NZ789147A

NZ789147A - Bioinformatics systems, apparatus, and methods for performing secondary and/or tertiary processing

Info

Publication number: NZ789147A
Application number: NZ789147A
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. ularly, a genomics analysis platform for executing a sequence analysis pipeline is provided. The genomics analysis rm 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 sing 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 al 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 sing engines to m the second set of genomic processing steps. A shared memory is also provided.

NZ 789147 BIOINFORMATICS SYSTEMS, APPARATUSES, AND METHODS FOR MING SECONDARY AND/OR RY SING Cross-Reference to Related Application The current application claims ty 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. 62/414,637, filed October 28, 2016, U.S. Application No. 15/404,146, filed y 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 application are incorporated herein by reference in their entireties.

Field ofthe Disclosure The subject matter described herein s to bioinformatics, and more particularly to systems, tuses, and methods for implementing bioinformatic protocols, such as performing one or more functions for ing genomic data on an integrated circuit, such as on a hardware processing platform.

Background to the Disclosure As described in detail herein, some major computational challenges for highthroughput DNA sequencing 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 analysis on a wide range ofsequencing data sets derived from such genomic data.

Keeping pace with such increased sequencing throughput generated by Next Gen Sequencers has typically been manifested as multithreaded software tools that have been ed on ever greater numbers of faster processors in computer clusters with expensive high availability storage that requires substantial power and significant IT support costs.

Importantly, future increases in sequencing throughput rates will translate into rating real dollar costs for these secondary processing ons.

The devices, s, 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 directed to s, s, and methods for employing 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 s, the devices, systems, and methods herein ed are configured for ming 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 particular embodiments, one or more secondary sing pipelines for processing genetic sequence data is provided. In other embodiments, one or more tertiary processing pipelines for sing genetic sequence data is provided, such as where the nes, and/or individual ts 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 is pipeline on genetic sequence data and/or other data d therefrom. In various ments, the system may include one or more of an electronic data source that provides digital signals representing a plurality of reads of genetic and/or genomic data, such as where each of the plurality of reads of genomic data include a ce of nucleotides. The system may further include 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 nce sequences, and one or more indices ofthe one or more genetic reference sequences.

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 integrated 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 additionally include a quantum computing processing unit, for use in implementing one or more ofthe methods disclosed herein.

In various embodiments, one or more of the plurality of electrical interconnects 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 indirectly such as via a wireless network connection (for instance, a cloud or hybrid cloud), with the electronic data source. Regardless of a tion with the sequencer, an integrated circuit of the disclosure may be ured for receiving the plurality of reads of genomic data, e.g., directly from the sequencer or from an ated memory. The reads may be digitally encoded in a standard FASTQ or BCL file format. ingly, the system may include an integrated circuit having one or more electrical interconnects that may be a physical interconnect that includes a memory interface so as to allow the integrated circuit to access the memory. ularly, the hardwired digital logic circuit 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 red 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 therefrom. 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 ated t is an FPGA, such steps in the sequence and/or further analysis process may involve the partial iguration ofthe FPGA during the analysis process.

Particularly, the set of sing 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 nce 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 nce sequences, e.g., as received from the mapping module and/or stored in the memory.

Further, the set of processing engines may include a sorting module so as to sort each aligned read according to the one or more ons in the one or more genetic nce sequences. Furthermore, the set of processing s 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 detailing the variations between the sequenced genetic 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 WO 14320 2017/036424 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 various embodiments, a system for executing a g analysis pipeline on a plurality ofreads of genetic data using an index of genetic reference data is provided. In various instances, the genetic sequence, e.g., read, and/or the genetic nce data may be ented by a sequence ofnucleotides, 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 al electrical interconnects may include a memory interface for allowing the ated circuit to access the memory. In more particular ments, the hardwired digital logic ts may be arranged as a set of processing engines, such as where each processing engine is formed of a subset of the hardwired digital logic circuits to perform one or more steps in the sequence analysis pipeline on the plurality ofreads ofgenomic data.

For instance, in one embodiment, the set of processing s may include a mapping module in a hardwired configuration, 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 ity ofphysical electrical interconnects, and for extracting 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 nce s, and/or one or more indexes, such an index built from the one or more reference s. 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 onboard or offboard , 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 smg 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 ured to receive one or more of the mapped positions for the read data via one or more of the ity of physical electrical interconnects. Then the memory (internal or al) may be accessed for each mapped on to retrieve a segment ofthe reference sequence/genome corresponding to the mapped position. An alignment of the read to each retrieved reference segment may be calculated along with a score for the alignment. Once calculated, at least one best-scoring ent of the read may be selected and output. In various instances, the alignment module may also implement a dynamic programming thm 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 algorithm. In ular instances, the calculating of the alignment may include first performing a gapless ent to each reference segment, and based on the gapless ent results, selecting reference segments with which to further perform gapped alignments.

In various embodiments, a variant call module may be provided for performing improved variant call functions that when implemented in one or both of software and/or hardware configurations te superior processing speed, better processed result cy, and enhanced overall efficiency than the methods, devices, and systems currently known in the art. Specifically, in one aspect, 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 provided. In r aspect, novel devices including an integrated circuit for ming such improved variant call operations, where at least a portion ofthe variant call operation is implemented in hardware, are provided.

Accordingly, in various instances, the methods disclosed herein may include g, 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 nce sequences. Additionally, the methods may include accessing, by the integrated and/or quantum circuits, e.g., by one or more of the plurality 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 genetic reference sequences; and aligning, by a second subset of the hardwired and/or quantum digital logic circuits, 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 physical electrical interconnects from a memory or a cache associated therewith, the aligned plurality of reads. In such an ce the method may e sorting, by a third subset of the hardwired and/or quantum l logic circuits, the aligned plurality of reads according to their ons in the one or more genetic reference sequences. In certain ces, the method may further include outputting, such as by one or more of the plurality of physical electrical interconnects ofthe integrated and/or quantum circuit, result data from the mapping and/or the aligning and/or the sorting, such as where the result data includes ons of the mapped and/or aligned and/or sorted plurality ofreads.

In some ces, the method may additionally include using the obtained result data, such as by a further subset ofthe hardwired and/or quantum l logic circuits, for the purpose of ining how the mapped, aligned, and/or sorted data, derived from the t's sequenced genetic sample, differs from a reference sequence, so as to produce a variant call file delineating the genetic differences n the two samples. Accordingly, in various embodiments, the method may further include accessing, by the integrated and/or quantum circuit, e.g., by one or more ofthe ity 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 function, e.g., an HMM or paired HMM operation, on the accessed reads, by a third or fourth subset of the hardwired and/or quantum 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.

Accordingly, 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 pipeline 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 ed, such as where the processing s may be present in a preconfigured and/or hardwired and/or quantum configuration on a processing rm 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 ming other tertiary processing on the s data.

In particular ces, the present devices, systems, and methods of ing the same in the performance of one or more genomics and/or bioinformatics secondary and/or ry processing protocols, have been optimized so as to deliver an ement in processing speed that is orders of magnitude faster than standard secondary processing pipelines that are implemented in software. Additionally, the pipelines and/or components thereof as set forth herein provide better sensitivity and accuracy on a wide range of sequence derived data sets for the purposes of genomics and bioinformatics processing. In various instances, one or more of these operations may be performed on by an integrated circuit that is part of or configured as a general purpose central processing unit and/or a cs processing unit and/or a quantum processing unit.

For example, genomics and bioinformatics are fields concerned with the application of information technology and computer science to the field of genetics and/or lar biology. In particular, bioinformatics techniques can be applied to process and analyze s 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 s practitioners in the development of prophylactic, eutic, and/or diagnostic methods for preventing, treating, ameliorating, and/or at least identifying diseased states and/or their potential, and thus, improving the safety, y, and effectiveness of health care on an individualized level. Hence, because of their focus on advancing personalized care, 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 ing the genetics, genomics, and/or bioinformatics technologies disclosed herein is that the qualitative and/or quantitative analyses of molecular biological, e.g., genetic, data can be performed on a r 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 various embodiments, the genomics and/or bioinformatics related tasks may form a genomics pipeline that includes one or more of a micro-array analysis pipeline, a genome, e.g., whole genome is pipeline, ping analysis pipeline, exome analysis pipeline, epigenome analysis ne, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analysis pipeline, including joint genotyping, ts analysis pipelines, 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 e CPU and/or GPU and/or may be ented in one or more quantum circuits of a quantum sing rm. However, common characteristics of traditionally configured software based bioinformatics s and systems is that they are labor intensive, 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 implemented in software by a CPU and/or GPU of quantum 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 ted herein, such as where the genomics 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 sing 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 presented herein are "just in time" storage and/or retrieval methods that optimize the storage of such data in a manner that tutes the speed of regenerating the data in exchange for the cost of storing such data collectively. Hence, the data generation, is, and "just in time" or "JIT" storage methods presented 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, therefore, are systems, apparatuses, and methods for implementing cs 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 purpose processor, such as for performing one or more bioanalytic operations in software and/or on re. 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 ming genetic analytic related tasks, such as mapping, WO 14320 PCT/0S2017/036424 aligning, variant calling, ssing, ressing, and the like, in an accelerated manner, and as such the integrated circuit may include a re accelerated configuration.

Additionally, in s instances, an integrated and/or quantum circuit may be provided such as where the circuit is part of a processing unit that is ured for performing one or more genomics and/or bioinformatics protocols on the generated mapped and/or aligned and/or variant called data.

Particularly, in a first embodiment, a first ated 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 ons in a genomics analysis pipeline, such as where the integrated circuit is ured as described herein above to include one or more digital logic circuits that are arranged as a set ofprocessing s, which are adapted to perform one or more steps in a mapping, aligning, and/or variant calling operation on the c data so as to produce sequence analysis results data. The first integrated circuit may r include an output, 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 ication with the memory via a communications interface. The second integrated 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 cs and/or bioinformatics functions of the genomic analysis pipeline on the mapped, aligned, and/or t called sequence 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 pipeline, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, epigenome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analyses pipelines, including joint genotyping, ts analyses pipelines, including structural variants, somatic variants, and GATK, as well as RNA cing analysis pipeline and other genetic analyses 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 ing the genome analysis pipeline for executing a whole genome analysis pipeline, such as a whole genome analysis ne that includes one or more of genome-wide ion analysis, whole-exome DNA analysis, whole transcriptome RNA analysis, gene function analysis, protein function analysis, n binding analysis, quantitative gene analysis, and/or a gene assembly analysis. In certain instances, the whole genome is pipeline may be performed for the purposes of one or more of ry is, personal medical y analysis, e diagnostics, drug discovery, and/or protein profiling. In a particular instance, the whole genome analysis pipeline is performed for the es of oncology analysis. In various ces, 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 software 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 ility that a given determined genotype is a true genotype. In other instances, the software may be configured for performing a metagenome analysis so as to produce metagenome result data that may in tum be employed in the performance of a microbiome analysis.

In certain instances, the first and/or second integrated t and/or the memory may be housed on an expansion card, such as a eral 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 board. 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 particular 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 instances, 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 genomic sequence data. In particular instances, 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. onally, each of the two memories may include a flip-flop configuration for storing a multiplicity of genetic sequence and/or processing result data.

Accordingly, in another aspect, the system may be configured for sharing memory resources amongst its component parts, such as in relation to ming 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 ng between the U/QPU and the FPGA, e.g., chip or PCie card. Such configurations may be particularly useful when distributing operations d to the processing of the large data structures ated with cs and/or bioinformatics analyses to be used and accessed by both the U/QPU and the associated integrated 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 ions 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 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. ingly, one or more of the various steps in the genomics and/or bioinformatics processing pipeline, or a portion thereof, may be med by one device, e.g., the CPU/GPU/QPU, and one or more of the various steps may be performed 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 ent 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 CPUs/GPUs/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.

Particularly, m vanous embodiments, a genom1cs analysis platform is provided. For instance, the platform may include a motherboard, a memory, and plurality of integrated and/or quantum circuits, 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 embodiments, the rm may include a first integrated and/or m circuit, such as an integrated circuit forming a central processing unit (CPU) or graphics processing unit (GPU), or a quantum circuit forming a quantum sor, that is responsive to one or more software or other thms that are configured to instruct 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 interconnects to connect with the motherboard. In various 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 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 ofthe one or more genetic reference sequences.

Additionally, the platform may include one or more of another integrated circuit(s), such as where each of the other integrated t forms a field programmable gate array (FPGA) having a second set of al 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 t is an FPGA, the FPGA 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, variant g, etc. Particularly, the red digital logic circuits of the FPGA may be arranged as a set ofprocessing s to perform one or more pre-configured steps in a sequence analysis pipeline of the genomics analysis, such as where the set(s) of processing engines include one or more of a g and/or aligning and/or variant call module, which s 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 s, and in various embodiments, an included processing 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 including a second set of processing engines, where the second set of sing engines 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 ofhardwired digital logic circuits may also be included such as where the third set of processing engines includes an aligning module configured to align the mapped read to one or more positions in the nce haplotype sequence. A fourth subset of hardwired digital logic circuits may additionally be included such as where the fourth set ofprocessing s includes 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 sequence analysis pipeline, such as on genetic data, is provided wherein the genetic data includes one or more of a genetic reference sequence(s), such as a haplotype or hypothetical haplotype sequence, an index of the one or more c 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 devices, and/or processed by a buted processing resource, such as a U/QPU and/or FPGA, which are coupled, e.g., tightly or loosely together. Hence, in various ces, the apparatus may include an integrated circuit, which ated circuit may e one or more, e.g., a set, of hardwired digital logic circuits, wherein the set of hardwired digital logic circuits may be interconnected, such as by one or a plurality of al electrical interconnects.

Accordingly, the system may be configured to include an ated circuit formed of one or more digital logic circuits that are onnected 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 U/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 processing engine may be configured for accessing the data stored in the cache and/or direct or ctly 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 ofdigital logic circuits, which is adapted to access in the memory, e.g., via the memory interface, at least some of the sequence of nucleotides in the read of genomic sequence data and the haplotype sequence data, and may also be ured to perform the HMM analysis on the at least some of the sequence of nucleotides in the read of c sequence data and the at least some of the sequence of nucleotides in the haplotype sequence data so as to produce HMM result data. Additionally, the one or more of the ity of physical electrical 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 executing a ce analysis pipeline such as on genetic ce data is provided. The genetic data may e one or more genetic reference or haplotype sequences, one or more indexes of the one or more c reference and/or haplotype sequences, and/or a plurality of reads of c data.

The method may include one or more of ing, accessing, mapping, aligning, sorting various iterations of the c sequence data and/or ing the results thereof in a method for producing one or more variant call files. For instance, in certain embodiments, the method may e receiving, on an input to an integrated circuit 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 arranged as one or more processing engines. In such an instance, a processing engine may be formed ofa subset ofthe hardwired l logic circuits that may be in a wired configuration. In such an instance, the sing engine may be configured to perform one or more pre-configured 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 f in a method for producing one or more variant call files. In some embodiments, the provided digital 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 , data for ming one or more ofthe operations detailed herein. In various instances, 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 ity of memories one of each coupled to the chip and a CPU/GPU/QPU that is itself coupled to the chip, e.g., loosely.

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

Hence, in various instances, implementations of various aspects of the disclosure may include, but are not limited to: apparatuses, systems, and methods including one or more features as described in detail herein, as well as articles that comprise a tangibly embodied machine-readable medium operable to cause one or more es (e.g., computers, etc.) to result in operations bed . Similarly, computer systems are also described that may include one or more processors and/or one or more es 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 residing in a single computing system or multiple 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 WO 14320 PCT/0S2017/036424 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 interconnect, or the like), via a direct tion between one or more of the multiple computing systems, etc. A memory, which can include a computer-readable e medium, may include, encode, store, or the like one or more programs that cause one or more processors to perform one or more ofthe operations associated with one or more ofthe algorithms described .

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 advantages of the subject matter described herein will be apparent from the description and drawings, and from the . While certain features of the currently disclosed subject matter are bed for illustrative purposes in relation to an enterprise resource software system or other business software on or architecture, it should be readily understood that such features are not intended to be limiting. The claims that follow this sure 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 t matter disclosed herein and, together with the description, help explain some of the principles associated with the disclosed implementations.

FIG. IA depicts a sequencing platform with a ity 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 s 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 transposition 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 architecture for electronically transposing the sequenced data. s an HMM 3-state based model rating 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 circuit of , showing an HMM r features in greater detail. depicts an overview of HMM related data flow hout the system including both software and hardware interactions. depicts exemplary HMM r collar connections. s a evel view of the major functional blocks within an exemplary HMM hardware accelerator. depicts an exemplary HMM matrix structure 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 simplifying assumptions of related 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 diagram showing the relation between GOP, GCP and transition probabilities. depicts an HMM Transprobs and Priors generation circuit to support the general state transition diagram of. s a simplified HMM state transition diagram showing the relation n GOP, GCP and transition probabilities. depicts a HMM Transprobs and Priors generation circuit to support the simplified state transition. s 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 B presents an exemplary method for computing a connection matrix such as in the pre-processing procedure ofA.

