, Biological Interpretation, and/or Specific Applications.

Hence, in various embodiments, an apparatus of the disclosure may e a computing architecture that achieves the high performance execution of algorithms, such as mapping and alignment algorithms, that operate on extremely large data sets, such as where the data sets exhibit poor locality of reference (LOR). These algorithms are designed to reconstruct a whole genome from millions of short read sequences, from modem so-called next generation sequencers, require multi-gigabyte data structures that are randomly accessed. Once reconstruction is achieved, as described herein above, further algorithms with similar characteristics are used to compare one genome to libraries of , do gene function is, etc.

There are two other typical ectures that in general may be constructed for the performance of one or more ofthe operations herein described in detail, such as including purpose multicore CPUs and general purpose Graphic Processing Units (GPGPUs). In such an instance, each CPU/GPU in a multicore system may have a classical cache based architecture, wherein instructions and data are fetched from a level 1 cache (LI cache) that is small but has extremely fast access. Multiple LI caches may be connected to a larger but slower shared L2 cache. The L2 cache may be connected to a large but slower DRAM (Dynamic Random Access Memory) system memory, or may be connected to an even larger but slower L3 cache which may then ted to DRAM. An advantage of this ement may be that applications in which ms and data exhibit locality of reference behave nearly as ifthey are executing on a computer with a single memory as large as the DRAM but as fast as the LI cache. Because full custom, highly optimized CPUs operate at very high clock rates, e.g., 2 to 4 GHz, this architecture may be essential to achieving good performance. Additionally, as discussed in detail with respect to , in various embodiments the CPU may be tightly coupled to an FPGA, such as an FPGA configured for running one or more functions d to the various operations bed herein, such as via a high bandwidth, low latency interconnect such as a QPI, CCVI, CAPI so as to further e performance as well as the speed and coherency of the data transferred throughout the system. In such an instance, cache ncy may be maintained between the two devices, as noted above.

Further, GPGPUs may be employed to extend this architecture, such as by implementing very large s of small CPUs, each with their own small LI cache, wherein each CPU executes the same instructions on different subsets ofthe data. This is a so called SIMD (Single Instruction stream, le Data stream) architecture. Economy may be gained by sharing the instruction fetch and decode logic across a large number ofCPUs. Each cache has access to multiple large external DRAMs via an interconnection network.

Assuming the computation to be performed is highly parallelizable, GPGPUs have a significant advantage over general e CPUs due to having large numbers of computing resources. Nevertheless, they still have a caching architecture and their performance is hurt by applications that do not have a high enough degree of locality ofreference. That leads to a high cache miss rate and processors that are idle while waiting for data to arrive from the al DRAM.

] For instance, in various instances, Dynamic RAMs may be used for system memory because they are more economical than Static RAMs (SRAM). The rule of thumb used to be that DRAMs had 4x the capacity for the same cost as SRAMs. However, due to declining demand for SRAMs in favor of DRAMs, which difference has increased considerably due to the economies of scale that favor DRAMs that are in high demand.

Independent of cost, DRAMs are 4x as dense as SRAMs laid out in the same n area because they only e one transistor and capacitor per bit compared to 4 transistors per bit to implement the SRAM's flip-flop. The DRAM represents a single bit of information as the presence or absence ofcharge on a capacitor.

A problem with this arrangement is that the charge decays over time, so it has to be refreshed periodically. The need to do this has led to ectures that organize the memory into independent blocks and access mechanisms that r multiple words of memory per request. This sates for times when a given block is unavailable while being refreshed. The idea is to move a lot of data while a given block is available. This is in contrast to SRAMs in which any location in memory is available in a single access in a constant amount of time. This characteristic allows memory accesses to be single word ed rather than block oriented. DRAMs work well in a caching architecture because each cache miss leads to a block of memory being read in from the DRAM. The theory of locality of reference is that ifjust accessed word N, then probably going to access words N+l, N+2, N+3 and so on, soon. provides an exemplary implementation of a system 500 of the disclosure, including one or more of the ions cards of , such as for bioinformatics processing 10. The system es a Bio IT processing chip 100 that is configured for ming one or more functions in a processing ne, such as base calling, error correction, mapping, alignment, sorting, assembly, variant calling, and the like as described herein.

The system 500 r includes a configuration manager that is adapted for configuring the onboard functioning of the one or more processors 100. ically, in various embodiments, the configuration manager is adapted to communicate instructions to the internal controller of the FPGA, e.g., firmware, such as by a suitably configured driver over a loose or tightly coupled interconnect, so as to configure the one or more processing functions of the system 500. For instance, the configuration manager may be adapted to configure the internal processing clusters 11 and/or engines 13 associated therewith so as to perform one or more d operations, such as mapping, ng, sorting, variant calling, and the like, in ance with the instructions received. In such a manner only the clusters 11 containing the processing s 13 for ming the requested processing operations on the data provided from the host system 1000 to the chip 100 may be engaged to process the data in accordance with the received instructions.

Additionally, in various embodiments, the configuration manager may further be adapted so as to itselfbe adapted, e.g., remotely, by a third party user, such as over an API connection, as described in greater detail herein above, such as by a user interface (GUI) presented by an App of the system 500. Additionally, the configuration r may be connected to one or more external memories, such as a memory forming or otherwise containing a database, such as a data base including one or more reference or individually sequenced genomes and/or an index thereof, and/or one or more previously , aligned, and/or sorted genomes or portions f. In s instances, the database may further e one or more genetic profiles characterizing a diseased state such as for the performance of one or more tertiary processing protocols, such as upon newly mapped, aligned genetic sequences or a VCF pertaining thereto.

The system 500 may also include a web-based access so as to allow remote communications such as via the internet so as to form a cloud or at least a hybrid cloud 504 communications platform. In such a manner as this, the processed information generated from the Bio IT processor, e.g., results data, may be encrypted and stored as an electronic health record, such as in an external, e.g., remote, se. In various instances, the EMR database may be searchable, such as with respect to the genetic ation stored n, so as to perform one or more statistical analyses on the data, such as to determine ed states or trends or for the purposes of analyzing the effectiveness of one or more prophylactics or treatments pertaining thereto. Such information along with the EMR data may then be further processed and/or stored in a further database 508 in a manner so as to insure the confidentiality ofthe source ofthe genetic information.

More particularly, illustrates a system 500 for executing a sequence analysis pipeline on genetic sequence data. The system 500 includes a configuration manager 502 that es a computing system. The ing system of the configuration manager 502 can include a personal computer or other computer workstation, or can be implemented by a suite ofnetworked computers. The uration manager 502 can further include one or more third party applications connected with the computing system by one or more APis, which, with one or more proprietary applications, generate a uration for processing genomics data from a sequencer or other genomics data source. The configuration r 502 further includes s that load the configuration to the genomics pipeline processor system 10. The genomics pipeline processor system 10 can output result data to, or be accessed via, the Web 504 or other network, for storage of the result data in an electronic health record 506 or other knowledge database 508.

As discussed in several places herein above, the chip implementing the genomics pipeline processor can be connected or integrated in a sequencer. The chip can also be connected or integrated, e.g., directly via an interloper, or indirectly, e.g., on an expansion card such as via a PCie, and the expansion card can by connected or integrated in a sequencer. In other implementations, the chip can be connected or integrated in a server computer that is connected to a sequencer, to transfer genomic reads from the sequencer to the server. In yet other implementations, the chip can be connected or integrated in a server in a cloud computing cluster of ers and servers. A system can include one or more sequencers connected (e.g. via Ethernet) to a server ning the chip, where genomic reads are generated by the multiple sequencers, itted to the server, and then mapped and aligned in the chip.

For instance, in general next generation DNA sequencer (NGS) data pipelines, the primary analysis stage processing is generally specific to a given cing technology.