A s an ary event n 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 ocessing procedure of FIGS. 17A and B. is an example of a bubble formation m a De Brujin graph m accordance with the methods of. is an example of a t pathway through an exemplary De Brujin graph. is a graphical representation ofan exemplary sorting function. is another example of a processing matrix for a pruned multi-region joint detection procedure. 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 ure. represents a selection of candidate solutions for the joint pile up of . represents a further selection of ate solutions for the pile up of , after a pruning on has been performed. represents the final candidates of , and their associated probabilities, after the performance ofa MRJD function. illustrates the ROC curves for MRJD and a conventional detector. rates the same results of displayed as a function of the sequence similarity ofthe nces.

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

B depicts an exemplary 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. s 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 sharing one or more memories and/or caches, wherein the CPUs are ured for communicating with one or more FPGAs that may also include a shared or common memory or . illustrates an exemplary method of data transfer throughout the system. s the embodiment of in greater detail. 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 s a block diagram for an exemplary genomic processing and analysis pipeline.

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

B depicts the block diagram of A rating r detail regarding the computing function for a genomic infrastructure for onsite and/or cloud based cs processing and analysis.

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

A depicts a block diagram illustrating a hybrid cloud configuration.

B depicts the block diagram ofA in greater , illustrating 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 ne as presented .

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

C es an exemplary tertiary processing methylation analysis for execution by the methods and s ofthe system herein.

D provides an exemplary tertiary processmg 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 provides an ary joint ping ry processing analysis for execution by the methods and devices ofthe system herein. depicts a flow diagram for an analysis ne ofthe disclosure. is a block diagram of a hardware sor architecture in accordance with an implementation ofthe disclosure. ] is a block diagram of a hardware sor architecture in accordance with another implementation. is a block diagram of a hardware processor architecture in accordance with yet another implementation. illustrates a genetic sequence analysis pipeline. illustrates processing steps using a genetic sequence analysis hardware platform.

] A illustrates an apparatus in accordance with an implementation ofthe disclosure.

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

Detailed Description ofthe Disclosure As ized above, the t disclosure 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, ng, sorting, and/or variant call protocol on data generated through a primary processing procedure, such as on genetic sequence data. For instance, in s aspects, the devices, systems, and s herein provided are configured for performing secondary 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 particular embodiments, one or more secondary processing pipelines for processing genetic sequence data is provided, such as where the pipelines, and/or individual elements thereof, may be ented in software, hardware, or a combination thereof in a distributed and/or an optimized fashion so as to deliver or sensitivity and improved accuracy on a wider range of ce derived data than is currently available in the art. Additionally, as ized above, the present disclosure is directed to devices, systems, and methods for employing the same in the mance 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 analysis protocol, exome analysis protocol, epigenome analysis protocol, metagenome analysis ol, microbiome analysis protocol, ping analysis protocol, including joint genotyping, variants analysis ols, 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 cing data. More particularly, a platform, or pipeline, of processing engines, such as in a software implemented and/or hardwired configuration, 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 analysis, such as a micro-array analysis, a genome, e.g., whole genome analysis, genotyping analysis, exome analysis, epigenome is, nome analysis, microbiome analysis, genotyping analysis, including joint genotyping analysis, variants analysis, including structural variants analysis, somatic variants analysis, and GATK analysis, as well as RNA cing analysis and other genetic analysis, such as with t to genetic based sequencing data, which may have been generated in an optimized format that delivers an improvement in processing speed that is magnitudes faster than standard pipelines that are implemented in known re alone. Additionally, the pipelines presented herein provide better sensitivity and accuracy on a wide range of sequence d data sets, such as on nucleic acid or protein derived ces.

As indicated above, in various 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 ols as well as for laxis 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 n profile derived therefrom and ing the same with predicted biological response from such drug administration.

Such bioinformatics processmg usually involves three well defined, but typically separate phases of information processing. The first phase, termed y processing, involves DNA/RNA sequencing, where a subject's DNA and/or RNA is obtained and subjected to various processes whereby 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 sing, involves performing one or more analyses on the subject's genetic makeup so as to determine therapeutically useful ation therefrom. ingly, 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 digitally encoded genetic sequence data obtained from the sequencer and/or sequencing protocol, such as by subjecting 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 n profile of an individual, such as where the individual's entire genetic makeup is determined, for instance, where each and every tide 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 s from that of the nt(s). This process is commonly 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 process can be very labor and time intensive, requiring many steps that may need to be performed one after the other and/or aneously, 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 generating 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 sing step, so as to e a multiplicity of read sequence segments ("reads") covering all or a portion of the individual's genome, such as in an oversampled . The end product generated by the sequencing device may be a collection of short sequences, e.g., reads, that represent small ts of the subject's genome, e.g., short genetic ces representing the dual's entire genome. As indicated, typically, the information represented 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 l ary processing protocol, a subject's genetic makeup is assembled by comparison 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 sequencer ning the raw sequenced read data. In order to e the subject's genome to that of the standard reference genome, it needs to be determined where each of these reads map to the nce genome, such as how each is aligned with respect 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 ons 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 subject's genetic code and that ofthe referent can be assessed.

For instance, reference based assembly in a typical 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 ces. Various mapping, aligning, sorting, 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 determine where on each chromosome each particular read is located. It is noted that these processes may be implemented in software or hardware, such as by the s and/or devices described in U.S. Patent Nos. 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 oning of these various algorithms and/or re 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 sequenced reads may be sed to determine the possible locations in the reference genome to which those reads could ly 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 ons 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 orm, which is used to map a selection of reads to a reference using a compression formula that compresses repeating sequences .

] 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 inserted by an algorithm 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 on 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 ce databases and calculates the statistical significance of matches. In such manners 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 sing ces and time in the performance of their functions, than would otherwise be the case, such as if the t's genome were being assembled by direct comparison, such as without the use of these algorithms.

] Additionally, an aligning 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 original 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 enting 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, representing 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 assigned position. Typical algorithms for performing alignment functions include Needleman-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 d of comparing 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 fying to which chromosome the read belongs and/or its offset from the beginning ofthat chromosome, the reads may be sorted by position. This may enable downstream analyses to take advantage of the oversampling procedures described herein. All of the reads that overlap a given position in the genome will be nt to each other after sorting and they can be organized into a pileup and readily examined to determine if the ty of them agree with the reference value or not. If they do not, a variant can be flagged.

For instance, in various ments, the s 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 A 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 variant 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 ions 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 ced and rebuilt prior to determining its variants. There are, however, several problems that may occur when attempting to generate such an assembly. For e, there may be problems with the chemistry, the sequencing machine, and/or human error that occur in the sequencing process. Furthermore, there may be genetic artifacts that make such reconstructions matic. For instance, a typical problem with performing such assemblies is that there are sometimes huge portions of the genome that repeat themselves, such as long ns of the genome that include the same strings of nucleotides. Hence, because any genetic sequence is not unique everywhere, it may be difficult to determine where in the genome an identified read ly 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 insertion or a deletion, such as where one or a multiplicity of bases have been added to or deleted from the subject's genetic 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. ingly, there are two main possibilities for variation. 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 tides 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 sed herein may be employed in a manner so as to compensate for these types of errors, and more particularly so as to distinguish errors in variation due to chemistry, machine or human, and real variations in the sequenced 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 ore to better ensure the accuracy of any call files generated so as to correctly identify true variants.

Hence, in particular embodiments, a platform of technologies for performing c analyses are provided where the rm 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 ce, in various aspects a ne may be provided wherein the pipeline includes performing one or more analytic functions, as described herein, on a genomic sequence of one or more individuals, such as data obtained in an image file and/or a digital, e.g., FASTQ or BCL, file format from an automated sequencer. A typical pipeline to be executed may include one or more of sequencing genetic al, such as a portion or an entire genome, of one or more individual ts, which genetic material may include DNA, ssDNA, RNA, rRNA, tRNA, and the like, and/or in some instances the genetic material may represent coding or noncoding s, such as exomes and/or episomes of the DNA. The pipeline may include one or more of performing an image processing procedure, a base calling and/or error tion 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 on on the genetic data. In certain instances, the pipeline may include ming one or more of a realignment, a deduplication, a base quality or score recalibration, a reduction and/or compression, and/or a decompression on the digitized genetic data. In certain instances the pipeline may include performing a t calling operation, such as a Hidden Markov Model, on the genetic data. ingly, 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 tructing a subject's consensus genomic sequence, comparing a subject's genomic sequence to a referent sequence, e.g., a reference or model genetic sequence, determining the manner in which the subject's genomic DNA or RNA differs from a nt, e.g., variant calling, and/or for performing a tertiary is on the subject's genomic sequence, such as for genome-wide variation analysis, gene function is, protein function analysis, e.g., protein binding analysis, quantitative and/or assembly analysis of genomes and/or transcriptomes, as well as for various diagnostic, and/or a prophylactic and/or therapeutic evaluation analyses.

As indicated above, in one aspect one or more of these platform functions, e.g., mapping, ng, g, realignment, duplicate marking, base quality score recalibration, variant calling, compression, and/or decompression functions is configured for implementation in software. In some aspects, one or more of these platform functions, e.g., mapping, aligning, sorting, local realignment, duplicate marking, base quality score recalibration, decompression, variant calling, ssion, and/or decompression functions is ured 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 ive and/or less time consuming manner and/or with greater percentage cy, e.g., the hardware implemented functionality is faster, less processing intensive, and more accurate.

For instance, in n ments, improved algorithms for performing such primary, secondary, and/or tertiary sing, as disclosed herein, are provided. The improved algorithms are ed 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 algorithms may be directed to more efficiently and/or more accurately ming one or more of local realignment, duplicate marking, base quality score recalibration, variant calling, compression, and/or decompression ons. Further, as described in greater detail herein below, in certain embodiments, these genetic analysis technologies may employ one or more algorithms, such as improved algorithms, that may be implemented by one or more of re and/or hardware that is run in a less processing intensive and/or less time consuming manner and/or with greater percentage accuracy than various traditional re implementations for doing the same. In various instances, improved algorithms for implementation on a m processing platform are provided.

Hence, in various s, 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 c 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, systems 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 include the performance of one or more of: g, ng, 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 tertiary sing platform.

Accordingly, in certain instances, methods, including software, firmware, re, and/or quantum processing algorithms for performing the methods, 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 mapping, aligning, sorting, nment, duplicate marking, base quality score recalibration, variant calling, ssion, decompression, and/or one or more tertiary processing protocols where the algorithm, e.g., including re, 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 software solution, the algorithm and/or its ant processes, has been optimized so as to be performed faster and/or with better accuracy for execution by that media. se, where the functions ofthe thm are to be implemented in a hardware solution, e.g., as firmware, the hardware has been designed 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 ion by that media.

Further, where the algorithm is to be implemented in a quantum processing on, 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 ce, can be ed such as in an iterative mapping, aligning, sorting, variant calling, and/or tertiary processing procedure. In another instance, systems and methods are provided for implementing the functions of one or more algorithms for the performance of one or more steps for ing 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 ically, in some instances, methods and/or machinery for implementing those methods, for performing secondary analytics on data ning to the genetic 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 involves 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 produce a prototypical, standard reference genome to which any individual's genetic material, e.g., DNA/RNA, may be compared, for example, so as to ine and reconstruct the individual's genetic sequence and/or for determining the difference between their genetic makeup and that ofthe standard reference, e.g., variant calling.

Particularly, a reason for performing a secondary analysis on a subject's ced A is to determine how the subject'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 ces 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 ry analysis ol, for instance, so as to t the potential for the occurrence of a diseased state, such as because of a genetic ality, 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 subject's DNA or the proteins ted 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 desirable, enhance the accuracy ofa variant calling protocol.

] Accordingly, in one , 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 diseases this person may or may have the potential for suffer from and/or for determining what ents or lifestyle changes this t 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 te 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 provide the subject with one or more therapeutic or prophylactic 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 c 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 is models, health related data and/or medical useful information, 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 clinical , and other such purposes. More particularly, 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 ormatics ry protocols, such as a micro-array analysis ol, a genome, e.g., whole genome analysis protocol, a ping analysis protocol, an exome analysis protocol, an epigenome analysis protocol, a metagenome analysis protocol, a microbiome analysis protocol, a genotyping analysis protocol, including joint genotyping, variants analyses protocols, ing structural variants, somatic variants, and GATK, as well as RNA cing protocols and other genetic analyses protocols.

As there are a finite number of diseased states that are caused by genetic malformations, in tertiary processing variants of a n 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. uently, in various instances, the methods herein sed may involve analyzing, e.g., scanning, the VCF and/or the ted sequence, against a known disease ce variant, such as in a data base of c markers therefore, so as to identify 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 s disclosed herein may entail the generation of one or more databases linking sequenced data for an entire genome and/or a variant call file pertaining thereto, e.g., such as from an individual or a plurality of individuals, and a ed state and/or searching the generated databases to determine if a particular t has a genetic composition that would predispose them to having such diseased state. Such searching may involve a comparison of one entire genome with one or more others, or a fragment of a genome, such as a fragment containing only the variations, to one or more nts 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 g and/or error tion operation and/or a mapping 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 genetic data.

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

Many of these s 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 r. Additionally, many of these modules and/or steps of the pipeline are optional and/or can be arranged in any logical order and/or omitted entirely. For instance, the re and/or hardware disclosed herein may or may not include an image processing and/or a base calling or sequence correction algorithm, such as where there may be a concern that such functions may result in a statistical bias.

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

Particularly, once the reads are ed a position relative to the reference genome, which may e identifying to which chromosome the read belongs and its offset from the beginning of that some, they may be licated and/or sorted, such as by position. This enables downstream analyses to take advantage of the s 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 ed 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 enting a portion or the entire genome of an dual. For instance, a first step in the secondary analysis nes, disclosed herein, is the receipt of genomic and/or bioinformatics data, such as from a genomics data generating 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 includes the base calls and quality scores made for each base of each sequence of the collection of sequences that compose at least a part of or the whole genome ofa subject. ionally, the sequencer generated BCL file is converted to a FASTQ file, which then may be transmitted to a secondary processing platform, such as disclosed , for r processing, such as to determine the genomics 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 encoded with a single ASCII character for brevity. Accordingly, within this and other s, it is the FASTQ file that is used for the purposes of further sing. 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 apparatus, 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 ly inputting such data into the present platform 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 insertion into a Next Gen sequencer to be sequenced in an iterative fashion, such that each sequence will be grown by the stepwise addition of one nucleotide after another. ically, 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 te sequences 6a are to be sequenced.

The platform 6 may be added to a flow cell 6c ofthe sequencer that is adapted for performing the cing reactions.

] As the cing reactions take place, at each step a nucleotide having a fluorescent tag 6d is added to the platform 6 of the flow cell 6c. If a hybridizing reaction occurs, fluorescence is observed, an image is taken, the image is then processed, and an appropriate base call is made. This is repeated base by base until all of the te sequences, e.g., the entire genome, has been ced and converted into reads, thereby producing the read data ofthe system. Hence, once sequenced, 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 ted 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 s and architectures for expedited BCL conversion into files that can be rapidly processed within the secondary processing system. More specifically, in particular instances, 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 ed for g 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 advanced into the g and aligning and/or other processing engines.

Particularly, 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 particularly, with respect to Next Gen Sequencing, the sequencing is typically med on glass plates 6 that form flow cells 6c that are d into the automated sequencer for sequencing. As can be seen with respect to FIG. IB, a flow cell 6c is a platform 6 composed of 8 al columns and 8 horizontal rows (front and back), together 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 ated 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 includes template genetic material 6a to be sequenced. The sequencing rm 6, ore, includes a number of template nucleotide sequences from the subject, which sequences are arranged in a grid like fashion oftiles on the platform. (See FIG. IB.) The genetic data 6 is then sequenced in an iterative n where each sequence is grown by the stepwise uction of one nucleotide after another into the flow cell, where each iterative growth step represents a cing cycle.

As indicated, an image is captured after each step, and the g 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 procedure 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 growing nucleotide strand being sequenced will have an x dimension 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 ntal axis, and the read ties, which represent 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 may be defined by a vertical and ntal axis, cycle by cycle, and the base by base data from each cycle for each read may be inserted into the matrix of FIG. ID, such as in a streaming and/or ned 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 l matrix of FIG ID is formed. Accordingly, there will be both a base call and a quality score entered into the matrix, where each tile from each cycle represents a separate file. It is to be noted that where the sequencing is performed on an integrated t, 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 determined 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 ofreads. 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 quality score may then be entered so as to fill out the matrix column by , 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 n reads, the matrix would contain about 1 n reads, which need to be organized and streamed into the secondary processing apparatus.

Accordingly, such organization is fundamental to rapidly and efficiently sing the data. Hence, in one aspect, 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 nes of the system herein disclosed. 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 millions of files generated, 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 transposed, 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 generated 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 , this data may be transposed, e.g., substantially aneously, so as to be represented, as seen within the l matrix, on a read by read, row by row basis, where each row represents an individual read, and each read is represented by a sequential number of base calls and quality , thereby fying both the sequence 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 l 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. Further, although the process may be implemented efficiently in software, it may be made even more efficiently and faster, by being ented 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 e, 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 associated 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 re where dedicated processing engines will queue up the column organized data and transpose that data from a column by column, cycle order configuration, to a row by row, read order uration, 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 hardware may include an array of registers 8a into which the cycle files may be dispersed and reorganized into individual read data, such as by writing one base from a column into registers that are zed into rows. More specifically, as can be seen with respect to FIG. IG, the hardware device 1, including the transposition processing 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 handling an increased amount of transactions per cycle, where the queued data is transmitted 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 l age 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 ed into associated r es, e.g., registers, in a different order whereby 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 l 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 er, 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 ers 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 accessed by one or more further processing engines of the secondary processing system for r processing, such as by a mapping, aligning, and/or variant calling engine, as described herein. It is to be noted, as bed 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. g, 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 locations 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 instance, 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 retrieving 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 s forms and queried in various manners. In some methods, the index may include a prefix and/or a suffix tree. In particular s, the index may be derived from a Burrows/Wheeler orm of the reference. Hence, atively, or in addition to employing a prefix or a suffix tree, a Burrows/Wheeler transform can be performed on the data. For instance, a Burrows/Wheeler transform may be used to store a tree-like data structure abstractly equivalent to a prefix and/or suffix tree, in a compact format, such as in the space allocated for storing the nce genome. In various instances, the data stored is not in a tree-like structure, but rather the reference sequence data is in a linear list that may have been scrambled into a ent 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 ively walk the "tree".

Additionally, in various instances, the index may include one or more hash tables, and the s disclosed herein may include a hash on that may be performed on one or more ns ofthe reads in an effort to map the reads to the reference, e.g., to the index of the reference. 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 portions of the read to determine where the one may match to the other. se, 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 required to find a unique n) per query to find a match, or a binary search method that takes ) probes where N is the number of seed ns in the table. Further, even though the hash function can break the reference genome down into segments s 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 initial key or address so as to access the hash table. For example, 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 checking the first key, e.g., if both the first and second keys arrive at the same cell in the hash table, then it is relatively 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 for confirming the first pass. ingly, for any seed, its original s 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 on.

And, as indicated above, in various instances, these two different 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 nce to the read, or ns thereof. However, using a brute force linear search to scan the reference genome for locations where a seed matches, over 3 billion 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 le 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 function can quickly determine where that read is going to show up in the reference genome. By using a hash table index, ore, 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 ces, 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 ted above. Alternatively, a trivial hash function mapper may be employed such as by simply dropping some of the 2k bits. r, in s instances, the CRC may be a stronger hash function that may better separate similar seeds while at the same time avoiding table congestion. This may especially be cial where there is no speed penalty when calculating CRCs such as with the dedicated hardware described herein.

In such instances, the hash record populated for each seed may include the reference position where the seed occurred, and the flag indicating whether it was e 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 . In various 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 s instances, it is not necessary for all portions ofall the reads to match exactly to all the portions of the reference .

As described herein, all of these operations may be performed via software or may be red, 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 perform more efficiently because of their implementation in such hardware. Additionally, one or more, e.g., two or all three, of these mapping functions may form a module, such as a mapping module, that may form part of a system, e.g., a ne, that is used in a process for determining an actual entire genomic sequence, or a portion thereof, ofan individual.

An advantage of implementing the hash module in hardware is that the processes may be accelerated and therefore med in a much faster . 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 instructions to be stored and/or fetched and/or read and/or interpreted, such as prior to execution. As indicated above, however, and described in greater detail herein, a chip can be red to perform these functions without having to fetch, interpret, and/or perform one or more of a sequence of instructions. Rather, the chip may be wired to perform such functions ly.

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

For example, in various ces, 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 g the hash module. Particularly, in various instances, the chip, such as an FPGA, may be configured so as to be tightly coupled to the host CPU, such as by a low latency onnect, 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 together 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 , e.g., containing an index of a reference genome, or a constructed or to be constructed 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 ce, reads, e.g., stored in FASTQ file format, may be sent by the host application to the d processing engines, e.g., a memory or cache or other register associated therewith, 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 ming 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 instances, a mapping module may be provided, such as where the mapping module is configured to perform one or more g functions, such as in a hardwired configuration. ically, the red 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 s-Wheeler Transform, and/or runs a hash function, for instance, a hash function that makes use of, or ise relies on, a hash-table indexing, such as of a reference, e.g., a reference genome sequence. In such instances, the hash function may be structured so as to implement a strategy, such as an zed 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 maximize the utility of the on-board or otherwise associated memory bandwidth, which may entally be constrained such as by space within the chip architecture.

Further, in certain instances, in order to make the system more efficient, the host U/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 wherein the integrated circuit has been pre-configured, e.g., prewired, 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 processing engines so as to form one or more s, such as a mapping .

] Accordingly, in various instances, a g module may be provided, such as in a first pre-configured wired, e.g., hardwired, configuration, where the mapping module is configured to perform various mapping functions. For instance, the g 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 subject's sequenced genetic sample, and/or a genetic reference 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 ace, 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 embodiments, the mapping algorithm and/or module ted herein, may be ed to build, or otherwise construct a hash table y the read, or a n thereof, of the sequenced genetic material from the subject may be compared 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 e, after it has been determined where all the possible matches are for the seeds against the reference genome, it must be determined which out of all the le locations a given read may match to is in fact the correct on 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 reference, if you take into account the position of the seed in the read. The actual alignment, ore, must be determined for each given read. This ination may be made in several different ways.

In one instance, all the reads may be evaluated so as to determine their correct alignment with t to the reference genome based on the positions indicated by every seed from the read that returned position information during the mapping, e.g., hash lookup, s. However, in various instances, prior to performing an alignment, a seed chain filtering function may be performed on one or more of the seeds. For instance, in certain instances, the seeds associated with a given read that appear to map to the same general place as against the reference genome may be aggregated into a single chain that references the same general region. 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 chain(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 evidence 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 er, 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 ent chain of seeds. Once these various seeds have been aggregated into one or more various seed chains, it may be ined 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 ly to be the correct one.

The outcome from performing one or more of these mapping, filtering, and/or g functions is a list of reads which es for each read a list of all the possible locations to where the read may matchup with the reference genome. Hence, a mapping 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 ion, you may not get an exact match to the reference and/or there may be l 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.

Accordingly, after mappmg and/or filtering and/or editing, the location positions for a large number ofreads have been determined, where for some ofthe individual reads a multiplicity of location 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 s reads align. Such aligning may be performed by one or more algorithms, such as a dynamic programming algorithm that matches the mapped reads to the reference genome and runs an alignment function thereon. An exemplary 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 , 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 determine 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 ndently and/or simultaneously according to the rules of dynamic programming applicable in the ent algorithm, such as Smith-Waterman, and/or Needleman-Wunsch, and/or related algorithms. ent 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 ed aneously 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 tide at each position in the read may be ed with the nucleotide at each position in the reference . As the wave front passes over the array, all potential ways of aligning the read to the reference window are considered, including 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 tides into one sequence, or deleting one or more nucleotides from one sequence.

An alignment score, representing the extent of the changes that would be required to be made to achieve an exact alignment, is generated, n this score and/or other ated 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 ent 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 nce 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 pipeline, that is used, such as in on with a mapping module, in a process for determining the actual entire genomic sequence, or a portion thereof, of an dual. For instance, the output ed from the performance of the g 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 ine the actual sequence ent of the subject's sequenced DNA.

Such an alignment function may at times be useful because, as described above, often times, for a variety of different s, the sequenced 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 deletion of one or more bases along one or more of the read sequences, which insertion or deletion is not present 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 because the reference differs from the true DNA sequence sampled, or because the read differs from the true DNA sequence d. The problem is to figure out how to correctly WO 14320 PCT/0S2017/036424 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 erent ways.

] In various instances, the input into an alignment function, such as from a mapping function, such as a prefix/suffix tree, or a Burrows/Wheeler transform, or a hash table and/or hash function, 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 derived, an alignment function may be performed, e.g., a Smith- Waterman gapped or gapless alignment, a Needleman-Wunsch ent, etc., so as to determine where in the genome one or more of the reads was actually d, such as by comparing all of the le ons 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 ured so as to be performed by software, however, in s instances, such as herein presented, one or more of these algorithms can be configured so as to be executed in hardware, as described in r 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, insertions, deletions, structural artifacts, 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 t the reference genome, 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 function may also be performed.

For example, a scoring function may be performed, e.g., as part of an overall alignment function, whereby as the alignment module performs its on and introduces one or more changes into a sequence being ed to r, 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 detracted 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 ined, such as where matches are detected the score is sed, and for each change introduced a penalty is incurred, and thus, the best fit for the possible ents can be determined, for example, by figuring out which of all the possible modified reads fits to the genome with the highest score. Accordingly, in various instances, the alignment on may be configured to determine the best combination of s that need to be made to the read(s) to e the highest scoring alignment, which alignment may then be determined to be the t or most likely alignment.

In view of the above, there are, therefore, at least two goals that may be achieved from ming an alignment function. One is a report of the best alignment, including position in the reference genome and a description 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 changes that were made to the reads so as to achieve their best fit alignment, e.g., ed alignment instructions indicating how the query actually aligns with the reference. Such a CIGAR string readout may be useful in further stages ofprocessing so as to better determine that for the given subject's genomic nucleotide sequence, the predicted variations as ed against a reference genome are in fact true variations, and not just due to machine, software, or human error.

As set forth above, in various 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 reference genomes, and further es genomic sequence data, such as from one or more memories, such as associated DRAMs, ning 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 ted ons and uses them to align the reads to the reference genome, such as by building a virtual array by which the reads can be compared 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 multiple le locations in the reference genome and scores the possibility that each on is the correct position. It then compares the best scores, e.g., the two best , 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. r, where the difference 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 genome. Ideally, the score ofthe best possible choice ofalignment is significantly greater than the score for the second best alignment for that sequence. There are many different ways an alignment scoring methodology may be ented, for ce, each cell of the array may be scored or a sub-portion of cells may be scored, such as in accordance with the methods disclosed herein. In s instances, g parameters for nucleotide matches, nucleotide mismatches, insertions, and deletions may have any s positive or negative or zero values. In various instances, these scoring ters may be modified based on ble information. For instance, accurate alignments may be achieved by making scoring parameters, including any or all of nucleotide match scores, nucleotide mismatch scores, 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 on. For example, score bonuses and/or penalties could be made smaller when a base quality score indicates a high probability a sequencing 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 entation 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 elements 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 ed for a new cell in the virtual alignment . Notionally, the various scoring cells are considered to be in various positions in the alignment matrix, corresponding to a g 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 sed 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 l alignment matrix, the notional positions ofthe scoring cells pondingly update each cell, for example, notionally "moving" a step to the right, or for example, a step downward in the alignment 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 g cells arrive in new notional positions, and compute alignment scores for the virtual ent matrix cells they have entered. In such an implementation, neighboring scoring cells in the linear array are coupled to communicate query (read) nucleotides, reference nucleotides, and previously ated 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 t 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 ght cell, and other reference nucleotides shift down-left through the wave front. This shifting of reference nucleotides may be the underlying reality of the notional movement of the wave front of scoring cells rightward through the alignment 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 g cell. Likewise, each time the wave front steps vertically, another query tide is fed into the bottom-left cell, and other query nucleotides shift up-right through the wave front. This ng of query nucleotides is the underlying y 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 typical alignment of query and reference ces without insertions or deletions, wave front steps may be commanded in alternating vertical and ntal directions.

Accordingly, neighboring g cells in the linear array may be d to communicate previously calculated alignment scores. In various ent scoring algorithms, such as a Smith-Waterman or Needleman-Wunsch, or such variant, the alignment score(s) in each cell of the virtual alignment matrix may be ated using usly 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 s) for another matrix position it has entered, it must ve such previously calculated scores corresponding to such other matrix positions.

These previously calculated scores may be obtained from storage 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 virtual alignment matrix (immediately left, above, and diagonally 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 immediately to the left in the matrix would have been scored by the current g 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 immediately above in the matrix would have been scored by the current scoring cell, if the most recent wave front step was vertical (downward), 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 different 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).

Accordingly, by considering information on the last one or two wave front step directions, a scoring cell may select the appropriate previously calculated scores, accessing them within itself, and/or within neighboring g cells, ing the coupling 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 extreme 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 directions, in order to notionally move the wave front in vertical and ntal, e.g., diagonal, steps, and coupling for accessing scores previously computed by neighboring cells in order to compute alignment score(s) in new virtual matrix cell positions entered by the wave front, it is accordingly le to score a band of cells in the virtual , the width of the wave front, such as by ding 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 oned 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 , and the wave front may then sweep rightward into the matrix by a series ofhorizontal steps, scoring a horizontal band of cells in the top-left region of the matrix. When the wave front reaches a predicted alignment relationship between the reference and query, or when matching is ed from increasing alignment scores, the wave front may begin to sweep diagonally down-right, by alternating al 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 ent matrix, the wave front may begin sweeping rightward again by successive horizontal steps, until some or all wave front cells sweep out of the ries of the alignment matrix, scoring a horizontal band ofcells 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 thm 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 similar manner. For instance, as set forth above, these alignment algorithms typically build the virtual array in a similar manner such that, in s instances, the horizontal top boundary may be configured to ent the genomic nce sequence, which may be laid out across the top row of the array according to its base pair composition. Likewise, the vertical boundary may be configured to represent the sequenced and mapped query sequences that have been positioned in order, downwards along the first column, such that their nucleotide sequence order is lly 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 ve 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 on may be determined.

With respect to a Needleman-Wunsch alignment on, 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 alignment matrix to the bottom edge.

When the wave front sweep is complete, the maximum score on the bottom edge of the alignment matrix (corresponding 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 instances disclosed herein, the reads can be any length long, can be any size, and there need not be ive read ters as to how the alignment is performed, 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 Smith-Waterman algorithm, which generates optimal local alignments, aligning the entire read sequence or part ofthe read sequence to some segment of the reference genome, this algorithm may be ured 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 matrix, such as if a very long read had only seeds in its middle mapping to the reference genome, but commonly the wave front may still score from top to bottom ofthe . Local ent is typically achieved by two adjustments. First, alignment 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 alignment. Second, the maximum alignment score produced in any cell in the matrix, not arily 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 position 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 h 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 possible 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 sed cells is determined (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 ine 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 truction or backtrace involves starting from a determined maximum 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 reached a particular cell in the alignment matrix, the next backtrace step is to the oring cell, immediately leftward, or above, or diagonally up-left, which buted the best score that was selected to construct the score in the current cell. In this manner, the evolution of the maximum score may be determined, y 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. Accordingly, it is such a back trace that identifies the proper alignment and thereby produces the CIGAR strand readout that represents how the sample c sequence d from the individual, or a portion f, 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 various reads and/or c nucleic acid sequence ofthe subject may be ed, such as by performing a back trace function moving rds up through 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 sure 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 ne 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 .

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

Performing backtrace then becomes a matter of ing the scoring vectors; when the ace 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; 3=backtrace diagonally up-left.

Such sconng vectors may be stored in a two-dimensional table arranged according to the ions of the alignment matrix, wherein only s corresponding to cells scored by the wave front are populated. Alternatively, to conserve memory, more easily record scoring vectors as they are generated, and more easily accommodate alignment matrices of various 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 ent ion. Additionally, for this option, a record may be kept ofthe directions ofthe s 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 sive 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 diagonal, the next scoring vector should be retrieved from two rows previous, because the wavefront had to take two steps to move from g any one cell to scoring the cell diagonally right-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 al extend flag, and a ntal 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 enting the coring alignment terminating in that cell, a 'vertical score' should be generated, corresponding to the maximum alignment score reaching that cell with a final vertical step, and a ontal score' should be generated, corresponding to the maximum ent score ng that cell with a final horizontal step; and when computing any of the three scores, a vertical step into the cell may be computed either using the primary score from the cell above minus a gap-open penalty, or using the vertical score from the cell above minus a gap-extend penalty, 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 penalty, 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 g, any time backtrace takes a al step upward from a given cell, if that cell's scoring vector's vertical 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 backtrace 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 te to support backtrace after concluding alignment scoring where the scoring wavefront took NR steps or fewer.