This primary is stage functions to translate physical signals detected inside the sequencer into "reads" of nucleotide sequences with associated quality (confidence) , e.g. FASTQ format files, or other formats containing sequence and usually quality information. y analysis, as mentioned above, is often quite specific in nature to the sequencing technology ed. In s sequencers, nucleotides are detected by sensing changes in fluorescence and/or electrical charges, electrical currents, or radiated light. Some primary analysis nes often include: Signal processing to amplify, filter, separate, and measure sensor output; Data ion, such as by quantization, decimation, averaging, transformation, etc.; Image sing or numerical processing to fy and enhance meaningful signals, and associate them with specific reads and tides (e.g. image offset calculation, cluster identification); Algorithmic processing and heuristics to compensate for sequencing logy artifacts (e.g. phasing estimates, cross-talk matrices); Bayesian probability calculations; Hidden Markov ; Base calling (selecting the most likely nucleotide at each position in the ce); Base call quality (confidence) estimation, and the like. As discussed herein above, one or more of these steps may be benefitted by implementing one or more of the necessary processing functions in hardware, such as implemented by an integrated circuit, e.g., an FPGA. Further, after such a format is achieved, secondary analysis proceeds, as described herein, to determine the content of the sequenced sample DNA (or RNA etc.), such as by mapping and ng reads to a reference , sorting, duplicate marking, base quality score recalibration, local re-alignment, and variant calling. ry analysis may then follow, to extract medical or research implications from the determined DNA content.

Accordingly, given the sequential nature of the above processing functions, it may be advantageous to integrate primary, secondary, and/or tertiary processing acceleration in a single integrated circuit, or multiple integrated circuits positioned on a single expansion card. This may be beneficial e sequencers produce data that typically requires both primary and ary analysis so as to be useful and may further be used in various ry processing protocols, and integrating them in a single device is most efficient in terms of cost, space, power, and resource sharing. Hence, in one particular aspect, the disclosure is directed to a system, such as to a system for executing a sequence analysis pipeline on genetic sequence data. In various instances, the system may include an electronic data source, such as a data source that provides digital signals, for instance, digital signals representing a plurality of reads of genomic data, where each of the plurality of reads of genomic data e a sequence ofnucleotides. The system may include one or more of a memory, such as a memory storing one or more genetic reference ces and/or an index of the one or more genetic reference sequences; and/or the system may include a chip, such as an ASIC, FPGA, or sASIC.

One or more aspects or features of the t matter described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or structured ASIC er hardware, re, software, and/or combinations thereof.

These various aspects or features can include entation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which can be special or general purpose, coupled to receive data and ctions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

These computer programs, which can also be referred to as programs, re, software applications, applications, components, or code, include machine instructions for a programmable sor, and can be ented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the term "machine-readable medium" refers to any computer program product, apparatus and/or device, such as for example magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable sor, including a machine-readable medium that receives machine instructions as a machine-readable . The term "machine-readable signal" refers to any signal used to e machine instructions and/or data to a programmable processor. The machine-readable medium can store such machine instructions nontransitorily , such as for example as would a non-transient state memory or a magnetic hard drive or any equivalent storage medium. The e-readable medium can alternatively or additionally store such machine ctions in a transient manner, such as for example as would a processor cache or other random access memory associated with one or more physical processor cores.

Additionally, due to the e growth in data production and ition in the 21 st Century, a need has developed for increased processing power that is capable of handling the ever-growing computationally intense analyses upon which modem development is founded. Supercomputers have been introduced, and have been useful for advancing technological development over a wide range of platforms. However, gh supercomputing is useful, it has proven to be insufficient for some of the very complex computing problems many of s technology companies face. Particularly, since the sequencing of the human genome, the technological advancement in the biological arts has been exponential. Nevertheless, in view ofthe high rate and increased complexity of the raw data produced every day, there has d a problematic bottleneck in the processing and analysis of the data generated. Quantum computers have been developed therefor to help resolve this bottleneck. Quantum computing represents a new frontline in computing, providing an entirely new ch to solving the worlds most challenging computational needs. m computing has been known smce 1982. For instance, in the International Journal of tical Physics, d Feynman theorized a system for performing quantum computing. Specifically, Feynman proposed a quantum system that could be configured for use in simulating other quantum systems in such a manner that the conventional functions of computer processing can be med more quickly and efficiently. See Feynman, 1982, International Journal of Theoretical Physics 21, pp. 467-488, which is hereby incorporated by reference in its entirety. Particularly, a quantum computer system can be designed so as to exhibit exponential time-savings in complex ations.

Such controllable quantum systems are commonly known as quantum computers, and have been successfully developed into general purpose processing computers that not only can be used to simulate quantum systems, but can also be adapted for running specialized quantum algorithms. More particularly, complex ms can be modeled in the form of an equation, such as a Hamiltonian, which may be represented in the quantum system in a manner that the behavior of the system provides information regarding the solution to the on. See Deutsch, 1985, Proceedings of the Royal Society of London A 400, pp. 97-117, which is hereby incorporated by reference in its entirety. In such instances, g a model for the behavior of the quantum system may be configured so as to involve solving a differential equation d to the wave-mechanical description of a particle, e.g., onian, of the quantum system.

In e, quantum computing is a computational system that uses quantummechanical phenomena, e.g., superposition and/or entanglement, to perform various calculations on large s of data extremely fast. As such, quantum computers are a vast improvement over conventional l logic computers. Specifically, conventional digital logic circuits function by using binary digital logic gates that are formed through the hardwiring of electronic circuitry on a conductive substrate. In a l logic circuit an "on/off' state of a transistor serves as a basic unit of information, e.g., a bit. Particularly, a common digital computer processor employs binary digits, e.g., bits, in an "on" or "off'state, e.g., as a O or 1, to encode data. Quantum computation, on the other hand, employs an ation device that uses superpositions of entangled states, called quantum bits or qubits, to encode data.

The basis for performing such quantum computations 1s an ation device, e.g., a unit, which forms the quantum bit. The qubit is analogous to the l "bit" in ional digital computers, except that the qubit has far more computational potential than a digital bit. Particularly, as described in greater detail herein, instead of only encoding one of two discrete states, like a "O" and a "1," as found in a digital bit, a qubit can also be placed in a superposition of "O" and "1." Specifically, the qubit can exist in both the "O" and "1" state at the same time. Consequently, the qubit can perform a quantum computation on both states simultaneously. In general, N qubits can be in a superposition of 2N states. m algorithms, therefore, can make use of this superposition property to speed up certain computations.

A qubit, therefore, is analogous to a bit in a traditional digital computer, and is a type of information device that exhibits coherence. Particularly, a quantum computing device is built up from a ity of information device, e.g., qubit, building blocks. For instance, the computing power of a quantum computer increases as the information devices that form its building blocks are coupled, e.g., entangled, er in a controllable manner.

In such an instance, the quantum state of one information device affects the quantum state of each ofthe other information devices to which it is coupled.

Accordingly, like the bit in classic digital computing, the qubit in quantum computing serves as the basic unit for the encoding of information, such as quantum information. Similar to a bit, the qubit encodes data in a two-state system, which in this instance is a m-mechanical system. Specifically, for the qubit, the two quantum states involve entanglement, such as ing the polarization of a single photon. Hence, where in a classical system, a bit has to be in one state or the other, in a quantum computing platform, the qubit may be in a superposition of both states at the same time, which property is fundamental to quantum processing. Consequently, the distinguishing feature between the qubit and the classical bit is that multiple qubits exhibit quantum entanglement. Such entanglement is a nonlocal property that allows a set of qubits to express higher correlation than is possible in a classical system.

In order to function, such information devices, e.g., m bits, must fulfill several ements. First, the information device must be reducible to a quantum two-level system. This means that the information device must have two distinguishable quantum states that may be used for ming computations. Second, the ation devices must be capable of producing quantum effects like entanglement and superposition. Additionally, in certain ces, the information device may be configured for g information, e.g., quantum information, such as in a coherent form. In such instances, the coherent device may have a quantum state that ts without significant degradation for a long period of time, such as on the order ofmicroseconds or more.

Particularly, quantum entanglement is the al phenomenon that occurs when a pair or a group of particles are generated or otherwise configured to interact in a manner that the quantum state of one particle cannot be described independently of another, despite the space that tes them. Consequently, instead of describing the state of one particle in ion of the others, a quantum state must be described for the system as a whole. In such ces, the measurements of various physical properties, such as position, momentum, spin, and/or polarization, performed on entangled particles are correlated. For example, if a pair of particles are generated in such a way that their total spin is known to be zero, and one particle is found to have clockwise spin on a certain axis, the spin of the other particle, measured on the same axis, will be found to be counterclockwise, as to be expected due to their entanglement.