For example, when aligning cleotide 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 tes, which may be very costly to include as local memory inside the integrated circuit. For such support, g 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 , may be excessive, which may bottleneck and ically reduce aligner performance. Accordingly, it is desirable to have a method for disposing of scoring vectors before completing alignment, so their storage ements can be kept bounded, e.g. to perform incremental backtraces, generating incremental partial CIGAR strings for example, from early ns of an alignment's scoring vector history, 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 tes, 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., comprising l 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 re entation, all the backtraces may be performed together. It is not necessary to extract alignment notations, e.g., CIGAR s, 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, ponding to the number of alignment cells, initialized e.g., all to '1 's, representing whether any of the backtraces pass through a corresponding on. For each step of simultaneous backtrace, scoring vectors ponding to all the current '1 'sin these registers, e.g. from one row ofthe scoring 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 positions, corresponding 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 information from the same cell. It has been observed, empirically and for theoretical reasons, that with high probability, all of the simultaneous backtraces merge into a singular backtrace path, in a relatively small number of backtrace steps, which e.g. may be a small multiple, e.g. 8, times the number of scoring cells in the wavefront. For example, with a 64- cell wavefront, with high ility, all backtraces from a given wavefront boundary merge into a single backtrace path within 512 backtrace steps. Alternatively, it is also le, 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 position, far enough back that they all either terminate or merge into a single backtrace path, e.g. in 512 ace steps or fewer. If they all merge together into a singular 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 l alignment information is possible. Further ace from the merge point, or any distance further back, is commenced, by normal singular backtrace methods, including recording the corresponding alignment notation, e.g., a partial CIGAR string. This ental backtrace, and e.g., l 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 entally 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 performed from a maximum scoring cell, if it terminates before reaching the g ry (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 ar backtrace, its alignment notation, e.g., CIGAR , may then be grafted onto the ental alignment notation, e.g., partial CIGAR string. Furthermore, in a very long alignment, the process of performing a simultaneous backtrace from a scoring ry, e.g., scored wave front position, until all backtraces terminate or merge, followed by a singular backtrace with alignment on extraction, may be repeated multiple times, from various successive scoring boundaries. The incremental alignment on, e.g. partial CIGAR string, from each successive incremental backtrace may then be grafted onto the lated previous alignment notations, unless the new simultaneous backtrace or singular backtrace terminates early, in which case lated previous alignment notations may be discarded. The eventual final backtrace likewise grafts its alignment notation onto the most recent accumulated alignment notations, for a complete ace description, e.g., CIGAR string.

Accordingly, in this manner, the memory to store scoring vectors may be kept bounded, assuming simultaneous backtraces always merge er in a bounded number of steps, e.g. 512 steps. In rare cases where simultaneous aces fail to merge or terminate in the bounded 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 alignment, 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 g boundary to commence a simultaneous backtrace. Optionally, a simultaneous backtrace may be ed to ate or merge within the single block, e.g. 512 steps. ally, 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 g to block N+1, a simultaneous backtrace may commence in block N, followed by a singular ace and alignment notation extraction in block N-1. If the speed of the simultaneous backtrace, the singular backtrace, and alignment scoring are all similar or identical, and can be performed aneously, e.g., in parallel hardware in an integrated circuit, then the singular ace 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 g 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 imately 64 kilobytes. In a variation, if the current maximum alignment score ponds to an earlier block than the current wavefront position, this block and the previous block may be preserved rather than ed, so that a final backtrace 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 another variation, to support overlapped alignments, the g wave front crossing gradually from one alignment matrix to the next as described above, additional , e.g. 1 or 2 additional blocks, may be utilized, e.g., 8 blocks total, e.g., imately 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 alignment function may have in some instances been described, such as with reference to an aligner, these ent functions may be performed sequentially by the same architecture, which has commonly been referenced in the art as an aligner. Accordingly, in various instances, both the mapping function and the aligning function, as herein bed 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 mapping on need first be performed.

In various instances, the devices, systems, and their methods of use of the present disclosure 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 ure may be performed on data to be sed, which gapless alignment procedure may then be followed by one or more of a gapped alignment, and/or by a selective Smith- Waterman ent 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 t 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 alignment on may be performed in order to determine 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 ming such an alignment function is determining where and how there are mismatches in the sequence in question versus the sequence of the nce genome. However, because ofthe great homology within the human genome, in theory, any given nucleotide sequence is going to largely match a entative reference sequence. Where there are mismatches, these will likely be due to a single nucleotide polymorphism, which is vely easy to detect, or they will be due to an ion 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 sequence 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 perform such analysis. Difficulties arise, r, where there are insertions or deletions in the sequence in question with respect to the reference sequence, e 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 indels, only a relatively smaller percentage of gapped alignment protocols need be performed as compared 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 ce of an indel in the sequence, and therefore will need a gapped alignment.

When an indel is indicated in a gapless ent procedure, only those ces get passed on to an alignment engine for further processing, such as an alignment engine ured for performing an advanced alignment function, such as a Smith Waterman alignment (SWA). Thus, because either a gapless 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 ent may be performed on a given selection of sequences, e.g., one right after the other, then the s are compared for each sequence, and the best result is chosen. Such an arrangement may be implemented, for instance, where an enhancement in accuracy is desired, and an sed amount oftime and resources for ming the required processing is acceptable.

] Particularly, in various instances, a first alignment step may be performed without ng a processing intensive Smith an function. Hence, a plurality of gapless alignments 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 sing 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 an alignments need be dedicated using less chip area, as it does not require as much chip area for the processing elements ed to perform gapless alignments as it does for performing a gapped ent. As the chip resource requirements go down, the more processing can be performed in a shorter period of time, and with the more processing that can be performed, 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 example, as disclosed herein, in various embodiments, an alignment processing engine is provided such as where the processing engine is configured for receiving digital signals, e.g., representing one or more reads of c 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 gapless ent function is performed on a contiguous portion of the read, e.g., employing a gapless aligner, and if the gapless alignment goes from end to end, e.g., the read is complete, a gapped alignment is not performed. However, if the results 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 ed alignment results may be used to determine if a gapped alignment is needed, for instance, where the ungapped 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 degree, 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 gapless 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 t 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, ed, and a lower score is indicative of the alignment not being te, and a gapped alignment needing to be performed. Hence, where a high score is attained a gapped alignment is not med, but only when the score is low enough is the gapped alignment performed. Of , in various instances a brute force ent approach may be employed such that the number of gapped and/or gapless aligners are ed in the chip architecture, so as to allow for a greater number of alignments to be med, 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 Waterman, aligner modules. In certain instances, these s may be configured so as to support global o-end) gapless alignment and/or local (clipped) gapped alignment, perform affine gap scoring, and can be configured for generating unclipped score bonuses at each end. Base-quality 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 anti-diagonal 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 wavefront may also be configured to support automatic ng, so as to track the best alignment through accumulated indels, such as to ensure that the alignment wavefront and cells being scored do not escape the g band. In the ound, logic engines may be ured to examine current wavefront scores, find the ms, flag the subsets of cells over a threshold 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 horizontally 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 y 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 ined where each read is mapped, and further determined where each read is aligned, e.g., each relevant read has been given a on 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 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 some location, so that the exact on 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 genomic sample form an individual, and mapping and/or ng that data, which data may then be sorted.

More particularly, 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 beginning of that chromosome, the reads may be sorted by position. g may be useful, such as in downstream es, 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 h the sorting , 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, ore, 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 e a pileup, such that all the reads that cover the same on are physically grouped together; and may r involve analyzing the reads of the pileup to determine where the reads may indicate an actual variant in the genome, as compared to the reference 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 sequencing 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 sequence of an individual, may be a local realignment module. For example, it is often difficult to determine insertions and ons that occur at the end ofthe read. This is because the Smith-Waterman or equivalent alignment process lacks enough context beyond the indel to allow the g to detect its presence. Consequently, the actual indel may be reported as one or more SNPs. In such an ce, the accuracy of the predicted 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 question 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 s 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 includes a substitution, at that position, then the indel may be inserted, such as into the reference, where it is not present, and the reads in the local pileup that overlap that region may be ned to see if collectively a better score is achieved then when the insertion and/or deletion was not there. e is an improvement, the whole set of reads in the pileup may be reviewed and if the score of the overall 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 ming a local realignment, one or more pileups where one or more indels may be positioned are examined, and it is determined if by adding an indel at any given position the overall alignment score may be ed. ] r module that may be included, for example, in a sequence analysis pipeline, such as for determining the genomic sequence of an individual, may be a duplicate marking . 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, mes multiple reads become attached to a single bead and this results in an excessive number of copies of the attached read. This enon 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 ant data. For instance, a typical variant call function or parts thereof may be configured so as to be ented in a software and/or hardwired configuration, such as on an integrated circuit. Particularly, variant 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 s aligned reads form a "pile up." Then the pileup of reads covering a given region of the reference genome are analyzed to determine what the most likely actual content of the sampled individual's DNA/RNA is within that region. This is then repeated, step wise, for every region of the genome. The determined content generates a list of differences termed tions" 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 genome. Next most common are insertions (into the reference) and deletions (from the reference), or "indels" tively. These are more common at shorter lengths, but can be of any length. Additional complications arise, however, because the tion of sequenced segments ("reads") is random, some regions will have deeper coverage than others. There are also more x variants that include multi-base substitutions, 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.

However, variant g is a difficult ure to implement 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 m one or more of the following tasks. For instance, they may come up with a set ofhypothesis genotypes (content of the one or two somes at a locus), use an 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 complex. Simpler variant callers look only at the column of bases in the aligned read pileup at the precise position of a call being made. More advanced variant callers 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 sequence, 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 ences are linked by appearing in the same read. Accordingly, a variant call protocol, as proposed herein, may implement one or more improved ons 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 operations 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 fication, such as for identifying places where le reads 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 region, all the overlapping reads may be led into a "De Bruijn graph" (DBG) . From this DBG, s paths h 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 ted 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, 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 ce intensive and time consuming operation to be performed, often requiring a pair HMM evaluation. Additionally, the constructing ofDe Bruijn graphs for each pileup of reads, with associated operations of identifying locally and globally unique K-mers, as described below may also be resource intensive and/or time consuming. Accordingly, m various embodiments, one or more of the various calculations involved in performing 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 indicated above, in vanous ments, a Haplotype Caller of the disclosure, implemented in software and/or in hardware or a ation thereof may be configured to e one or more of the ing operations: Active Region Identification, Localized Haplotype Assembly, Haplotype Alignment, Read Likelihood Calculation, and/or Genotyping. For instance, the devices, systems, and/or s of the disclosure may be configured to perform one or more of a mapping, aligning, and/or a g operation on data obtained from a t's sequenced A to generate mapped, d, 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 sing engines for performing a variant call ion, including one or more ofthe aforementioned 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 subjected to r sing 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 ion on the obtained results data. Active region identification involves identifying and determining places where multiple reads, e.g., in a pile up of reads, disagree with a reference, and further involves generating one or more windows around the disagreements ("active regions") 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 d to the regions in the reference genome where they are expected to have originated in the subject'sgenetic sequence. r, 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 variant 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 difference 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 e one or more SNPs, ng 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. ingly, 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 sing should be limited to those instances where a perceived mismatch is non-trivial, e.g., a non-noise ence. In determining the significance of a mismatch, places where le reads in a pile up disagree 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 ted 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 mismatch quality scores, such as above a given old 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 old for r processing may be deemed to not have been met, and the non-agreeing ) 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 disagreement may be statistically significant enough to warrant further processing, and an active region around the identified (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 t 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 t every region in a pile up of sequences to further processing, an active region can be identified whereby it is only those s where extra processing may be needed to y determine if a true variance or a read error has occurred that may be determined as needing of further processing. And, as indicated above, it may be the size of the supposed variance that determines the size ofthe window of the active region. For instance, in various instances, 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 , the size of the active window can be any suitable length so long as it provides the context to determine the statistical 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 cluster 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 ce, it may be desirable to analyze any and all the ences that might occur in clusters, so as to analyze them all in one or more active regions, because to do so can provide supporting information about each individual difference and will save processing time by decreasing the number of active windows engaged. In s instances, the active region ries may be determined by active probabilities 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 procedure 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 overlapped 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 ty of ts are to be determined. In various instances, as indicated above, the active region can be extended, e.g., by ing 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 t nding a difference may be desired. Accordingly, 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 pileup, 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 s s of various reads within a pileup are employed by the DBG in ining whether a variant actually is present or not for any given locus in the sequence within the active region. As it is within the active window that this determination is being made, it is those portions of any given read within the s 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 on in the sequence, it is determined based on the collection of overlapping reads for that position, whether there is a match or a mismatch 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 ng, and from stringing or ing the matching reads together, as determined by their points of matching, it can be established for each position within that segment, r and to what extent the reads at any given on 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; r, 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 pathways 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 difference resulting in two divergent strings that will continue along two different path lines until matching between the two ces resumes. Each vertex may be given a weighted score identifying 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 multiplicity of reads, e.g., where each k-mer has the same sequence pattern, they may be accounted for in the graph by sing the count for that pathway where the k-mer overlaps an y existing k-mer pathway. Hence, where the same k-mer is generated from a multiplicity of overlapping reads having the same sequence, 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 pondence therewith. In such an instance, the pattern is only recorded for the first instance ofthe k-mer, and the count is incrementally sed for each k-mer that repeats 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 , which can be generated from each given read by sequentially walking the length ofthe read in ten base ts, 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 . The generated k-mers may then be aligned with one another such that areas of identical matching between the ted 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 d and the percentage of matching and ching may be determined. Particularly, the reference and any previously 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 overlaps any portion of a previously ted 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 c 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 nce 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 compared to the reference genome. However, if only a limited number of k-mers from a limited number of reads evidence the artifact, it is likely to be caused by machine and/or processing and/or other error and not indicative ofa true variation at the position 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 branching 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 ent 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 sequence 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 generally and will be linear, but post the point of ence, a new pathway through the WO 14320 PCT/0S2017/036424 graph will emerge to accommodate the ence represented in the ce of the newly graphed k-mer. This divergence being ented 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 s 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 pathway through the graph represented by the k-mers of the reference sequence. For ce, naturally after a while the read(s) that support the branched 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 te for about 10 nodes, because there are 10 bases per k-mer that overlap that locus of mismatching between the read and the reference. 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 variant. As the graph is processed, bubbles within bubbles within bubbles may be formed along the reference backbone, so that they are stacked up and a plurality of pathways through 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 divergence, all within a given window, each y 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 represent 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 e that occurs when preparing the DNA sample, 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 y 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 ce, if the graph es a y 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 individually need to be extracted, which extracted paths are termed as candidate haplotypes. Such ate ypes represent theories 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 represents a way to reliably extract a good set ofhypotheses to test.

For instance, in performing 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 sing. onally, localized haplotype ly may take place, such as where, for each given active , all the overlapping reads in the pile up may be assembled into a "De Bruijn graph" (DBG) matrix. From this DBG, various paths through 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 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 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 ype, to estimate a probability of observing the read assuming the haplotype was the true original DNA sampled. y, a genotyping operation may be implement, and a variant call file produced. As indicated above, any or all of these operations may be configured so as to be ented in an optimized manner in software and/or in hardware, and in various instances, because of the resource intensive and time ing nature of building a DBG matrix and ting 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 engagement 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 rated manner by an integrated circuit as described herein. In various instances, these tasks may be configured to be ented by one or more quantum circuits such as in a quantum ing device. ingly, in various instances, the devices, systems, and methods for performing the same may be configured so as to perform a haplotype alignment and/or a read likelihood calculation. For instance, as indicated, each extracted ype may be aligned, such as Smith-Waterman aligned, back to the reference genome, so as to determine what variation(s) from the reference it implies. In various exemplary instances, scoring may take place, such as in accordance with the following ary scoring 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 assembled 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 ated with a given haplotype may be ated 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 d 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 oned 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 generated by traversing the extracted paths through the graph and calculating the probabilities that any given path is the true path.

Hence, in such an instance, a ence in this ent protocol from a typical SW alignment 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 probability for alignment of the reads to the haplotype.

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

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

In such instances, the HMM evaluation may employ a dynamic programming method to calculate the total probability of any series of Markov state transitions arriving at the observed read in view of the possibility that any divergence 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 cing cts.

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 es 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 t pathway that matches the subject's actual genetic code for that region must be calculated. Consequently, each pathway 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 imate model for how a true ype in the sampled DNA may transform into a possible ent detected read. It has been observed that these types of transformations are a ation 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 ation 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 3-state 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 e base model tions 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 on 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 alignments, as herein described, as well. ingly, such a 3-state model, as set forth herein, may be employed in a SW and/or NW process thereby ng 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 ng the transition probabilities to vary by position. For instance, the probabilities of all M tions 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 translate to a probability of a sequencing SNP error. When the two bases match, the prior ility is taken as one minus this error probability, 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 ct "Markovish" model. In s ces, the maximum-likelihood transition sequence may also be determined, which is termed herein as an alignment, and may be performed using a man-Wunsch or other dynamic programming algorithm. But, in various instances, in performing a variant calling function, as disclosed herein, 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 possible transition paths h the graph, from read on zero at any haplotype position, to the read end position, at any ype position, each component path probability being simply the product of the various constituent tion probabilities.

] Finding the sum of pathway probabilities may also be performed by employing a virtual array and using a dynamic programming algorithm, as bed above, such that in each cell of a (0 ... N) x (0 ... M) matrix, there are three probability values calculated, corresponding 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 states, and 0.0 in the I and M states; and the rest of the left column (haplotype position 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 later.) This 3-to-1 computation dependency restricts 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 ard. 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 position, may be ured 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 probability of0.0 may be used there, so as to avoid double counting.

As described herein, in various ces, 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 haplotypes may be aluated 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 ypes 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 instances, floating point operations may be used.

There are three parallel 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 pipeline 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 ilities each cycle, such as from one or more already computed 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 latency.

As indicated above, in ming a variant call function, as disclosed herein, a De Bruijn Graph may be formulated, and when all ofthe reads in a pile up are identical, the DBG will be . However, where there are differences, the graph will form "bubbles" that are indicative of regions of ences resulting in multiple paths diverging from matching the reference ent and then later re-joining in matching ent. 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 ed such as where the possible diploid combinations of the candidate haplotypes may be formed, and for each of them, a conditional probability of ing the entire read pileup may be calculated. These results may then be fed into a Bayesian formula module to calculate 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 described herein, in various ces, a genotyping operation may be performed, which genotyping operation may be configured so as to be implemented in an optimized manner in re and/or in hardware and/or by a quantum processing unit. For ce, the possible diploid combinations of the ate haplotypes may be formed, and for each combination, a conditional probability 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 ype or variant call operation on generated and/or supplied data so as to produce a variant call file with respect thereto. Specifically, as described herein above, in particular ces, a t call file may be a digital or other such file that encodes the difference n one sequence and another, such as a the difference between a sample sequence and a reference sequence. Specifically, in various instances, the variant call file may be a text file that sets forth or otherwise details the genetic and/or structural ions in a person's genetic makeup as compared 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 ing and y inherited together. Particularly, a haplotype can refer to a ation 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 ining the identity of a person's genome it is important to know which form of various different possible alleles a specific person's genetic sequence codes for. In ular instances, a haplotype 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 thm employing a ype call program, e.g., GATK, for simultaneously determining SNPs and/or insertions and/or deletions, i.e., indels, in an individual's genetic sequence. In particular, the algorithm may involve one or more haplotype assembly protocols such as for local de-novo assembly of a haplotype in one or more active regions of the genetic ce 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 assumed 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 teristics or is otherwise assumed to be a Markov process with unobserved (hidden) states. In particular instances, the model may involve a simple dynamic Bayesian 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 segment 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 t's DNA/RNA must be sequenced, e.g., via a Next Gen Sequencer ("NGS"), to produce a readout or " 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 d, as herein bed 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, therefore, it is assumed that the read potentially representing a given gene of a subject is a representation of the prototypical haplotype albeit with s SNPs and/or indels that are to presently be determined.

Specifically, in particular aspects, devices, s, and/or methods for practicing the same, such as for performing a haplotype and/or variant call function, such as deploying an HMM function, for instance, in an accelerated haplotype caller is provided. In various instances, in order to me 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 entations. For instance, in a particular , 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 variant call function, such as employing an HMM protocol. ularly, the HMM function may be med in hardware, software, or via one or more quantum circuits, such as on an accelerated device, in accordance with a method described herein. In such an ce, 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 software-controlled CPU or GPU or even a QPU, to a haplotype caller implementing an accelerated HMM, which haplotype caller may be deployed on a microprocessor chip, such as an FPGA, ASIC, or structured ASIC or implemented by one or more quantum circuits. The method may further include the steps for processing the data to produce HMM result data, which s 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 provided. 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 platform. Specifically, provides a high-level view of an HMM interface structure. In particular ments, the variant call system 1 is configured to accelerate at least a portion of a variant call operation, such as an HMM ion. Hence, in various instances, the variant 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 related to the sequencing and/or processing of genetic ation, such as for comparing a sequenced genetic sequence to one or more nce ces.

Additionally, the system 1 includes a peripheral device 2, such as an expansion card, that includes a hip 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 CPU/GPU/QPU 1000 with the chip 7. For instance, the device 2 may comprise a peripheral component onnect, such as a PCI, PCI-X, PCie, or QPI (quick path interconnect), and may include a bus interface 4, that is adapted 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 ular ces, the interface may be a peripheral component interconnect s (PCie) 4 that is ated with the hip 7, which microchip includes an HMM accelerator 8. For example, in particular instances, the HMM accelerator 8 1s configured for ming an accelerated HMM function, 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 exemplary 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 variant call function, such as including an HMM task. Accordingly, the HMM accelerator 8 may be composed of a data butor 9, e.g., CentCom, and one or a multiplicity of processing clusters 11 - lln+l that may be zed as or otherwise include one or more instances 13, such as where each instance may be ured 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 otherwise transferring that data to one or more ofthe licity ofHMM sing clusters 11.

] Particularly, in certain embodiments, the distributor 9 may be positioned logically between the on-board PCie interface 4 and the HMM accelerator module 8, such as where the interface 4 communicates 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 adapted 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 ise include an array of clusters 1la-1 ln+l, such as where each HMM cluster 11 may be ured as or otherwise includes a cluster hub 11 and/or may include one or more ces 13, which instance may be configured as a processing engine 13 that is adapted for performing one or more operations on data received 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 directing data to a plurality ofthe processing s 13a - 13m+1 within the cluster 11.

In various instances, the HMM accelerator 8 is configured for comparing each base of a subject's ced genetic code, such as in read , with the various known or generated candidate haplotypes of a nce sequence and determining the probability 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 resulting in a variation of the base at the on being considered. Particularly, in various embodiments, WO 14320 PCT/0S2017/036424 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 re, 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 hip 7, such as positioned on the peripheral expansion card or board 2. In various embodiments, this onality may be implemented partially as software, e.g., run by the CPU/GPU/QPU 1000, and partially as re, implemented on the chip 7 or via one or more quantum processing circuits. Accordingly, in various embodiments, the chip 7 may be present 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 include or otherwise be associated 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 ments, 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., included on the motherboard of a er, or indirectly d thereto, such as being included as part of a peripheral device 2 that is operably coupled 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 tood that although configured as a microchip, the accelerator could also be configured as one or more quantum circuits of a quantum processing unit, wherein the quantum circuits are configured as one or more processing engines for performing one or more ofthe functions disclosed herein.

Hence, the peripheral device 2 may e 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 expansion bus 4 may be a Peripheral Component Interconnect s (PCie) that is ured to communicate with or otherwise include the microchip 7, such as via tion 5. As described herein, the microchip 7 may at least lly 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 ation therewith, and is configured 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 variant 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 partially configured as an HMM accelerator 8. The CPU/GPU/QPU 1000 communicates with the microchip 7 over an interface 5 that is adapted to tate the communication between the CPU/GPU/QPU 1000 and the HMM accelerator 8 of the microchip 7 and therefore may communicably 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 includes 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 configured for receiving and processing the data, such as by running an HMM protocol thereon, computing final values, outputting the results thereof, and repeating the same. In various instances, the mance of an HMM protocol may include ining one or more transition probabilities, as described herein below. ularly, each HMM engine 13 may be configured for performing 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 s or is different from a corresponding base in a hypothetical ype sequence, as described herein below. presents a detailed depiction of the HMM cluster 11 of . In various embodiments, each HMM r 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 sor. Particularly, a HMM cluster may be provided, where the cluster is configured as a cluster hub 11. The cluster hub 11 takes the data ning to one or more jobs 20 from the distributor 9, and is r 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 transfer ofdata throughout the system may be relatively low dth process, and once a job 20 is received, the system 1 may be configured for completing the job, such as without having to go off chip 7 for memory. In various embodiments, one job 20a is sent to one processing engine 13a at any given time, but l jobs 20a-n may be distributed by the cluster hub 11 to several ent processing engines 13a-13m+l, such as where each of the processing engines 13 will be working on a single job , e.g., a single comparison between one or more reads and one or more haplotype sequences, in el and at high speeds. As described below, the performance of such a job may typically involve the generation of a virtual matrix whereby the subject'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 licity 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 n base pairs, there may be on the order of 1 to 2 billion different 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 , each HMM instance 13 may be adapted so as to perform an HMM protocol, e.g., the generating and processing of an HMM matrix, on sequence data, such as data received thereby from the CPU/GPU/QPU 1000. For example, 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 t, with scores between 20 to 45 generally being acceptable as relatively accurate).

Particularly, as indicated above, a Phred quality score is a quality tor that measures the quality of the identification of the nucleobase identities ted by the sequencing processor, e.g., by the automated DNA/RNA sequencer. Hence, each read base includes its own y, e.g., Phred, score based on what the sequencer evaluated the y 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 l Phred score prior to the performance of an HMM protocol thereon, such as by taking into account neighboring scores and/or fragments ofneighboring DNA and allowing such factors to influence the Phred score ofthe base, e.g., cell, under examination.

In such instances, as can be seen with respect to the system 1, e.g., computer/quantum re, may determine and identify various active regions 500n within the sequenced genome that may be explored and/or otherwise ted to further processing as herein described, which may be broken down into jobs 20n that may be elized amongst the various cores and available threads 1007 throughout the system 1. For instance, such active regions 500 may be identified as being sources of variation between the sequenced and reference genomes. ularly, the CPU/GPU/QPU 1000 may have multiple threads 1007 running, identifying active regions 500a, 500b, and 500c, compiling and aggregating 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 threads t the less active time spent g.

Once identified, compiled, and/or aggregated, 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 interface 4, e.g., in a fire and forget manner, and will then move on to a different process while waiting for the HMM 8 to send the output data back so as to be d back up to the corresponding active region 500 to which it maps and/or aligns. The data distributor 9 will then distribute the jobs 20 to the various 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 ce, in various embodiments, raw jobs data and processed job results data may be sent through 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 h 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 cluster 11, such as where one 4K page is sent to one cluster 11. r, 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 cluster 11 approach as presented here efficiently distributes ng 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 received data may then be sent over the PCie bus 5 to the CentCom butor 9 of the variant caller microchip 7. The distributor 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 ace 4 is adapted to provide 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 g up with the output of the processed 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 accelerator instances 13 may directly add to the overall sing time ofthe system 1. Particularly, when analyzing a human genome, there may be on the order of two or more billion 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.

Accordingly, sets forth an overview of an exemplary data flow throughout the software and/or re of the system 1, as described generally above. As can be seen with respect to the system 1 may be configured in part to transfer data, such as n the PCie interface 4 and the distributor 9, e.g., CentCom, such as over the PCie bus 5. onally, 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 r buses 10. Hence, in various embodiments, the HMM rator 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 function. In such an instance, there is an interface, such as a cluster bus 10, that connects the CentCom 9 to the HMM cluster 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 module. As can be seen with respect to each HMM cluster 11 may be configured to communicate with, e.g., e data from and/or send final result data, e.g., sum data, to the CentCom data distributor 9 through a dedicated r bus 10. Particularly, any le interface or bus 5 may be provided so long as it allows the PCie interface 4 to communicate with the data distributor 9.

More particularly, the bus 5 may be an interconnect that es the interpretation logic useful in talking to the data distributor 9, which interpretation logic may be configured to accommodate any protocol employed 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 domains 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 ed, such as for communications, e.g., to and from the cluster 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 ations described herein.

Particularly, in various embodiments, as can be seen with t to the system 1 may be set up such that, in a first instance, as the data distributor 9 leverages 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 instance, an HMM cluster bus 10 may communicably and/or operably connect the U 1000 to the various clusters 11 of the HMM accelerator module 8. Hence, as can be seen with t 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 ted its currently assigned task. It is to be noted that each HMM rator engine 13 can handle ping and pong inputs (e.g., can be working on one data set while the other is being loaded), thus minimizing downtime between jobs. Additionally, the HMM cluster collar 11 may be configured to automatically take the input job 20 sent by the data butor 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 Accordingly, in view of the above, the system 1 may be streamlined 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 re HMM accelerator 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 e 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 e interface logic that communicates with the cluster hub 11, which hub is in communications with the distributor 9, which in tum is communicating with the PCie interface 4 that communicates with the variant call re 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 es, e.g., the HMEM and RMEM. This configuration may also be implemented in one or more quantum circuits and adapted accordingly.

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 loaded, 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 ular instances, two haplotypes and two read sequences may be loaded into their WO 14320 PCT/0S2017/036424 respective memories, which would allow the four sequences to be processed together in all nt 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 r ease the bandwidth when desired.

Additionally, 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 described herein and below. Accordingly, this leaves the pong side of the memory available 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 sing 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 sequences, 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 s ation related to "Priors" data, as explained below, which in tum feeds the Prior results data into the M, I, and D state calculator block 17b, for use when calculating transition probabilities. One or more scratch RAMs 17c may also be ed, such as for holding the M, I, and D states at the ry of the swath, e.g., the values ofthe bottom row of the processing swath, which as indicated, in various instances, 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 immediately be transmitted back to the variant call software of the CPU/GPU/QPU 1000. It is to be noted that this configuration may be adapted 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 ne steps functioning in concert to make an overall 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 employed in determining whether the particular read being examined s the haplotype reference 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 ed to a particular processing engine 13 for processing. These data are then used by the M, I, and D calculator 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 on; and/or if there is a variation, whether such variation is the likely result of a true ility in the ype 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 analysis includes the MID calculator 17 ining the transition probabilities from one base to another 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 ion between the read and the nce is a true variation and not just some machine or processing error. For these purposes, the Phred score for each base being considered is useful in determining the transition probabilities in and out of the match state, such as going from a match state to an insert or deletion, e.g., a gapped, state m the comparison. Likewise, the transition probabilities of uing 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 y.

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

] Accordingly, as ed with respect to FIGS. 7 and 8 each ofthe M, I, and D state values are computed for each possible 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 position in the read and haplotype reference.

Hence, if the read and haplotype sequences are each 100 bases in length, the matrix 30 will include 100 by 100 cells, a given n of which may need to be processed in order to determine the likelihood and/or extent to which this particular read matches up with this particular reference. Hence, once virtually formed, the matrix 30 may then be used to determine the various 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 parallel and/or sequential fashion when traversing the matrix with the control logic 15. For ce, as depicted 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 clearly be seen. Hence, for determining a given M state of a t cell being sed, the Match, Insert, and Delete states of the cell diagonally above the t cell need to be pushed into the present cell and used in the calculation of the M state of the cell presently 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 processed (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 respect 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 sing approach ses the processing efficiency and hput 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 instance, depicts 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 ion of the cells may be populated with an M, I, and D probability state, and the transition probabilities of transitioning from the present state to a neighboring state. In such an instance, as described in greater detail above, a move from a match state to a match state s in a forwards diagonal progression through the matrix 30, while moving from a match state to an insertion state s 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 sequential nature of the processing (s) that are configured so as to produce a sing 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. ingly, in certain instances, it may be desirable to calculate the ties of each cell in a downwards and diagonal manner, as explained above, rather than simply calculating each cell along a al or horizontal axis exclusively, although this can be done if desired. This is due to the increased wait time, e.g., latency, that would be required WO 14320 PCT/0S2017/036424 when processing the virtual cells of the matrix 35 individually and sequentially along the vertical or horizontal axis alone, such as via the hardware uration.

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 y 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 complete 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 s computational delays of the pipeline associated with the M, I, and D state calculations 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 quantum 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 ated the entire matrix 30, may be a bottom row (e.g., Row of) ning M, I, and D states, where the Mand I states may be summed (the D states may be ignored at this point having already led their function in processing the calculations above), so as to produce a final sum value that may be a single probability that estimates, for each read and ype index, the probability of observing the read, e.g., assuming the ype 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 probability that the read is an actual representation of that haplotype. This probability 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 s 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 haplotype, and thereby allows the system to better determine how the t's genetic sequence, e.g., en masse, may differ from that of a reference sequence. For instance, many haplotypes 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 l of its surrounding ors that have already been calculated. Particularly, as shown in greater detail with t 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 probability (gap open penalty) of going from a match state to either an insertion or a deletion, e.g., gapped, state. r, when in either a gapped insertion or gapped deletion state there may be only a 0.1 probability (gap extension or continuation penalty) of g in that gapped state, while there is a .9 probability of ing 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 . onally, the state transition probabilities may in part be derived from the "Phred" quality score that anies each read base. These transition 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 ated. 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 penalties may be variable and therefore programmable within the system, albeit by employing additional hardware resources dedicated to determining such variable tion 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 ned below.