Hence, one particle of an entangled pair simply " what measurement has been performed on the other, and with what outcome, even though there is no known means for such information to have been communicated between the particles, which at the time of measurement may be separated by arbitrarily large distances. Because of this relationship, unlike cal bits that can only have one value at a time, lement allows multiple states to be acted on simultaneously. It is these unique entangled onships and quantum states that have been capitalized upon for the development of quantum computing.

] Accordingly, there are various kinds of physical operations employing pure qubit states that can be performed. For instance, a quantum logic gate can be formed and configured to operate on the basic qubit, where the qubit undergoes a unitary transformation, such as where the unitary transformations corresponds to rotations, or other quantum phenomena, of the qubit. In fact, any two-level system can be used as a qubit, such as photons, electrons, r spins, coherent light states, optical lattices, Josephson junctions, m dots, and the like. Specifically, a quantum gate is the basis for a quantum circuit operating on a small number of qubits. For instance, a quantum circuit is comprised of quantum gates that act on fixed numbers of qubits, such as two or three, or more. Qubits, therefore, are the building blocks of m circuits, like classical logic gates are for conventional digital circuits. Specifically, a m circuit is a model for quantum computation where the computation is a sequence of quantum gates that are reversible transformations on a quantum mechanical analog of an n-bit er. Such analogous structures are referred to as n-qubit ers. Hence, unlike classical logic gates Quantum logic gates are always reversible.

Particularly, as described herein, a digital logic gate is a physical, wired device that may be implemented using one or more diodes or stors that act as electronic switches for performing logical operations, e.g., Boolean functions, on one or more binary inputs, so as to produce a single binary output. With amplification, logic gates can be cascaded in the same way that Boolean functions can be composed, allowing the construction of a physical model of all of n logic, and therefore, all of the algorithms and mathematics that can be described with Boolean logic can be performed by digital logic gates. In a like manner a cascade of quantum logic gates can be formed for the performance ofBoolean logic operations.

Quantum gates are y represented as matrices. In vanous implementations, a quantum gate acts on k qubits that may be represented by a 2k x 2k unitary matrix. In such instances, the number of qubits in the input and output of the gate should be equal, and the action of the gate on a ic quantum state is found by multiplying the vector that represents the state by the matrix representing the gate. Hence, given this configuration quantum computational operations may be executed on a very small number of m bits. For instance, there are m algorithms that are configured for running much more complex computations faster than any possible probabilistic classical algorithm. Particularly, a quantum algorithm is an algorithm that runs on a quantum circuit model ofcomputation.

Where a cal algorithm is a finite sequence of step-by-step instructions or procedures that may be performed by digital logic circuits of a classic computer; a quantum algorithm is a step-by-step procedure, where each of the steps can be performed on a quantum er. However, even though quantum algorithms exist, such as Shor's, Grovar's, and Simon's algorithms, all classical algorithms can also be performed on a quantum computer with the correct configurations. Quantum algorithms are usually used for those algorithms that are inherently quantum, e.g., such as involving superposition or quantum entanglement. m algorithms may be stated in various models of quantum computation, such as the Hamiltonian oracle model.

Accordingly, as a classical computer has a memory made up of bits, where each bit is ented by either a "1" or a"O"; a quantum computer supports a sequence of qubits where a single qubit can represent a one, a zero, or any quantum osition ofthose two qubit states. Consequently, a pair of qubits can be in any quantum osition of 4 states, and three qubits can be in any superposition of 8 states. In l, a m computer with n qubits can be in an arbitrary superposition of up to 2n different states simultaneously, which compares to a normal computer that can only be in one of these 2n states at any one time. ore, qubits can hold exponentially more information than their classical rparts. In action, a quantum er operates by setting the qubits in a drift that solves the problem by manipulating those qubits with a fixed sequence of quantum logic gates. It is this sequence of quantum logic gates that forms the operations of m algorithms. The calculation ends with a measurement, collapsing the system of qubits into one ofthe 2n pure states, where each qubit is "O" or "1", thereby decomposing into a classical state. Hence, traditional algorithms may also be performed on a m computing platform, where the outcome is typically n classical bits of information.

In standard notation, the basic states of a qubit are referred to as the "O" and "1" states. However, during quantum computation, the state of a qubit, in general, may be a superposition of the basic or basis states such that the qubit has a nonzero probability of occupying the "O" basis state and a simultaneous nonzero probability of occupying the "1" basis state. Accordingly, the quantum nature of the qubit is largely derived from its ability to exist in a coherent osition of basis states, and for the state of the qubit to have a phase.

A qubit will retain this ability to exist as a coherent superposition ofbasis states as long as the qubit is sufficiently isolated from sources ofdecoherence.

Consequently, to te a computation using a qubit, the state of the qubit is measured. As indicated above, when a measurement of the qubit is done, the quantum nature of the qubit may be arily lost and the superposition of the basis states may collapse to either the "O" basis state or the "1" basis state. Thus, in such a manner as this, the qubit regains its similarity to a conventional digital "bit". However, the actual state of the qubit after it has sed will depend on the various probability states present immediately prior to the measurement operation. Thus, qubits may be employed to form quantum circuits, which themselves may be configured to form a quantum computer.

There are l general approaches to the design and operation of a quantum computer. One approach that has been put forth is that of a circuit model for quantum ing. Circuit model quantum computing requires long quantum coherence, so the type of information device used in quantum computers that support such an approach may be the qubit, which by definition has long coherence times. ingly, the circuit model for quantum computing is based upon the premise that qubits can be formed of and be acted on by logical gates, much like bits, and can be programmed using quantum logic in order to perform calculations, such as Boolean computations. ch has been done to develop qubits that can be programmed to perform quantum logic functions in this manner. For example, see Shor, 2001, arXiv.org:quant-ph/0005003, which is hereby incorporated by reference in its entirety. se, a computer sor may take the form of a quantum processor such as a superconducting quantum processor.

A superconducting quantum processor may include a number of qubits and associated local bias devices, for instance, two, three, or more superconducting qubits. ingly, gh in various ments, a computer processor may be configured as a non-traditional onducting processor, in other embodiments, it the computer sor may be configured as a superconducting processor. For instance, in some embodiments, a non-traditional superconducting processor may be configured so as to not focus on quantum effects such as superposition, entanglement, and/or m tunneling, but may rather operate by emphasizing different principles, such as those principles that govern the operation of classical computer processors. In other embodiments, the computer processor may be configured as a traditional superconducting processor such as by being adapted to process through various quantum effects, such as superposition, entanglement, and/or quantum tunneling.

] Accordingly, in various instances, there may be certain ages to the implementation ofsuch superconducting processors. Particularly, due to their natural physical properties, superconducting processors in general may be capable ofhigher switching speeds and r computation times than non-superconducting processors, and therefore it may be more practical to solve certain problems on superconducting processors. Further, detail and embodiments of exemplary m processors that may be used in conjunction with the present devices, systems, and the methods of their use are described in USSNs: 11/317,838; 12/013,192; 12/575,345; 12/266,378; 13/678,266; and 14/255,561; as well as the s divisionals, continuations, and/or uation in parts thereof; including US Patent Nos. 7,533,068; 7,969,805; 9,026,574; 9,355,365; 9,405,876; and all of their foreign counterparts, which are hereby incorporated by reference in their entireties.

] Further, in addition to the above quantum devices and systems, methods for their use in solving complex computational problems are also presented. For instance, the quantum devices and systems herein sed may be employed for controlling the quantum state of one or more ation devices and/or systems, in a coherent manner, so as to perform one or more steps in a bioinformatics and/or genomics processing pipeline, such as for the performance of one or more operations in an image processing, base calling, mapping, ng, sorting, variant calling, and/or other cs and/or bioinformatics ne. In particular embodiments, the one or more operations may include ming a burrowwheelers , smith-waterman, and/or an HMM operation.

Particularly, solving complex cs and/or bioinformatics computational problems using a quantum computing device may include generating one or more qubits and using the same to form a quantum logic circuit representation of the computational problem, encoding the logic circuit representation as a discrete optimization problem, and solving the discrete optimization problem using the quantum processor. The representation may be an arithmetic and/or geometric problem for solution by an addition, subtraction, multiplication, and/or divide circuit. The discrete optimization problem may be composed of a set of miniature optimization problems, where each miniature optimization m encodes a respective logic gate from the logic circuit representation. For instance, a mathematical circuit may employ binary representations of factors, and these binary representations may be decomposed to reduce the total number of variables required to ent the mathematical circuit. Accordingly, in accordance with the ngs herein, a computer processor may take the form of a l and/or an analog processor, for instance, a quantum processor such as a onducting quantum processor. A superconducting quantum processor may include a number of qubits and associated local bias s, for instance two or more superconducting qubits, which may be formed into one or more quantum logic circuit representations.

More ularly, in various embodiments, a superconducting integrated t may be provided. Specifically, in particular embodiments, such a superconducting integrated circuit may include a first superconducting current path that is disposed in a metal, e.g., first, metal layer. A dielectric, e.g., first dielectric, layer may also be included, such as where at least a portion of the dielectric layer is associated within and/or carried on the first metal layer. A second superconducting t path may also be included and disposed in a second metal layer, such as metal layer that is carried on or otherwise associated with the first dielectric layer. In such an embodiment, at least a portion of the second superconducting current path may overlay at least a portion of the first superconducting current path.

Likewise, a second dielectric layer may also be included, such as where at least a portion of the second dielectric layer is associated with or carried on the second metal layer.

Additionally, a third superconducting current path may be included and disposed in a third metal layer that may be associated with or carried on the second dielectric layer, such as where at least a portion of the third superconducting t path may overlay at least a portion of one or both of the first and second superconducting current paths. One or more additional metal layers, dielectric layers, and/or current paths may also be included and configured accordingly.

Further, a first superconducting tion may be positioned between the first superconducting t path and the third superconducting current path, such as where the first superconducting connection extends through both the first dielectric layer and the second dielectric layer. A second superconducting connection may also be included and positioned between the first superconducting current path and the third superconducting t path, such as where the second superconducting tion may extend through both the first dielectric layer and the second dielectric layer. onally, at least a portion of the second superconducting current path may be led by an outer superconducting current path that may be formed by at least a portion of one or more of the first superconducting current path, at least a portion ofthe second superconducting current path, and/or the first and second superconducting connections. Accordingly, in such instances, the second superconducting current path may be configured to couple, e.g., inductively couple, a signal to the outer superconducting current path.

In some embodiments, a mutual inductance between the second superconducting current path and the outer superconducting current path may be sub-linearly proportional to a ess of the first dielectric layer and a thickness ofthe second dielectric layer. The first and the second superconducting connections may also each include at least one respective superconducting via. Further, in various embodiments, the second superconducting current path may be a portion of an input signal line and one or both the first and the third superconducting current paths may be coupled to a superconducting programmable device. In other embodiments, the second superconducting current path may be a portion of a superconducting programmable device and both the first and the third superconducting t paths may be coupled to an input signal line. In particular embodiments, the superconducting programmable device may be a superconducting qubit, which may then be coupled, e.g., quantumly coupled, to one or more other qubits so as to from a quantum circuit, such as ofa quantum processing device.

Accordingly, provided herein are devices, systems, and methods for g ational problems, especially ms related to resolving the genomics and/or bioinformatics bottleneck described herein above. In various ments, these devices, systems and methods introduce a technique y a logic circuit entation of a computational problem may be solved directly and/or may be encoded as a discrete zation problem, and the discrete zation problem may then be solved using a computer processor, such as a quantum processor. For instance, in particular embodiments, solving such discrete optimization problems may include executing the logic circuit to solve the original computational problem.

] Hence, the devices, systems, and methods described herein may be implemented using any form of computer processor such as including traditional logic circuits and/or logic circuit representations, such as configured for use as a quantum processor and/or in super conducting processing. Particularly, various steps in performing an image processing, base calling, mapping, aligning, and/or variant calling bioinformatics pipeline may be encoded as discrete zation problems and as such may be particularly well-suited to be solved using the quantum processors, disclosed herein. In other instances, such computations may be resolved more generally by a er sor that harnesses m effects to achieve such computation; and/or in other instances, such computations WO 14320 PCT/0S2017/036424 may be performed using a dedicated integrated circuit, such as an FPGA, ASIC, or structured ASIC, as described herein in detail. In some embodiments, the discrete optimization problem is cast as a problem by configuring the logic ts, qubits, and/or couplers in a quantum processor. In some embodiments, the quantum processor may be specifically adapted to facilitate solving such te optimization problems.

As disclosed throughout this specification and the appended claims, reference is often made to a "logic circuit representation", e.g., of a computational problem. Depending on the context, a logic circuit may incorporate a set of logical inputs, a set of logical outputs, and a set c gates (e.g., NAND gates, XOR gates, and the like) that transform the logical inputs to the logical outputs through a set of intermediate logical inputs and intermediate l outputs. A complete logic t may include a representation of the input(s) to the computational problem, a representation ofthe output(s) ofthe computational problem, and a representation ofthe sequence of intermediate steps in between the s) and the output(s).

Thus, for various purposes of the present devices, systems, and methods, the ational problem may be defined by its input(s), its output(s), and the intermediate steps that orm the s) to the output(s) and a "logic circuit representation" may include all of these elements. Those of skill in the art will appreciate that the encoding of a "logic circuit representation" of a computational problem as a discrete optimization m, and the uent mapping of the discrete optimization problem to a quantum processor, may result in any number of layers involving any number of qubits per layer. Furthermore, such a mapping may implement any scheme of inter-qubit coupling to enable any scheme of inter-layer coupling (e.g., ng between the qubits of different layers) and layer coupling (e.g., ng between the qubits within a particular layer).

Accordingly, as indicated, in some embodiments, the structure of a logic circuit may be stratified into layers. For example, the logical s) may represent a first layer, each sequential logical (or arithmetic) operation may represent a respective additional layer, and the logical output(s) may represent another layer. And as previously described, a logical operation may be executed by a single logic gate or by a combination of logic gates, depending on the specific logical operation being executed. Thus, a "layer" in a logic circuit may include a single logic gate or a combination of logic gates depending on the particular logic circuit being implemented.

Consequently, in various embodiments such as where the structure of a logic t stratifies into layers (for example, with the logical input(s) representing a first layer, each sequential logical operation representing a respective additional layer, and the logical output(s) representing another , each layer may be embodied by a tive set of qubits in the quantum and/or superconducting processor. For e, in one ment of a quantum processor, one or more, e.g., each, row of qubits may be programmed to ent a respective layer of a m logic circuit. That is, particular qubits may be programmed to represent the inputs to a logic circuit, other qubits may be programmed to represent a first l operation (executed by either one or a plurality of logic gates), and further qubits may be programmed to represent a second logical operation (similarly executed by either one or a plurality of logic gates), and yet further qubits may be programmed to represent the outputs ofthe logic circuit.

Additionally, with various sets of qubits enting s layers of the problem, it can be advantageous to enable independent dynamic control of each respective set. Further, in various embodiments, various serial logic ts may be mapped to the quantum processor, and the respective qubits mapped to facilitate the onal interactions for quantum processing in a manner suitable to enable independent control thereof. From the above, those of skill in the art will appreciate how a similar objective function may be defined for any logic gate. Thus, in some embodiments, the problem representing a logic circuit may essentially be comprised of a plurality ofminiature optimization problems, where each gate in the logic circuit corresponds to a particular miniature optimization m.

Hence, exemplary logic circuit representations may be generated using systems and methods that are known in the art. In one example, a logic circuit representation of the computational problem, e.g., the genomics and/or bioinformatics m, may be generated and/or encoded using a classical digital computer processor and/or a quantum and/or onducting processor as described herein. Accordingly, a logic t representation of the computational problem may be stored in at least one computer- or processor-readable storage medium, such as a computer-readable non-transitory storage medium or memory (e.g., volatile or non-volatile). Therefore, as discussed herein, the logic circuit entation of the computational problem may be encoded as a discrete optimization problem, or a set of optimization objectives, and in various embodiments, such as where a classical digital computer processing paradigm is configured to solve the problem, the system may be configured so that bit strings that satisfy the logic circuit have energy of zero and all other bit strings have energy greater than zero, where the discrete optimization problem may be solved in such a manner as to establish a on to the original computational problem.