Accordingly, there is a licity of ations 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 atical computations 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 non-trivial.

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 te the ions.

For instance, these computations may be run at high speeds inside the processor 7, such as at about 300MHz. This may be achieved such as by pipelining the FPGA or ASIC heavily with registers so little mathematical computation occurs between each flipflop.

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 ents one clock cycle.

Hence, the number ofM, I, and D state calculations may be limited. In such an ce, the processing engine 13 may be configured in such a manner that a grouping, e.g., swath 35, of cells in a number ofrows ofthe matrix 30 may be processed as a group (such as in a down-and-left-diagonal fashion as illustrated by the arrow in before ding 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 described herein, may be adapted so as to make the overall system more efficient, as described above.

Particularly, sets forth an exemplary computational ure for performing the various state processing calculations herein bed. 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 implemented 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 calculating the match state 15a of the t cell being sed, all ofthe previous match, insert, and delete states ofthe adjoining cells along with s "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 tive calculations. Hence, as can be seen with respect to in calculating a match state, three state multipliers, as well as two , 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 sing each new cell in the HMM matrix 30 uses the knowledge or pre-computation ofthe following values, such as the "previous" 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 nce base at a given cell in the matrix 30 match or are different. Such information is particularly useful when determining a match state. ically, as can be seen with respect to in such instances, there are basically seven "transition probabilities" (M-to-M, I-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., ofseeing 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 previous state, e.g., Match-to-Match, -to-Insert, Delete-to-Delete.

The state values (e.g., in any cell to be processed in the HMM matrix 30), Priors, and transition ilities are all values in the range of [0,1]. Additionally, there are also known starting conditions 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. tively, along with the priors multiplier this sums to a total of eight multiplications and four addition operations for the M, I, and D state ations 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 red to the U/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 coverage, 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 ations may then be ed 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 reference s. 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 compute the final sum value, such as by including a mux into a ion 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% overhead. In alternative embodiments, dedicated hardware resources can be used for performing such calculations. 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 leading to the summing values.

] In certain instances, these calculations and relevant processes may be configured so as to correspond to the output of a given sequencing platform, such as ing an ensemble of sequencers, which as a collective may be capable of outputting (on average) a new human genome at 30x coverage every 28 s (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 t calling operations are configured to fit within such a cing 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 image/BCL/FASTQ file results from the sequencer and perform the steps of mapping and/or aligning the genome, e.g., equencer processing. That leaves about 18 minutes of the sequencing time period for performing the variant calling step, h the HMM operation is the main ational component, such as prior to the nucleotide sequencer sequencing the next genome, such as over the next 28 minutes. Accordingly, in such instances, 18 s may be budgeted to computing the 20 on 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 ions (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 ained throughput. ] sets forth the logic blocks 17 of the processing engine of including 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 ce 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 le 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 associated resource usage, and/or in a further effort to include as many HMM engine instances 13 on the chip 2 as desirable and/or possible, simplifications may be made with regard to the logic blocks 15a'-c' ofthe processing instance 13 for computing 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 ding I and D states before multiplying by a common Indel-to-M transition probability. For instance, in various instances, if the I and D state calculations are d to be the same, then the state calculations per cell can be fied 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.

Additionally, 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 summing calculation. In such an instance, the final logic, ing 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 liers 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 related to transition probabilities, as well as the effect of sharing various M, I, and/or D ces, 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 compensate for delays in the actual hardware implementations of the multiply and addition operations, and/or to fy the control logic, e.g., 15.

As shown in FIGS. 9 and 10, these respective liers and/or adders may be floating point multipliers and adders. However, in various 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. ts what log domain ation would look like if all the multipliers turned into , e.g., 15a", 15b", and 15c", such as occurs when employing a log domain computational configuration. Particularly, all of the multiplier logic turns into an adder, but the adder itself turns into or otherwise includes 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 ce lengths as well as the values typically seen for read quality (Phred) 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, s 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 r than 80 powers of 10, thereby requiring the variant call re to bump up to double-precision (64-bit) ng 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 compute pipeline resource requirements will need to be increased, thereby occupying greater chip space, and/or slowing . In such instances, a fixed-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 ble. Accordingly, in such instances, oint-only log-domain number representation may be implemented, as described herein.

] In such a scheme, as can be seen with respect to , d of representing the actual state value in memory and computations, the ase-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 ly operations go away in the log-domain. Rather, they are replaced by add operations, and the add operations are morphed into a function that can be expressed 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 between the two values being summed in the log-domain. Such a tion factor can be either computed or generated from the look-uptable.

Whether a correction factor computation or look-up-table entation is more efficient to be used depends on the required precision (bit width) on the difference between the sum values. In particular instances, therefore, the number of log-domain bits for state representation can be in the neighborhood of 8 to 12 integer bits plus 6 to 24 fractional bits, depending on the level of quality desired for any given implementation. This implies somewhere 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 ping-pong structure may also be used in the read ce 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 rt memory (such as one port for write, one port for read, and/or te clocks for write and read .

Particularly, 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 corresponding base in one or more reference genomes, a virtual matrix 30 is formed, wherein the reference genome is theoretically set across a horizontal axis, while the ced reads, representing the sample genome, is tically 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 sing 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 traversal 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 grouping (swath) down in the matrix 30. In various instances, the system 1 may be configured to allow up to 1008 length haplotypes and/or reads in the HMM accelerator 8, and since the numerical entation employs W-bits for each state, this implies a 1008word x W-bit memory for M, I, and D state e.

Accordingly, as indicated, such memory could be either a -port or double-port memory. Additionally, a cluster-level, h 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 ured for a per-engine-instance 13 basis. In particular HMM implementations, multiple engine instances 13a-(n+l) may be grouped into a cluster 11 that is serviced by a single connection, e.g., PCie bus 5, to the PCie interface 4 and DMA 3 via CentCom 9. Multiple clusters lla-(n+l) can be tiated so as to more efficiently utilize PCie bandwidth using the existing CentCom 9 functionality.

Hence, in a typical configuration, somewhere between 16 and 64 engines 13m are instantiated within a cluster 1ln, and one to four clusters might be tiated in a typical SIC 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 hardware.

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 r 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 theory, this FIFO could be very small (on the order of two 32-bit , 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 ed 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 appropriately configured function efficiently to avoid high-bandwidth 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 ented so as to avoid the development of other necks within the system.

WO 14320 PCT/0S2017/036424 Particularly, the system 1 may be configured so as to maximize the process of efficiently feeding 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 s the presentation 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 architecture 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 ations may be simplified and the resulting values generated. Thus, increasing throughput, efficiency, and reducing the possibility of a bottleneck forming at this stage in the s.

Additionally, 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 probability= lW'(-(read Phred/10)). Then the Prior can be ed based on whether the read base matches the esis haplotype base: If the read base and esis ype 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 re accelerator 8 as 6-bit values. The equations to derive the Priors, then, have 64 possible es 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 quantity formed by enating the Phred value with a single bit that indicates whether, or not, the read base s 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 transition, and the Match-to-Delete state transition, as ted above. This equates to the M2I and M2D values in the state transition 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 lW'(-(read GOP(I)/10)) and M2D transition probability= lW'(-(read GOP(D)/10)). Similar to the Priors derivation in hardware, a simple 64 word look-up-table can be used to derive the M2I and M2D values. If GOP(I) and GOP(D) are inputted to the HMM re 8 as potentially different values, then two such look-up-tables (or one resource-shared look-up-table, potentially clocked at twice the ncy ofthe rest ofthe circuit) may be utilized.

Furthermore, with respect to determining match to match transition probabilities, in s instances, 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 on above can be ented in hardware in a manner so as to increase overall ency and throughput, such as by reworking the equation to be: M2M tion probability= (0.5 - M2I transition ility) + (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 respect 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 rator 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 ility= lW'(-(read GCP(D)/10)). Similar to some of the other transition probabilities discussed above, the 121 and D2D values may be efficiently implemented in hardware, and may include two look-up-tables (or one ceshared p-table), such as having the same form and contents as the Match-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 probabilities, the I2M and D2M transition probabilities are functions of the gap continuation probability (GCP) values and may be computed as: I2M transition ility = 1 - 121 transition probability, and D2M tion probability= 1 - D2D transition ility, 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 another 64 word look-up-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 operations 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 various ces, a simple HMM hardware accelerator architecture 17a is presented, which accelerator may be configured to e separate GOP values for Insert and Delete transitions, and/or there may be te GCP values for Insert and Delete transitions. In such an ce, the cost of generating the seven unique transition probabilities and one Prior each clock cycle may be configured 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 presented herein, may be configured so as to fit as many HMM engine instances 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 ed 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 s in several simplifications to the re, as indicated above.

In such instances, only one 64 word look-up-table may be employed so as to generate a single M2Indel value, replacing both the M2I and M2D transition probability values, whereas two tables are typically 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 Indel2Indel transition probability may be calculated instead ofseparate 121 and D2D values, using one 64 word look-up-table instead of two tables; and single Indel2Match transition probability may be calculated 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 ecture with 121, D2D, I2M, and D2M transition probabilities can be eliminated altogether. In various of these instances, the static Indel2Indel and Indel2Match ilities 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 bitstream-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 m may look like when implementing these s simplifying assumptions. Specifically, sets forth the simplified HMM state transition diagram ing the relationship between GOP, GCP, and transition probabilities with the simplifications set forth above.

Likewise, sets forth the circuitry 17a,b for the HMM transition ilities and priors generation, which supports the simplified state transition diagram of . As seen with t to , a circuit realization of that state tion diagram is provided. Thus, in various instances, for the HMM hardware rator 8, the cost of generating the transition probabilities and one Prior each clock cycle reduces to: Two 64 word p-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 l 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 control 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 ion, the worst-case latency 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 tood 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 ations of the cell in the HMM matrix that is immediately to the right (e.g., having the same read base as the given cell, but having the next haplotype base). se, the M and I state information 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 ations of cells in the next diagonal of the HMM cell matrix. rly, 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 actually two als 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 beneficial result for hardware design.

Particularly, given these urations, I and D state ations may be adapted to take half as long (e.g., 10 cycles) as the M state calculations (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 computations for a cell in the HMM matrix 30 will all complete 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 ne 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 ations 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 ted 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 ers 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, natively 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. Particularly, in various embodiments, operations start at cell (0,0), with M state calculations beginning 10 clock cycles before I and D state ations 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 calculations 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 ive 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). However, there is a ten cycle latency after the start of I and D calculations before the results from cell (1,0) will be available. The hardware, therefore, inserts 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 computations 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 se may be cell (3,0). However, there is a ten-cycle latency after the start of I and D calculations before the results from cell (2,0) will be available. The hardware, therefore, inserts seven dead cycles into the e 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 ting 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 ht away to start the D state computations of cell (3,0). And, so, computation for another ten cells in the diagonal 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 ations before the results from cell (12,1) will be available.

The re may be configured, therefore, to start operations associated 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 traverse should be cell (13, 2). However, as before there may be a ten-cycle latency 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 sed 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 sed, 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 coordinates (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 associated 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 ted 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 ately 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 e 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 ofpipeline 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 independent 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 tage ofHMM matrix evaluation 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 ined that traversing the HMM matrix in the manner described above results in less than 10% overhead for the whole genome processing.

Further methods may be ed to reduce the amount of overhead cycles ing: 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 operations 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 increased 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, ing about twenty five rows to about thirty rows of cells 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 computation 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 ad index, 10) = 0, retrieve the previously saved M, I, and D state information from the appropriate place in the M, I, D state storage 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 accelerator 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 , an additional row may be added to the bottom ofthe HMM matrix 30, and the clock cycles ated 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 ing 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 various reads originated, such as with respect to a given on, e.g., chromosomal location.

In such instances, the variant caller may be configured to detect the underlying sequence at that location, such as independently 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. ] ularly, though DNA cing has improved ically, variant calling remains a difficult problem, largely due to the genome's ant structure. As disclosed herein, however, the complexities presented by the genome's ancy may be overcome, at least in part, from a perspective driven by short read data. More particularly, the devices, systems, and methods of employing the same as disclosed herein may be configured in such a manner so as to focus on Homologous or Similar regions that may otherwise have been characterized by low t calling accuracy. In certain instances, such low variant calling accuracy may stem from difficulties observed in read mapping and alignments with respect to homologous regions that typically may result in very low read MAPQs.

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

For instance, many regions of the genome are homologous, e.g., they have dentical copies located elsewhere in the , 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 process 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 susceptible 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 te 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 multi-region joint detection operation as herein described. For ce, instead of ering each region in isolation and/or instead of performing and analyzing long read sequencing, multi-region joint detection (MRJD) methodologies may be employed, such as where the MRJD protocol ers 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 ence and/or certainty scores.

For example, for a d organism with statistically uniform coverage, a brute force Bayesian calculation, as described above, may be performed in a t call analysis. However, in a brute force MLRD computation, the complexity of the calculation grows rapidly with the number of s N, and the number of candidate ypes 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 ntial. For instance, as described in greater detail below, in a brute force implementation, the number of candidate haplotypes includes the number of active positions, which if a graph-assembly technique is used to generate the list of candidate haplotypes in a variant call operation, such as in the ng 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 an 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 respect to the methods disclosed herein, variant calling, e.g., the process of reconstructing a subject's genome from the reads a cer produces, remains a difficult m, largely due to the genome's redundant structure. The devices, systems, and methods disclosed herein therefore are configured to reduce the complexities presented by the genome's redundancy from a ctive driven by short read data in contrast to long read sequencing. In particular, provided herein are methods for performing very long read detection that accounts for homologous and/or similar regions of the genome that are usually characterized by low variant calling accuracy without necessarily having to perform long read sequencmg.

For instance, in one embodiment, a system and method for performing multi region joint detection is provided. ically, in a first instance, a l variant calling operation may be med such as employing the methods disclosed herein. Particularly, a l 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 sequencer, a subject's genome may be broken down into subsequences, e.g., reads, lly 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 comparison to the subject's actual genome, it may be determined to what extent, and how the subject'sgenome s from the reference , 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 ces comparing the one with the other(s) to determine ifthey agree or disagree. Accordingly, each base within the sequences represents a position to be called, such as represented by position A in A.

Specifically, for every position A of the reference to be called with respect to the t's genome, a pile up of ces, 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 nucleotides 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 represents the active region of bases in the sample being called, where the calling es comparing each base at a given position, 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, provides ce for the true identity of the base at that position being called.

For this purpose, based on the relevant MAPQ confidence score d for each read segment, it may be lly determined, within a n 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 configured to perform one or more multi-region joint detection operations, as herein described, which may be employed to give greater confidence in the achievable s. For instance, in such an instance, the t caller may be configured to analyze the various regions in the genome so as to ine particular regions that appear to be similar. For example, as can be seen with respect to A, there may be a reference region A, and a reference region B, where the referenced sequences are very similar to one another, 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 on may appear to match. Because ofthe extent of similarities, these two regions, e.g., RefA and RefB, will typically be ered 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 similar, 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 n reference regions C and D, but may, in certain instances, have difficulties calling out the differences n the highly homologous regions of reference regions A and B, e.g., because of their high homology. Particularly, because of the extent ofthe dissimilarity between nce sequences A and B to nce 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 gy, 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 distinguish and/or account for the difference between homologous regions, such as when performing a mapping, aligning, 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 pped to the wrong place, and as such, to effectuate better accuracy, when performing the t calling operations disclosed herein, these gous regions, and the reads mapped and aligned thereto, should be considered together, such as in a joint detection protocol, e.g., a 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 various 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 ons that appear to be homologous. Making such joint calls is advantageous because 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 on actually map and align, and that is ntly uncertain, and the very problem being solved by the proposed joint detection. Hence, because 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 region. 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 reference, e.g., B, could belong to either Ref. A or Ref. B.

] Consequently, where desired, an MRJD protocol may be implemented on addition to the variant call algorithm implemented in the devices, systems, and methods herein. For instance, in one iteration, a variant call algorithm takes the ce presented in the mapped and/or aligned reads for a given region in the sample and nce genomes, analyzes 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 difference, 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 regions, based on a percentage of correspondence between the sequence in a ity 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 some. Hence, where diploid nces 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 example, 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 t to Ref. B, another copy of the subject's chromosome has an "A" for both alleles at on A. This also becomes more complicated, where the sample being analyzed contains a mutation, e.g., at one of those naturally occurring le 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 e reads that indicate there is heterozygosity at position A, such as where some ofthe reads e a "C" at this on, 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 on A indicate gosity, such as where all the reads in the pileup have an "A" at that on (e.g., Hb1 = "A", Hb2 = "A"). However, MRJD overcomes these difficulties by making a joint call simultaneously, by analyzing 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 s homologous regions are fied, the next step is to ine the correspondence between the homologous reference 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 homologous regions may be discarded, and rather, all of the reads in any of the pileups in these homologous regions may be considered collectively together, knowing that any of these reads may belong to any of the homologous s 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 regions 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 e 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 homologous regions are useful for non-homologous regions as well, because 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 cy of the system. Likewise, for a erence region, e.g., a homologous region, as described above, what is being called is an HA1 and HA2 for the first , and an HA1 and HA2 for the second region (which is equivalent two strands for each chromosome and two regions for each strand = 4 diploidtypes, generally.