Further, in other embodiments, the discrete optimization problem may be solved using a er processor, such as a quantum sor. In such an instance, solving the discrete optimization problem may then involve, for example, evolving the quantum sor to the configuration that minimizes the energy of the system in order to establish a bit string that satisfies the zation objective(s). Accordingly, in some embodiments, the act of solving a te optimization problem may include three acts. First, the discrete optimization problem may be mapped to a computer processor. In some embodiments, the computer processor may include a quantum and/or super conducting processor and mapping the discrete optimization problem to the computer processor may include programming the elements (e.g., qubits and rs) of the quantum and/or superconducting processor.

Mapping the discrete optimization m to the computer processor may include the discrete optimization problem in at least one computer or processor-readable storage medium, such as a computer-readable non-transitory storage medium or memory (e.g., volatile or atile Accordingly, in view of the above, in various instances, a , system, and method for executing a sequence analysis pipeline, such as on genomics material, is ed.

For instance, the genomics material may include a plurality ofreads of genomic data, such as in an image file, BCL, FASTQ file, and the like. In various embodiments, the device and/or system may be employed for executing a sequence analysis on genomic data, e.g., reads of genomic data, such as by using an index of one or more genetic reference sequences, e.g., stored in a memory, for example, where each read of genomic data and each reference sequence represents a sequence ofnucleotides.

Particularly, in various embodiments, the device may be a quantum computing , such as formed of a set of quantum logic circuits, e.g., hardwired quantum logic ts, for instance, where the logic ts are interconnected with one r. In various instances, the quantum logic circuits may be interconnected by one or more superconducting connections. Additionally, one or more of the superconducting connections may include a memory interface, such as for accessing the memory. Together the logic circuits and interconnects may be configured to process information represented as a quantum state that is itself ented as a set of one or more qubits. More particularly, the set of hardwired quantum logic circuits may be arranged as a set of processing s, such as where each processing engine may be formed of a subset of the hardwired quantum logic circuits, and may be configured to perform one or more steps in the ce analysis pipeline on the reads of genomic data.

] For instance, the set of processing engines may be configured so as to include an image processing, base calling, mapping, aligning, sorting, variant calling, and/or other genomics and/or bioinformatics processing module. For example, in various embodiments, a g module, such as in a first red configuration, may be included. Additionally, in further embodiments, an ent module, such as in a second red configuration, may be included. Further, a sorting module, such as in a third hardwired configuration, may be included. And, in additional embodiments, a variant calling module, such as in a fourth hardwired configuration, may be included. Further still, in various embodiments, an image sing and/or base calling module may be included in further hardwired configurations, such as where one or more of these hardwired configurations may include hardwired quantum logic circuits may be arranged as a set ofprocessing engines.

More particularly, in particular instances, a quantum computing device and/or system may include a mapping module, where the mapping module comprises a set of quantum logic circuits that are ed as a set ofprocessing engines, one or more ofwhich are configured for performing one or more steps ofa mapping procedure. For instance, one or more quantum processing engines may be configured to receive a read of genomic data, such as via one or more of a plurality of superconducting connections. Further, the one or more quantum sing engines may be ured to extract a portion of the read to generate a seed, such as where the seed may represent a subset of the sequence of nucleotides represented by the read. Additionally, one or more ofthe quantum processing engines may be configured to calculate a first address within the index based on the seed, and access the s in the index in the memory, so as to receive a record from the address, such as where the record represents position information in the genetic nce ce. Further more, the one or more m processing engines may be configured to ine, e.g., based on the record, one or more matching positions from the read to the genetic reference sequence; and output at least one ofthe matching positions to the memory via the memory interface.

Further still, the mapping module may include a set of quantum logic circuits that are arranged as a set of processing engines configured for calculating a second address within the index, e.g., based on both of the record and of a second subset of the sequence of nucleotides that is not contained in the first subset of the ce of nucleotides. The processing engine(s) may then access the second address in the index in the memory so as to receive a second record from the second address, such as where the second record, or a subsequent record, includes position ation in the genetic reference ce. The processing engine may further be configured for determining, based on the position information, the one or more matching positions from the read to the genetic reference sequence.

Additionally, in various instances, a quantum computing device and/or system may include an alignment module, where the alignment module comprises a set of m logic circuits that are arranged as a set of processing s, one or more of which are configured for performing one or more steps of an alignment procedure. For instance, one or more quantum processing engines may be configured to e a plurality of mapped positions for the read from the memory, and to access the memory to retrieve a segment of the genetic reference sequence corresponding to each of the mapped positions. The one or more processing engines formed as an alignment module may further be configured to calculate an alignment ofthe read to each retrieved segment ofthe genetic reference sequence so as to generate a score for each alignment. r, once one or more scores have been generated at least one best-scoring alignment of the read may be selected. In particular instances, the quantum computing device may include a set of quantum logic circuits that are arranged as a set of processing engines that are configured for performing a gapped or gapless alignment, such as a Smith Waterman alignment.

Further, in n instances, a quantum computing device and/or system may include a variant g module, where the variant calling module comprises a set of quantum logic circuits that are ed as a set of processing s, one or more ofwhich are configured for performing one or more steps of a variant calling procedure. For instance, the quantum computing variant calling module may include a set of quantum logic circuits that are adapted for executing an analysis on a plurality of reads of genomic data, such as using one or more candidate haplotypes, e.g., stored in a , where each read of c data and each candidate haplotype represent a sequence ofnucleotides.

Specifically, the set of quantum logic circuits may be formed as one or more quantum processing engines that are configured to receive one or more of the reads of genomic data and generate and/or e the one or more ate ypes, e.g., from the memory, such as via one or more of a plurality of superconducting connections. Further, the one or more quantum processing engines may be configured to receive one or more ofthe reads of genomic data and the one or more candidate haplotypes from the memory, as well as to compare nucleotides in each of the one or more reads to the one or more ate haplotypes, so as to determine a probability of each candidate haplotype representing a correct t call. Additionally, one or more of the m processing engines may be configured to generate an output based on the determined probability.

Additionally, in various instances, the set of quantum logic circuits may be formed as one or more quantum processing engines that are ured to ine a probability of observing each read of the plurality of reads based on at least one candidate haplotype being a true ce of nucleotides, e.g., of a source organism of the plurality of reads. In ular instances, with respect to determining probability, the one or more quantum processing engines may be configured for executing a Hidden Markov Model. More particularly, in additional embodiments, the one or more quantum processing s may be configured for merging the plurality of reads into one or more contiguous nucleotide sequences, and/or for generating the one or more candidate ypes from the one or more contiguous nucleotide ces. For instance, in various embodiments, the g of the ity of reads includes the one or more quantum processing engines constructing a De Bruijn graph.

Accordingly, in light of the above, a system for ming vanous computations in solving problems related to genomics and/or bioinformatics processing is provided. For ce, the system may include one or more of an onsite automated sequencer, e.g., NGS, and/or a processing server either or both of which may include one or more CPUs, GPUs, and/or other integrated circuits, such as including an FPGA, ASIC, and/or structured ASIC that are configured as herein described for performing one or more steps in a sequence analysis pipeline. Particularly, the Next Gen Sequencer may be configured for sequencing a plurality of nucleic acid sequences so as to generate one or more image, BCL, and/or FASTQ files representing the sequenced nucleic acid sequences, which nucleic acid sequences may be a DNA and/or an RNA sequence. These sequence files may be processed by the sequencer itself or by an associated server unit, such as where the sequencer and/or the associated server includes an integrated circuit, such as an FPGA or ASIC, configured as herein described for performing one or more steps in a secondary sequence analysis pipeline. r, in various instances, such as where the automated sequencer and/or an associated server is not configured for performing a secondary sequence analysis on the data generated from the sequencer, the generated data may be transmitted to a remote server that is configured for performing a secondary and/or tertiary sequence analysis on the data, such as via a cloud mediated interface. In such an instance, the cloud accessible server may be configured for receiving the generated ce data, such as in image, BCL, and/or in FASTQ form, and may further be configured for performing a primary, e.g., image sing, and/or a secondary and/or tertiary processing analysis, such as a sequence analysis pipeline, on the ed data. For instance, the cloud accessible server may be one or more servers including a CPU and/or a GPU one or both ofwhich may be associated with an integrated circuit, such as an FPGA or ASIC, as herein bed. Particularly, in n instances, the cloud accessible server may be a quantum computing server, as herein described.

Specifically, the cloud accessible server may be configured for performing a primary, secondary, and/or tertiary genomics and/or bioinformatics analysis on the received data, which analyses may include performing one or more steps in one or more of an image processing, base calling, mapping, aligning, sorting, and/or variant calling protocols. In certain instances, some of the steps may be performed by one processing platform, such as a CPU or GPU, and others may be med by another processing rm, such as an associated, e.g., tightly d, integrated circuit, such as an FPGA or ASIC, that is specifically configured for performing various of the steps in the sequence analysis pipeline.

In such ces, where data and the results of is are to be transferred from one platform to r, the system and its components may be configured for compressing the data prior to transfer, and decompressing the data once transferred, and as such the system components may be configured for generating one or more of a SAM, BAM, or CRAM files, such as for transfer. Additionally, in various embodiments, the cloud accessible server may be a m computing platform that is configured herein to perform one or more steps in the sequence analysis pipeline, as described herein, and may include the performance of one or more secondary and/or tertiary sing steps in accordance with one or more of the methods disclosed herein.

Further, with respect to quantum ing, detail and embodiments of exemplary quantum processors and the methods of their use that may be employed in conjunction with the present devices, systems, and methods are described in U.S. Patent Nos. 7,135,701; 7,533,068; 7,969,805; 8,560,282; 8,700,689; 8,738,105; 9,026,574; 9,355,365; 9,405,876; as well as the various counterparts thereto, which are hereby incorporated by reference in their entireties.

] Additionally, with respect to the artificial intelligence module set forth above, in one aspect, a cloud accessible artificial intelligence module is provided, and is configured for being communicably and operably d to one or more ofthe other ents ofthe BioIT pipeline disclosed herein. For instance, the A/I module may work y with the WMS so as to efficiently direct and/or control the various processes of the system sed herein. Accordingly, in various ments, an A/I module is provided, wherein the A/I module is configured for acting as an interface between the genomic world and the clinical world.

For instance, in various instance, the BioIT system may be configured for receiving clinical data. In such an instance, the workflow manager system may be configured for ing the clinical data, and other such data, and implementing one or more deterministic rule s, so as to derive results data pursuant to its analysis of the clinical data. For example, in certain embodiments, the various databases of the system may be configured so as to have a relational architecture.

These constructions may be represented by one or more table structures. A series of tables, for instance, may then be employed by which correlations may be made by the WMS in an iterative n. For example, in various use models a first correlation may be made with t to a subject's name with a medical condition. Another table may then be employed to correlate the subject's medical condition with their medicine. Likewise, a further table may be used to correlate the progress of the medicine with respect to the alleviation of symptoms and/or the disease itself. A key may be used to correlate the tables, which key may be accessed in response to question prompt or d. The key may be any common identifier, such as a name, a , e.g., a social security number, tax identification number, employee number, a phone number, and the like, by which one or more of the tables may be accessed, correlated, and/or a question answered. Accordingly, without the key it becomes more difficult to build correlations between the information in one table with that ofanother.

] However, in other instances, the A/I module may be configured to provide a more comprehensive analysis on ted and/or provided data. For example, the A/I module may be configured so as to implement one or more machine learning protocols on the WO 14320 PCT/0S2017/036424 data of the system that are devised to teach the AI module to make correlations between the genomic data, e.g., generated by the system, and a clinical deposition ofone or more subjects, such as in view ofEMR and other clinically relevant data input into the system.

Specifically, the A/I module may include programing directed at training the system to more rapidly, e.g., instantly, recognize how an output was achieved based on the type and characteristics ofthe input received. The system therefore is configured for learning from the inputs it receives, and the results it outputs, so as to be able to draw ations more rapidly and accurately based on the initial input of data received. Typically, the input data may be of two general types. In a first instance, the data may be of a type where the output, e.g., the answer, is known. This type of data is may be input into the system and used for training purposes. The second type of data may be that where the answer is n, and therefore, must be determined, this data will likely be genomic data, upon which analysis is to be made, or clinical data to which a clinically nt results are to be determined.

Specifically, these methods may be used to enhance the A/I modules ability to learn from the first type of input data, so as to better t the e for the second kind of input data.

Specifically, based on historical evidence, the A/I module may be configured to learn to predict outcomes based on previously observed data.

More specifically, a clinical genomics platform is presented herein, wherein the clinical genomics platform is configured to correlate clinical es of diseases with genomics data. In such an instance, the clinical profiles of subjects may be input into the system and may be assessed along with their determined genomic profile. Particularly, in combining these two datasets, the A/I module is configured for determining the various interrelationships n them. Accordingly, in a first step, a graph se or knowledge graph may be constructed. For example, in this instance, the knowledge graph may be composed of three typical elements, which lly include a subject, a predicate, and an object, these may form nodes, and the relationship between the nodes must be ined.

Any particular data point may be selected as a node, and nodes may vary based on the queries being performed. There are several different types of relationships that can be determined.

For instance, onships may be determined based on their effects, e.g., they are effect based; or they may be determined based on nces, e.g., relationships that are unknown but determinable.

Accordingly, with respect to constructing the knowledge graph, any particular data point may form a node. For instance, on one side of the graph a disease condition may form a node, and on the other side ofthe graph a genotype, e.g., a ce ofvariances, may form a node. In between these two nodes may be a third node, e.g., a series of third nodes, such as one or more symptoms, one or more medications, one or more allergies, one or more other conditions or phenotypic traits, e.g., blood pressure, cholesterol, etc. Additionally, in between these nodes are the relationships that may be ined.

Specifically, when building the knowledge graph, clinical data input into the system, such as from a medical s facility, e.g., onic medical records, family history of medical conditions, etc. that may be encrypted and securely transferred electronically. se, genomic data from the subject may be sequenced and ted in accordance with the secondary processing steps set forth herein. Further, once these two nodes have been established one or more third nodes may be input into the system, from the ce ofwhich the relationship(s) between the two original nodes may be determined.

For instance, in one example, a first node may be represented by the medical records of a person or a tion of people, and a second node may be represented by a disease characteristic. In such an instance, one or more third nodes may be input to the system and generated within the graph, such as where the third node may be a medication; a physical, biological, mental, ion and/or characteristic; an allergy; geographical region; diet, a food item and/or ingredient; an environmental condition; a geographical condition; powerlines, ar towers; and/or the like. A series of relationships may then be determined by analyzing various points of connection between these three items. Particularly, in a particular instance, one node may represent a patient suffering from a disease condition, a second node may be the patient's genomic data, and among the third nodes may be the patient's genomic variations, e.g., the subject's mutations, chromosome by chromosome, their medication, physiological conditions, and the like. Likewise, this process may be repeated for le subjects having the same diagnosis and/or condition. Hence, in a manner such as this the correlation between the clinical and cs worlds may be determined.

] Accordingly, a step in building a clinical genom1cs graph is to define the anchor nodes, these represent the two bounding elements between which all the various commonalities are defined and explored. Hence, a further step is to define all the possible known correspondences between the two anchor nodes, which may be represented in the graph as a third node. These known correspondences may be built around detailing the s caused by and/or the characteristics of one node or the other. These may form the known WO 14320 PCT/0S2017/036424 and/or able relationships between the nodes. From these known relationships, a second type of relationship may be explored and/or determined which relationships may be built on inferences. Further, to better determine causal and/or predictable outcomes the various different relationships may be weighted, such as based on the degree of nty, number of commonalities, number of instances sharing the node, number of common onships, and the like.