Accordingly, MRJD may be employed to determine an initial answer, with respect to one or more, e.g., all, homologous regions, and then single region detection may be applied back to one or more, e.g., all, single or non-homologous s, e.g., employing the same basic analysis, and thus, better accuracy may be achieved. Hence, single region nt detection 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 on that s from the nce forms a divergent pathway or "bubble" in the graph, from which haplotypes are extracted, where each extracted haplotype, e.g., divergent pathway, forms a potential esis for what might be on one of the two strands of the chromosomes at a particular location ofthe active region under ation.

However, if there are a lot of divergent pathways, e.g., a lot of s 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 haplotypes may be considered.

In such an instance, instead of extracting complete source to sink haplotypes from start to finish, e.g., from the beginning of the sequence to the end, only the sequences associated with the individual bubbles need be extracted, 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 , all of the ent hypothetical haplotypes for analysis may be derived from mixing and ng the sequences pertaining to all of the various bubbles in different ations. In a manner such as this, all of the haplotypes to be extracted do not need to be enumerated. These methods for performing multi-region joint detection, are described in greater detail herein below.

Further, 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 calculations are made, and it s apparent that various ent candidate hypotheses are simply too improbable. Hence, as the tree grows and is pruned, not all ofthe hypothesized haplotypes need to be calculated.

Specifically, with respect to the growing of the tree function, when there is disagreement between two nces, or between the references and the reads, as to what base is t at given positions being resolved, it must be determined which base actually belongs in which on, and in view of such disagreements it must be determined which differences may be caused by SNPs, Indels, or the like, versus which are e errors. ingly, when growing the tree, e.g., extracting bubbles from the De Bruijn graph, such as via SW or NW aligning, and oning them within the emerging tree graph, each bubble to be extracted becomes an event in the tree graph, which represents possible SNPs, Indels, and/or other differences from the reference. See C. ] ularly, in a DBG, the bubbles represent mismatches from the reference, e.g., representative 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 rized as , and a list of the various events, e.g., bubbles, is generated, ore, the determination then becomes: what combination of the possible events, e.g., of possible SNPs and , 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, forming a root Go (representing 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 respect to a growing the tree function, as can be seen with respect to D, the calculation begins with determining the haplotypes, e.g., HA1, HA2, HB 1, HB2 (for a diploid organism), where the initial haplotypes are considered to all be unresolved with respect 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 unresolved. Then a particular event, e.g., an initial bubble, is selected as the origin for determination, whereby the initial event is to be resolved for all of the ypes, 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 on 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 question 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 ilities for resolving this position. Hence, the calculation moves immediately from the root to 16 branches, representing the potential resolutions for the event at position one.

Therefore, as can be seen with respect to D, all of the potential 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 hood 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 e all events for all haplotypes. Hence, the probabilities may be recalculated for observing any particular read given the various potential haplotypes.

Particularly, for each node, there may be four haplotypes, and each haplotype may be compared against each read in the pileup. For instance, in one ment, the SW, NW, and/or HMM engine, analyzes each node and considers each of the four haplotypes for each node. Consequently, generating 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 ype for one read for each ofthe haplotypes and each ofthe reads for all ofthe viable nodes.

Hence, if for exemplary purposes of this e, 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 probability, because 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 existence of a "C" at this position in the reads in question is ce of a sequencing or other ific 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 ted by a ty 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 ined, and the ses herein described may then be repeated for that new position with t to any ofthe surviving nodes that have not heretofore been pruned.

Particularly, event two may be ed from the existing available nodes, and that event can serve as the G1 root for ining the likely identity of the base at position two, such as by once again ng the new haplotypes, e.g., 4, as well as their various branches, e.g., 16, explaining the le variations with respect to position 2. Hence, through repeating this same process, event 2 may now be resolved. Therefore, as can be seen with respect to D, once position 1 has been determined, a new node for on 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 already been resolved, with respect 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 resolved, e.g., including all children nodes and children of en 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 represents the joint diplotype, e.g., which sequence has the highest probability of being true. Therefore, in this manner, because ofthe pruning 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 ntially more than performing non-joint diplotype calling, e.g., single region calling. Accordingly, the present analytics modules are able to determine and resolve two or more regions of high homology with a high degree of cy, e.g., employing joint diplotype analysis, where ional 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 is on regions ofhigh homology. The t iterations me these and other such problems in the field. More ularly, the present devices, systems, and their methods ofuse may be configured so as to consider a greater proportion, e.g., all of the haplotypes, despite 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 function, as herein described.

A benefit of these configurations, e.g., joint-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 hundred of bases being processed to a few thousands, or even tens or hundreds of nds of bases can be processed together, such as in one contiguous active region. This increase in size ofthe active window of is allows for more evidence to be ered when determining the identity of any particular 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 r context allows for supporting evidence to better be chained together when ing one or more reads covering one or more regions having one or more deviations from the reference. Hence, in such a , 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 respect to a given particular event presently under consideration may be made, thereby allowing evidence from farther away, e.g., hundreds to thousands ofbases or more away, to be informative 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 e thousands, to tens of thousands, even hundreds of thousands 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 , once it becomes clear they are no longer viable they may be pruned, and for those that remain viable, ng may be employed so as to improve the accuracy of the eventual variant calls being made. This is all made possible by quantum and/or Hardware computing. 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 ed by the De Bruijn graph. However, 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 assessed. ingly, as can be seen with t to C, from the ted De Bruijn graph, once multi-region joint detection, and/or pruning has occurred, a set of ial haplotypes will result, and then these haplotypes will be tested against the actual reads of the subject. Specifically, each horizontal cross section represents a haplotype, e.g., BI, that may then be ted to another HMM protocol so as to be tested against the reads so as to determine the probability ofa particular read given the ype 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 ofvariants in the unresolved positions. Therefore, this further facilitates the iterative growing of the tree function, where the actual growing of the tree, e.g., the ming 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 calculations 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 ating pruned nodes so as to process only those possibilities that have the greatest probability for successful characterizing the true genotype.

Accordingly, when determining the probability of a ic base actually being present at any one position, the identity of the base at that on may be determined based on the identity at that position on each region of each chromosome, e.g., each haplotype, that represents a viable candidate. Hence, for any ate, what is being determined is the identity of the given base at the on in question in each of the four ypes simultaneously. Particularly, what is being determined is the probability of observing the reads of each of the s given the ined 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 diploid 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 sequences in the pileups assuming that they are in fact the truth. This initial ination may be performed by an HMM haplotype 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 ons to ine, assuming that the Ha1 haplotype is the truth, what is the likelihood of actually ing the given read sequence in question.

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 sequencing or sequencing ery error, and not an actual variation. Hence, the HMM calculation is a function ofthe error models. Specifically, it asks what is the probability ofthe necessary ation 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 aneously (e.g., instead of considering at most possibly two haplotypes at one region, now what is being considered is simultaneously the possibility offour haplotypes for any given position at any given region, simultaneously, using all ofthe reads data from all of the regions in question. These processes, e.g., g the tree, multi-region joint detection, and PD-HMM, will now be described in greater detail.

Specifically, as can be seen with t to FIGS. 17 and 18, a high-level sing chain is provided, such as where the processing chain may include one or more of the following steps: Identifying and inputting homologous regions, performing preprocessing of the input homologous regions, performing a pruned very long read (VLRD) or multi region joint detection (MJRD),S and outputting a variant call file. Particularly with respect to fying homologous s, a mapped, aligned, and/or sorted SAM and/or BAM file, e.g., a CRAM, may be used as the y input to a region joint ion processing engine implementing an MRJD algorithm, as described herein. The MJRD processing engine may be part of an integrated circuit such as a CPU and/or GPU and/or Quantum computing platform, running software, e.g., a quantum algorithm, or implemented within an FPGA, ASIC, or the like. For instance, the above disclosed 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 y alignments. These primary and secondary reads may then be used to identify a list of homologous s, which homologous regions may be ed 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 configured MRJD module.

Accordingly, in the pre-processing stage, for every set ofhomologous regions, a joint-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 candidate variants may be processed and evaluated 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 ypes (P(GilR)) may be calculated. From these eriori probabilities a genotype matrix may be computed. Next, N diplotypes with the lowest a-posteriori probabilities may be pruned so as to reduce the computational complexity of the calculations. Then the next ate t 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, WO 14320 PCT/0S2017/036424 the candidate ts from one or more, e.g., all, regions in the gous 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 y 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 led joint pile-up is loaded, a candidate variant list is then created from the assembled joint pile up, and a connection matrix is ed. Particularly, m various instances, a preprocessing methodology may be performed, such as prior to performing one or more variant call ions, such as a multiple read joint detection ion. Such operations may include one or more preprocessing blocks, including: steps pertaining to the loading of joint pile-ups, generating a list of variant candidates from the joint pileups, and computing a connection matrix. Each of the blocks and potential steps associated therewith will now be discussed in r 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 reference s 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. Further, all the primary reads corresponding to each region may then be extracted from the input SAM or BAM file and be mapped to the universal coordinates. This g may be done, as described , such as by using the alignment information (CIGAR) present 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 ce, , a candidate 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 various candidate variants (SNPs/Indels) that may be identified from the joint pileup. Once the DBG is produced the various bubbles in the graph can be mined so as to derive a list of variant candidates. ularly, given all the reads, a graph may be generated usmg each reference region as a backbone. All of the identified candidate variant positions can then be aligned to universal coordinates. A connection matrix may then be computed, where the matrix s the order ofprocessing of the active positions, which may be a function of the read length and/or insert size. As referenced herein, shows an example of a joint pileup oftwo gous regions in chromosome 1. Although this pileup is with reference to two homologous regions of chromosome 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 some.

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 s. The construction of the DBG is performed in such a manner as to te bubbles, indicating variations, representing alternate pathways through the graph where each alternate path is a ate 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 aligned to universal coordinates. Specifically, illustrates a flow chart setting forth the process of generating a DBG and using the same to e candidate haplotypes. More specifically, the De Bruijn graph may be employed in order to create the candidate t 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 ne and all ofthe reads corresponding 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 events. However, when employing an MRJD pre-processing ol, as described herein, N regions may be jointly sed, 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 y. Accordingly, in order to reduce the computational xity, instead of extracting entire haplotypes, only the bubbles need be extracted from the graphs that are representative ofthe candidate variants.

An example 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 s candidate haplotypes. Specifically, for each bubble a SW alignment may be performed on the ate 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 instances, 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 , such as to a universal coordinate , 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 extracting 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. ically, once the representative bubbles have been extracted, the global ent, e.g., Smith-Waterman alignment, of the bubble path and the corresponding reference backbone may be performed to get the candidate variant(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 duplicate candidates, if any, may be removed, and the unique candidate variant positions may be mapped to the sal nate system ed from the joint pile-up. This results in a final list of candidate t positions for the N regions that may act as an input to a "Pruned" MRJD algorithm.

In particular preprocessing blocks, as described 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 WO 14320 PCT/0S2017/036424 computed so as to define the order of processing 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 process first. This connection , therefore, may be a function of the mean read length and the insert size ofthe -end reads. Accordingly, 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 candidate variants at other positions. This is because these candidate variants are more likely to provide ce for the current variant 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 on, exemplary steps of a pruned MRJD algorithm, as referenced above, is set forth in . For instance, the input to the MRJD platform and thm is the joint pileup ofN regions, e.g., all the ate variants (SNPs/ INDELs), the a-priori probabilities based on a mutation model, and the connection matrix.

Accordingly, the input into the pruned MRJD processing rm may be the joint pile-up, the identified active positions, the generated tion matrix, and the a-posteriori ility model, and/or the s 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, ate joint diplotypes may be generated. For instance, a joint diplotype is a set of 2N haplotypes, where N is the number of regions being jointly processed. The number of candidate joint diplotypes M is a function of the number of regions being jointly processed, number of active/candidate variants 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 y processed; M = 22-N.P = 24 = 16 candidate diplotypes Hence, for a single candidate active position, given all the reads and both the reference regions, let the two haplotypes 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' , '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 ate joint diplotype set. This may be done using a HMM algorithm, as described 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 ype (P(rilGm)) using the results from the modified HMM. This may be done using the formula below.

For the case of2-regionjoint detection: Gm= [Sll,m, B12,m, B21,m, B22,m], wherein Sij,m, i is the region and j is the phase m) = P(rilB11,m)+ P(rilB12,m)+ P(rilB21,m)+ P(rilB22,m) P(RIGm) = IL P(rilGm). Given P(rilGm), it is htforward 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 a-priori probabilities (P(Gi)).

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

Further, an intermediate genotype matrix may be calculated for each region given the eriori probabilities for all the candidate joint diplotypes. For each event ation in the genotype 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 mediate" because not all the ate variants supporting the current candidate have been ed. However, as seen earlier, 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 aposteriori 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. Finally, the final genotype matrix may be updated based on a efined 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 current candidates being processed using the tion matrix.

Once all of the candidates have been included, the processing of the current candidate is done. Other stopping criteria for processing ate variants are also possible. For example, the process may be stopped when the confidence has stopped increasing as more candidates variants are added. This analysis, as ified in , may be restarted and repeated in the same manner for all other candidate variants in the list thereby resulting in a final t call file at the output of MRJD. Accordingly, instead of considering 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 disclosed 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 ated 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 processed. And let H1c be a candidate haplotype, k = 1...K, each of which may include various SNPs, ions and/or deletions relative to a reference sequence. Each ype 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 t 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 haplotype 1c), 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 ...rNR}: ) = Tif=Rl P(rilGm).

Next, the a-posteriori probability is calculated of each ate 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 on, 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 reported as a quality score on a phred scale: QUAL(Vj) = -10log10 P(VilR) .

P(ref I R) An exemplary process for performing various variant g operations is set forth herein with respect to where a conventional and MRJD detection process 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 bed 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 reference. These regions are referred to as the active positions. All other ons can be ignored, as they don'taffect the calculation.

Accordingly, as can be seen with respect to , in a tional or, 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 reference 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 t calling in region #1. As can be seen, all of these reads match the reference for region #1, so no variants will be called. However, read pairs 24-32 map y 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 ed employing conventional methods. The relevant calculations are set forth below. In this instance N = 2 regions. 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 position). If all ations 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 brute-force calculation where all ations of all candidate haplotypes are considered, the number of candidate solutions is M = K2N = g2·2 = 4096, and P(Gm/R) may be calculated for each candidate 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 ts (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 assumed that only base call errors are present and all base call errors are y 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. Accordingly, it may be assumed that Pe = 0.01, which corresponds to a base y 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 P(rilGm2), with the product at the bottom. shows that 1) = 3S 30 15 and 2) = 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 te this example, a simple independent identically distributed (IID) model may be assumed, such that the a-priori probability of a ate solution 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, WO 14320 PCT/0S2017/036424 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 y score ofphred 50.4 can be seen.

Accordingly, as can be seen, there is an immense computational complexity for performing a brute force variant calling operation, which complexity can be d by ming multiple region joint detection, as described herein. 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 solutions for which to calculate ilities is M = K2N. In a brute force implementation, the number of candidate haplotypes is K = 2Np' where Np is the number of active ons (e.g., as exemplified above, if graph-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 expensive 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 uncommon to have values ofNp much higher than this.

Consequently, because a brute force Bayesian calculation can be prohibitively complex, the following description sets forth further methods for reducing the complexity of such calculations. For ce, in a first step of another embodiment, starting with a small number of positions Ni (or even a single position 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 sed 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, le candidates at the new position(s), such as in a growing the tree function. These steps of (1) performing the Bayesian calculation, (2) pruning the tree, and (3) g the tree, may then be ed, 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 on was or was not found). This process is illustrated in the flow chart set forth in .

WO 14320 PCT/0S2017/036424 It is to be tood that there are a variety of possible variations to this approach. For instance, as indicated, the pruning threshold may be adaptive, such as based on the number of surviving candidates. For instance, a simple entation may set the threshold to keep the number of candidates below a fixed number, while a more sophisticated implementation may set the threshold based on a cost-benefit analysis uding additional candidates. Further, a simple ng criteria may be that a result has been found with a sufficient level of ence, or that the confidence on the initial position has stopped increasing as more positions are added. Further still, a more sophisticated 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 reads).