Hence, the construction and implementation of a dynamic knowledge graph is at the heart of the clinical genomics processing platform. As indicated, the various processing rms of the global system may be coupled together, so as to seamlessly er data between its various ents. For instance, as indicated, the mapping, aligning, and/or variant calling pipelines may be configured for transmitting its data, e.g., s data, to the artificial intelligence module. ularly, the A/I module may be configured for receiving inputs of data from one or more of the secondary processing rm components, and/or one or more of the other component of the system. More ularly, the A/I module is configured for receiving mapping, aligned, and/or variant called data from the mapper, aligner, and/or variant calling processing engines, and for taking that data and using it to generate one or more nodes within the knowledge graph. Further, as indicated, the A/I module may be configured for receiving input data from one or more other sources, such as from a medical office, a health care service provider, a research lab, a records storage facility, and the like, such as where the records include data pertaining to the physical, , and/or emotional well-being of one or more subjects, and for taking that data and using it to generate one or more nodes within the knowledge graph. onally, once the knowledge graph architecture has been constructed, it can continually be updated and grown by adding more and more pertinent data into the dge structure, building more and more potential nodes and/or relationships. In such an instance, the bounding nodes may be of any combination of nodes, and as such, in certain instances, may be user selectable. For instance, in various embodiments, the system may be configured for being accessible by a third party. In such an instance, the user may access the A/I module, e.g., via a ly configured user interface, upload pertinent information into the system and/or determine the relevant nodes by which to bound an inquiry, e.g., by clicking on or drag and dropping them, and may formulate a relevant question to be answered by the A/I module. Accordingly, the user may review and/or select the bounding nodes, and then allow the system to generate an appropriate knowledge map employing the selected nodes, and determine the relationships between the nodes, from which relationships various inquiries may be queried and answered, or at least be inferred, e.g., by the A/I system.

For example, in one use model, a user may be a physician who desires to know how a certain drug dosage is affecting a patient with respect to a given e.

Consequently, the physician may upload the patient's EMR, the disease condition, and the drug dosage, and with this data the A/I module may generate a suitable knowledge graph (and/or add to an already ng knowledge graph), from which knowledge graph the bounding nodes may be selected and relationships determined. Further, in various instances, the user may upload the patient's genetic data, which data may be subjected to secondary processing, and the results thereof, e.g., mapped, aligned, and/or variant call result data, and uploaded into the A/I module. In such an instance, the disease and/or EMR and/or family medical y data may be correlated with the genomic data from which data various relationships may be determined, inferences assessed, and predictions made.

Specifically, a subject's VCF may be entered into the , e.g., all of the determined somal properties may be uploaded, for instance, as a constellation of nodes, which nodes may be used to determine various relationships pertinent to the subject, such as by querying the system and allowing it to generate the appropriate tions from which an answer may be inferred. More specifically, one or more subject's phenotypical characteristics, e.g., the human phenotype ontology, may be uploaded into the system, so as to generate a further constellation of nodes. For instance, when the genomic and/or medical histories of two people are d into the system, any relationships between them may be determined by the A/I module, such as with respect to common genotypes, phenotypes, conditions, environments, geographies, allergies, ethnic-cultural backgrounds, tions, and the like. r, relationships between two or more characteristics in a subject, or between subjects, may be determined. For example, a relationship between a subject's systolic and diastolic blood pressure may be determined by the system. Specifically, a series of ic systolic and lic readings may be entered into the system, whereby the machine learning platform of the system may analyze the readings, and/or determine one or more relationships n the two, such that if a given systolic input is entered into the system, the predicted diastolic output may be given, taking the predictive s between the two into account. It is to be noted that gh the preceding e was given with respect to blood pressure, within a single subject, the same will apply to any to given nodes that are in a mathematical relationship to one another, such as with respect to a multiplicity of subjects and/or a variety ofconditions.

Additionally, although in some instances, the relationships may be configured in a linear array, such as to form a neural network of information, in various other instances, the relationships may be formed in a multiplicity of stages, such as in a deep learning protocol. For instance, the A/I system may be adapted so as to process information in a layered or multi-staged n, such as for the purpose of deep learning. Accordingly, the system may be configured to evaluate data in stages. Specifically, the A/I module may be adapted such that as it examines various data, such as when performing a learning protocol, stage by stage, each connection between data gets weighted by the system, e.g., based on historical evidence and/or characteristics of onships.

The more stages of learning that are ted within the system the better the weighting between junctions will be, and the deeper the learning. Further, uploading data in stages allows for a greater convergence of data within the system. Particularly, various feature extraction paradigms may also be employed so as to better organize, weight, and analyze the most salient features of the data to be uploaded. Additionally, in order to better correlate the data, one or more users may input and/or te basic weighting functions, while the system itself may employ a more advanced weighting function based on active learning protocols.

To provide for interaction with a user, one or more aspects or es of the t matter described herein can be implemented on a er having a display device, such as for example a e ray tube (CRT), a liquid crystal display (LCD) or a light ng diode (LED) monitor for ying information to the user and a keyboard and a pointing device, such as for example a mouse or a all, by which the user may provide input to the computer. Other kinds of s can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, ing, but not limited to, acoustic, speech, or tactile input. Other possible input s e, but are not limited to, touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive trackpads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.

The subject matter described herein can be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. The implementations set forth in the foregoing ption do not represent all implementations consistent with the subject matter described herein. Instead, they are merely some examples consistent with aspects d to the described subject . Although a few ions have been described in detail above, other modifications or additions are possible. In particular, further features and/or ions can be provided in addition to those set forth herein. For example, the implementations described above can be directed to various combinations and subcombinations of the disclosed features and/or ations and subcombinations of several further features disclosed above. In addition, the logic flows depicted in the accompanying figures and/or described herein do not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope ofthe following claims.

Claims

1. An integrated circuit for executing a sequence analysis pipeline on a read of genomic data, derived from a genetic sequence of a subject, using one or more of a reference genetic sequence and a candidate haplotype sequence to ine a probability of observing the read of genomic data assuming the candidate haplotype sequence is a true representation of the t's genetic sequence, each read of genomic data, the genetic reference sequence, and the candidate haplotype sequence enting a sequence of nucleotides, the integrated circuit being formed of a set of reconfigurable hardwired digital logic circuits that are interconnected by a plurality of physical electrical interconnects, one or more of the plurality of physical electrical interconnects comprising a memory interface for the integrated circuit to access a memory, the reconfigurable hardwired digital logic ts being arranged as a first set of processing engines, the first set of processing engines to perform a plurality of steps in the sequence analysis pipeline on the read of genomic data and the candidate ype ce, the first set of sing engines comprising a ate haplotype generation and analysis module in a first hardwired uration to: receive from the memory, via at least one of the plurality of physical electrical interconnects, a plurality of aligned reads of genomic data; form a pile up out of the plurality of aligned reads of genomic data; generate a graph of potential candidate haplotype sequences from the pile up of reads of genomic data to produce a generated graph; extract a candidate haplotype sequence from the generated graph of potential haplotype sequences to e an extracted contiguous candidate haplotype sequence; select a read of genomic data for sequence analysis; compare the sequence of nucleotides of the uous candidate haplotype sequence to the sequence of nucleotides of the read of genomic data; m a Hidden Markov Model (HMM) operation on each of the nucleotide sequences of the read of genomic data and the contiguous candidate haplotype sequence; and determine a ility of observing the read of genomic data assuming the candidate ype sequence is a true representation of the subject's genetic sequence.

2. The integrated circuit in accordance with claim 1, wherein the extraction of the candidate haplotype sequence further comprises an active region identification to identify one or more positions within a region of the pileup that disagrees with the reference genetic sequence.

3. The ated circuit in accordance with claim 2, wherein the active region identification comprises generating a virtual window around the active region so that only a sequence of nucleotides within the virtual window is selected for extraction of the candidate ype sequence.

4. The ated circuit in accordance with claim 3, n the extracting of the contiguous sequence comprises localized haplotype assembly.

5. The integrated circuit in accordance with claim 1, wherein the generated graph is a De Bruijn graph from which graph the uous candidate haplotype sequence is extracted.

6. The integrated circuit in accordance with claim 5, wherein each extracted contiguous candidate haplotype sequence is d back to the reference genetic sequence to produce a CIGAR .

7. The integrated circuit in accordance with claim 6, wherein the extracted contiguous candidate haplotype sequence is smith-waterman aligned.

8. The integrated circuit in accordance with claim 5, wherein the reconfigurable hardwired l logic circuits are further arranged as a second set of sing s, the second set of processing engines comprise an HMM module, the HMM module configured to: e both the extracted uous candidate haplotype sequence and the read of genomic data; uct a computational matrix, the computational matrix comprising a first axis and a second axis, the first axis comprising the candidate haplotype sequence, and the second axis comprises the read of genomic data, the computational matrix for comparing the sequence of nucleotides of the read of genomic data to the sequence of nucleotides of the candidate haplotype sequence; and traverse a pathway through the computational matrix by performing an HMM calculation using the input values for each of the computational cells in the pathway to thereby determine a final probability of observing the sequence of the read of genomic data assuming the candidate haplotype sequence is a true representation of the subject's genetic sequence.

9. The integrated circuit in accordance with claim 8, wherein a plurality of candidate haplotype sequences is extracted from one or more pathways through the computational matrix, where each path through the matrix comprises a single candidate haplotype sequence.

10. The integrated circuit in accordance with claim 9, wherein each read of the pile up of reads is tested against each extracted candidate haplotype sequence ted by the candidate haplotype generation and analysis .

11. The integrated circuit in accordance with claim 10, wherein the comparing comprises performing a read likelihood ation to produce a maximum likelihood alignment sequence.

12. The integrated circuit in accordance with claim 11, wherein the comparing further comprises genotyping.

13. The ated circuit in accordance with claim 11, wherein the maximum likelihood alignment ce is subjected to a variant calling operation.

14. The integrated circuit in accordance with claim 13, wherein the reconfigurable hardwired digital logic circuits are additionally arranged as a third set of processing engines, the third set of processing engines comprise a Variant Calling module, the t Calling module configured to receive the m likelihood alignment sequence and perform one or more Variant Calling operations on the m hood alignment sequence to produce one or more Variant Calls.

15. An ated circuit for executing a sequence analysis pipeline on a read of genomic data, derived from a genetic ce of a subject, using one or more of a reference genetic sequence and a candidate haplotype sequence to determine a probability of observing the read of c data assuming the candidate haplotype sequence is a true representation of the subject's genetic sequence, each read of c data, the reference genetic sequence, and the candidate haplotype sequence representing a sequence of nucleotides, the ated circuit being formed of a set of reconfigurable hardwired digital logic circuits that are interconnected by a plurality of physical electrical interconnects, one or more of the plurality of physical electrical onnects comprising a memory interface for the integrated circuit to access a memory, the reconfigurable hardwired digital logic circuits being arranged as a first set of processing engines, the first set of sing engines to perform a plurality of steps in the sequence analysis pipeline on the read of genomic data and the candidate haplotype sequence, the first set of processing engines comprising a candidate haplotype generation and is module in a first hardwired configuration to: e, via at least one of the plurality of physical electrical interconnects, a plurality of reads of genomic data; form a pile up out of the plurality of reads of genomic data; te a graph of potential candidate haplotype sequences from the pile up of reads of genomic data to produce a generated graph; extract a candidate haplotype sequence from the generated graph of potential haplotype sequences to produce an extracted candidate haplotype ce; and test a read of the pile up of reads t the extracted candidate haplotype sequence so as to determine a probability of observing the read of c data assuming the candidate haplotype sequence is a true representation of the subject's genetic sequence.

16. The integrated circuit in accordance with claim 15, wherein the extraction of the candidate haplotype ce further comprises an active region identification to identify one or more positions within a region of the pileup that disagrees with the reference genetic sequence.

17. The integrated circuit in ance with claim 16, wherein the active region identification comprises generating a virtual window around the active region so that only the ce within the virtual window is ed for extraction of the candidate haplotype sequence.

18. The integrated circuit in accordance with claim 17, wherein the ting of the candidate haplotype sequence comprises zed haplotype assembly.

19. The integrated circuit in accordance with claim 18, wherein the generated graph is a De Bruijn graph from which graph the candidate haplotype sequence is extracted.

20. The integrated t in accordance with claim 19, wherein the reconfigurable hardwired digital logic circuits are further arranged as a second set of processing s, the second set of processing engines comprise an HMM module, the HMM module configured to: receive both the extracted candidate ype sequence and the read of genomic data; construct a computational matrix, the computational matrix comprising a first axis and a second axis, the first axis comprising the extracted candidate haplotype sequence, and the second axis comprises the read of genomic data, the computational matrix for comparing the sequence of nucleotides of the read of genomic data to the sequence of tides of the extracted candidate haplotype sequence; and traverse a pathway through the computational matrix by ming an HMM calculation using the input values for each of the computational cells in the pathway to thereby determine a final probability of observing the sequence of the read of genomic data assuming the extracted candidate haplotype sequence is a true entation of the subject's genetic sequence.

21. An integrated circuit for generating a candidate haplotype ce using one or more of a read of genomic data, derived from a genetic ce of a subject, and a reference genetic sequence, each read of genomic data and the reference genetic sequence representing a sequence of nucleotides, the integrated t being formed of a set of reconfigurable hardwired digital logic circuits, the reconfigurable hardwired digital logic circuits being arranged as a first set of processing engines, the first set of processing engines comprising a candidate haplotype tion module in a first hardwired configuration to: receive a plurality of reads of c data; form a pile up out of the plurality of reads of genomic data; generate a graph of potential candidate haplotype sequences from the pile up of reads of genomic data to produce a generated graph; extract a candidate haplotype sequence from the generated graph of potential haplotype ces to produce an extracted candidate haplotype sequence.

22. The integrated circuit in accordance with claim 1, wherein the extraction of the candidate haplotype sequence r comprises active region identification to identify one or more positions within a region of the pileup that disagrees with the reference genetic sequence.

23. The integrated circuit in accordance with claim 22, wherein the active region identification comprises generating a virtual window around the active region so that only a sequence within the virtual window is selected for extraction of the candidate haplotype sequence.

24. The integrated circuit in accordance with claim 23, wherein the generating of the ate haplotype sequence ses localized haplotype assembly.

25. The integrated circuit in ance with claim 24, wherein the generated graph is a De Bruijn graph from which graph the candidate haplotype sequence is extracted.

26. A system for executing a sequence analysis on a read of genomic data, derived from a c ce of a subject, using one or more of a reference genetic ce and a candidate haplotype sequence to determine a probability of observing the read of genomic data assuming the candidate haplotype sequence is a true representation of the subject's genetic sequence, each read of genomic data, the genetic reference sequence, and the candidate haplotype ce representing a sequence of nucleotides, the system comprising: a based server, the cloud based server comprising one or more of a central processing unit (CPU) or a graphics processing engine (GPU); and an integrated circuit connected with the cloud-based server, the integrated circuit being formed of a set of reconfigurable hardwired digital logic circuits that are interconnected by a plurality of physical electrical interconnects, the reconfigurable hardwired digital logic circuits being arranged as a first set of processing engines, the first set of processing engines to perform a plurality of steps in the sequence analysis on the read of genomic data and the candidate haplotype sequence, the first set of processing engines comprising a candidate haplotype generation and analysis module in a first hardwired configuration to: receive a plurality of reads of genomic data; form a pile up out of the ity of reads of genomic data; generate a graph of potential candidate haplotype ces from the pile up of reads of genomic data to produce a generated graph; extract a candidate haplotype sequence from the ted graph of ial haplotype sequences to produce an extracted candidate haplotype sequence.

27. The integrated circuit in accordance with claim 26, wherein the extraction of the candidate haplotype sequence further comprises active region identification to identify one or more ons within a region of the pileup that disagrees with the reference genetic sequence.

28. The integrated circuit in accordance with claim 27, wherein the active region fication comprises generating a virtual window around the active region so that only a sequence within the virtual window is selected for extraction of the candidate ype sequence.

29. The integrated circuit in accordance with claim 28, wherein the generating of the candidate haplotype sequence comprises localized haplotype ly.

30. The integrated circuit in accordance with claim 29, wherein the ted graph is a De Bruijn graph from which graph the candidate haplotype sequence is extracted. WO 14320 W0 14320 PCT/USZOl