A useful feature of this algorithm is that the probability that the true solution wasn't found can be quantified. For instance, a useful estimate is ed by simply g the probabilities of all pruned branches at each step: Ppruned = Ppruned + Lmi;pruned set P(G/n IR). Such an estimate is useful for calculating the confidence of the resulting variant 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 teristic (ROC) curves. This is a key advantage of this pruning method over other ad hoe xity 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 pruning threshold ofphred 60 on each iteration: Let {cfn, m=1...Mi} represent the candidate solutions on the j-th iteration. shows the candidate ons on the first iteration, representing all ations 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 old (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.

WO 14320 PCT/0S2017/036424 Next, as can be seen with t 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 candidates is shown in , again shown in order of decreasing probability. Again, the probabilities can be calculated and ed to the pruning threshold, and in this instance 5 candidates survive. y, all combinations of the surviving candidates from iteration #2 and the candidate bases in position #3 (A and T) can be determined. The final ates and their associated ilities are shown in . Accordingly, when calculating the probabilities of individual variants, it is determined that a heterozygous A-+C SNP at position #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 result, 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 variations in deciding which regions to include. For example, prior to running a Region Joint Detection, the variant caller may be configured to determine whether 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 offline, such as based on a search ofthe reference genome.

] 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 influence the probability of bases ted by far more than the total read length (e.g., the paired read length or long le length). As such, it must be d which positions to include in the MRJD calculation, and the number tions is not unconstrained (even with pruning). For example, some implementations may process overlapping 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 determining 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 ons based on the distance to the already-included positions, or the degree of connectivity with these positions (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 ility threshold, but in general the pruning threshold may be adaptive or based on the number of surviving candidates. For ce, a simple implementation may set the threshold to keep the number of candidates below a fixed number, while a more ticated implementation may set the threshold based on a costbenefit analysis ofincluding additional 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 mirror-image representations across regions (in addition to ).

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 cial. 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 instance, if only a subset of the base positions (e.g. when processing overlapping blocks) is being solved for, the s may stop when the result for the subset has been found with a sufficient level of confidence, or when the ence has stopped increasing as more positions are added. A more sophisticated implementation, however, may perform some type of enefit analysis, weighing the computational cost against the potential value of adding more positions. ri ilities may also be useful. For instance, in the es 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 positions where the references differ. Therefore, 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 ity, and this knowledge is still ng. As such, some implementations may update the a-priori models as better information becomes available. This may be done automatically as more results are produced. Such updates may be based on other ical s or other regions of the genome for the same sample, which learnings can be applied to the methods herein to further e a more rapid and accurate analysis.

Accordingly, in some instance, an iterative MJRD process may be implemented. Specifically, 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 system. For instance, the output of the calculation at one location can be used as an input a-priori probability for the calculation at a nearby location. Additionally, some implementations may use a combination ofpruning and iterating to achieve the desired performance/complexity tradeoff.

Further, sample preparation may be implemented 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 variation in the insertion size could significantly improve the performance for MRJD. For example, the sample may be prepared to intentionally introduce a bimodal distribution, a multi-modal distribution, or bell-curve-like distribution with a higher ce than would typically be implemented for conventional detection. illustrates the ROC curves for MRJD and a conventional detector for human sample 8 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 approx. 400. As can be seen with respect to , MRJD offers dramatically improved sensitivity and specificity over these s than conventional detection s. 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 rity 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 . onally, in various ces, this methodology may be extended to allow message passing n related regions to further reduce the complexity and/or increase the detection performance. For instance, the output ofthe ation 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 ular instances, as indicated above, prior to running region joint detection, the variant caller may determine whether a given active region should be processed dually or y with other s. Additionally, as indicated above, some implementations may rely on a list of secondary alignments provided by the mapper to make such a decision. Other implementations may use a database of homologous regions, 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 separately te 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 homologous regions in diploid chromosomes, each joint diplotype will include four haplotypes. In such instances, all or a portion of the possible haplotypes may be considered, such as by being constructed, for instance, by modifying 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 (combinations of variants) becomes exponentially large, and the number ofjoint diplotypes nations ofhaplotypes) may be astronomical. uently, 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 lly-determined joint diplotypes. In such an instance, each node of the tree may be a partially ined joint diplotype that includes a partially determined haplotype per ploidy per homologous reference region. In this instance, a partially determined haplotype may include a reference region ed by a partially ined subset of the possible variants. Accordingly, a partially determined subset of the possible variants may include an indication, for each possible variant, of one of three states: that the variant is determined and present, or the t is determined and absent, or the variant is not yet determined, 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 further from the root have sively more variants determined as present or absent in each haplotype ofeach node'sjoint diplotype.

Further, in the t 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 ype 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 ined; e.g., several or many variants are still not determined as present or absent from the ypes 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 probability P(RIH) of observing read R assuming the true haplotype H* is consistent with partially determined haplotype H. Consistent in this context means that some specific true haplotype H* agrees with partially determined haplotype H with respect to all ts whose presence or absence 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 lly 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 ype 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 consider PCR indel errors, it is useful to use a pHMM-like calculation ng through all determined ts 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 tide sequence ofread R, the base quality scores (e.g., phred scale) ofthe called nucleotides ofR, a baseline haplotype HO, and a list ofundetermined variants (edits) from HO. The undetermined ts may include single-base substitutions (SNPs), 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 ion may be represented by first editing the insertion into the baseline haplotype, then indicating the corresponding undetermined deletion which would undo that insertion.

Restrictions may be placed on the undetermined deletions, to facilitate hardware engine implementation with limited state memory and logic, such as that no two undetermined deletions may overlap (delete the same baseline haplotype bases). If a partially determined haplotype must be tested with undetermined variants violating such restrictions, this may be resolved by converting one or more rmined variants into determined variants in a larger number of PD-HMM operations, covering 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 m ) among all haplotypes H* that can be formed by editing HO with any subset of the undetermined variants. The maximization may be done locally, buting to the pHMM- like dynamic programming in a given cell as if an nt undetermined variant is present or absent from the haplotype, whichever scores better, e.g., contributes the greater l probability. Such local maximization during dynamic programming may result in larger estimates of the m P(RIH*) than true maximization over individual pure H* haplotypes, but the difference is generally inconsequential.

Undetermined SNPs may be incorporated into PD-HMM by allowing one or more matching nucleotide values to be specified for each haplotype on. 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 transition to an 'M'state results in multiplying the path probability by the probability ofa t base call (ifthe haplotype position matches the read position) or by the probability of a specific base call error (if the haplotype position ches the read position); for PD-HMM this is modified by using the correct-call probability if the read position matches either possible ype base (e.g. 'C'or 'T'),and the base-call-error probability otherwise.

] Undetermined haplotype deletions may be orated into PD-HMM by flagging optionally-deleted haplotype positions, and modifying the dynamic programming of pHMM to allow alignment paths to skip horizontally across undetermined deletion ype ts 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 without being reduced by ordinary gap-open or gap-extend probabilities.

In one particular embodiment, ype 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" al probability representations) for each cell of the HMM matrix (haplotype horizontal/ read vertical), each PD-HMM cell may further include 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 extending into the or 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 ofadjacent 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 ified 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 represented in FI through F2 columns, and zing M/BM, I/BI, and D/BD multiplexers may be shown in an F2 , these components may be present 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 on. It is further to be noted that the PD-HMM matrix of cells may be depicted as a schematic representation of the l M, I, D, BM, BI, and BD state calculations, but in a hardware implementation, a smaller number of cell calculating logic ts 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 logical dependencies of the PDHMM calculation.

Thus, in this embodiment, the pHMM state values in one column may be immediately left of an undetermined deletion which may be captured and itted rightward, unchanged, to the rightmost column of this undetermined deletion, where they substitute into pHMM calculations whenever they beat normal-path scores. Where these maxima are chosen, the "bypass" state values BM, BI, and BD represent the local dynamic programming results where the undetermined deletion 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 another embodiment, a single bypass state may be used, such as a BM state receiving from an M state in FI flagged columns, or receiving a sum of M, D, and/or I . In another embodiment, rather than using "bypass" states, gap-open and/or gap-extend penalties are eliminated within columns of undetermined deletions. In r embodiment, bypass states contribute additively to dynamic programming rightward of undetermined deletions, rather than local maximization being used. In a r embodiment, more or fewer or differently defined or differently located haplotype 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 haplotype are supported, rather than, or in addition to, rmined ons. 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 substitutions are supported as atomic ts (all present or all absent). In a further embodiment, undetermined length-varying substitutions are supported as atomic variants. In another embodiment, rmined variants are penalized with fixed or configurable ility 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 re engine architecture for "ordinary" pHMM ation or may be implemented by one or more quantum circuits in a quantum computing platform. In addition to an engine pipeline logic to calculate, it, and store M, I, and D state values for various or successive cells, parallel ne 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 values. Flags such as FI and F2 may be stored in memories along with associated haplotype bases. le 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 tide value. Cell calculation dependencies in the pHMM matrix are unchanged in PD-HMM, so order and pipelining of multiple cell calculations can remain the same for PD-HMM.

However, the latency in time and/or clock cycles for complete cell calculation increases at 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 ation, 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 t dependency .

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 ation, and may require about twice the hardware resources for an equivalent throughput engine embodiment. r, a PD-HMM engine has exponential speed and efficiency advantages 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 partially determined haplotype has 30 undetermined variants, each of which may be independently t or , 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 reference. For instance, even though the occurrence of mutations may be random, there are instances n the hood of their occurrence appears to be potentially predictable to some . 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 ons 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 on 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 performing genomic variation analyses, better and more accurate genomic analysis results may be obtained, such as with more te demarcations of c mutation.

Such mutation models may give an account for the frequency and/or location of s known mutations and/or ons that appear to happen in conjunction with one another or otherwise non-randomly. For instance, it has been determined that toward the ends of a given some variations occur more predominantly. Thus, known models of mutations can be generated, stored in a se 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 accordance with the machine learning principles disclosed herein. Specifically, machine learning may be implemented on the tive data sets, especially with respect to the ions 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 configured 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 accurately determine the presence 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 ness 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 nce genome out of a multiplicity of reference genomes may be a better nce genome by which a particular sample is to be analyzed. Further, in various instances, the mutation model and/or try may be employed so as to better select the reference genome to be employed in the system processes, and thereby enhance the accuracy 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 , 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 nce genome to apply.

It is to be noted that when ming 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 system, such as in a process of biological contextually sensitive mutation model. More particularly, since DNA codes for genes, which genes code for proteins, 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 es on the subject's genome.

For example, tertiary analysis, such as on a sample set of genes coding for mutated proteins, may be informative 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 on models used herein for achieving better accuracy and efficiency when performing the various secondary analysis operations disclosed . Specifically, ation about mutated proteins, e.g., contextual tertiary analysis, can be used to update the mutation model when ming secondary analysis of those regions known to code for the proteins and/or to potentially include such mutations Accordingly, in view of the above, for embodiments involving FPGA- accelerated mapping, alignment, sorting, and/or variant calling applications, one or more of these functions may be implemented in one or both of software and hardware (HW) sing components, such as software running on a ional 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 sed in a next step on the other device.

For instance, where a mapping function is run, the ng 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 WO 14320 PCT/0S2017/036424 CPU may build the data structure, store it in an associated , such as a DRAM, which memory may then be accessed by the processing engines running on the FPGA.

For instance, in some embodiments, communications between the CPU and the FPGA may be ented by any suitable interconnect 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 between 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 (s) accessed are non-cacheable, because there is no ty to maintain cache coherency 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 n the devices so they don't slow each other down waiting for high latency operations. This slows down the various processing operations disclosed herein. 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 CPU/FPGA integrations, it is generally necessary to have "centralized" software control over the FPGA interface. In such instances, the s software threads may be processing various data units, but when these s generate work for the FPGA engine to perform, the work must be aggregated in "central" buffers, such as either by a single ator software thread, or by multiple threads locking ation access via semaphores, with transmission of aggregated work via DMA packets managed by a l 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 s waiting software worker threads. However, this centralized software control of communication with HW FPGA logic is cumbersome and expensive in resource usage, reduces the efficiency 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 ation 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. Likewise, the CPU 1000 may be icably coupled to its own DRAM 1014, such as by a ly configured bus 1006. As indicated above, the FPGA 7 may be configured to include one or more processing engines 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 memories and/or processing engines.

Many of the operations disclosed , to be performed by the FPGA 7 for genomic processing, require large memory es for the performance of the underlying operations. Specifically, due to the large data units involved, e.g. 3+ billion nucleotide reference genomes, 100+ billion 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 mapping the seeds from a ced DNA/RNA query to a 3Gbp reference genome, and/or for fetching candidate segments, e.g., from the reference genome, to align against.

Accordingly, in various entations ofthe system herein sed, 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 g ion. 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 instances, 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 ations. In such instances, in on 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 package (which may even need to be larger so as to have enough pins for several external memory busses) and several 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 utilize an expansion card 2, containing the FPGA 7, its memory 14, and other ting 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 ated 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 d 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 INTELS® Quick Path Interconnect (QPI) or HyperTransport (HT).

Accordingly, as seen with t to B, a system 1 is provided n the system includes both a CPU 1000 and a processor, such as an FPGA 7, wherein both devices are associated with one or more memory modules. For ce, as depicted, the CPU 1000 may be coupled, such as via a suitably configured bus 1006, to a DRAM 1014, and likewise, the FPGA 7 is communicably 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 interconnect, e.g., PCie interface, the CPU 1000 is coupled to the FPGA 7 by a low latency, hyper transport 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 uration, one or more cashes 14a associated with the devices may be ured so as to be coherent with t to one another.

Some key properties of such a tightly coupled CPU/FPGA interconnect include a high bandwidth, e.g., 12.8GB/s; low latency, e.g., 0ns; an adapted protocol designed for allowing efficient remote memory es, 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 le 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 ity of interconnects 1003, such as native CPU-CPU interconnects 1003a and 1003b. However, in this instance, as ed in A, a CPU 1000 is configured so as to be coupled to the interconnect 1003a, but rather than another CPU being coupled ith 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 associated with the tive devices 1000, 7 may be made so as to accessible to each other, such as in a high-bandwidth, cache coherent manner.

Likewise, as can be seen with respect to B, the system can also be configured so as to e es 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 d one with the other such as via a tight coupling interconnect 3. Further, as can be seen with respect to , in various instances, a package 1002a may be provided, wherein the package 1002a includes a CPU 1000 that has been fabricated 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 r CPU, and y operate in a cache-coherent shared-memory environment with one or more CPUs, just as multiple CPUs would on a multi-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 ed. Thus, in such a configuration, rapid, short, random es are supported efficiently by the interconnect 3, such as with low latency. This makes it practical 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 g and/or aligning and/or variant calling function, as described herein, in a shared . Particularly, in one step (1) a data structure may be generated or ise 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 reference genome, such as for the performance ofmapping and/or aligning and/or t 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 genome(s), and/or an index f.

Particularly, in a step (3), e.g., in an exemplary mapping ion, the FPGA 7 may access the data structure, e.g., the sequenced reads and/or reference sequences, so as to produce 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 ces may be employed for the purposes of performing a hash on 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 re-configured, as an alignment engine, which may then access the stored mapped data structure so as to perform an ng function n, 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 production of a De Brujin Graph ("DBG"), which DBG may then be stored in its associated memory. se, 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 particular 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 te caches to be coherent, such as with respect to the transitionary data, e.g., results data, stored thereon, such as results from the mance of one or more functions herein. In a manner such as this, data may be shared substantially seamlessly between the tightly coupled devices, thereby allowing a ne 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 external memory 14 attached, and hence, due to such a tight ng configuration, the stored reads, the reference genome, 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 ncy configurations, as well as other component configurations discussed herein, allow smaller, lower-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 process, and the same or another thread 20b, may be configured to then process the large queue of results data generated thereby, 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 re 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 coherent . ularly, given the relationship between the structures provided herein, the granularity of the re/hardware cooperation can be much finer, with much smaller, lower level ions being allocated so as to be performed by various hardware engines 13, such as function calls from s allocated software threads 20. For e, in a loose CPU/FPGA interconnect platform, for efficient acceleration of DNA/RNA read mapping, alignment, and/or variant calling, a full mapping/aligning/variant calling pipeline may be constructed as one or more re and/or FPGA engines, with unmapped and unaligned reads being streamed from software to hardware, 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 configurations herein described, this can be very fast.

However, in various instances, such a system may suffer from limitations of flexibility, complexity, and/or mmability, 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 mmable than software, and may therefore be limited to less algorithmic complexity.

By contrast, using a tight CPU/FPGA interconnect, such as a QPI or other interconnect in the configurations disclosed herein, several resource ive discrete operations, such as seed generation and/or mapping, rescue scannmg, s alignment, gapped, e.g., Smith-Waterman, alignment, etc., can be ented as ct tely accessible hardware engines 13, e.g., see , and the l g/alignment and/or variant call algorithms can be implemented 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 rd hardware implemented operations.

Furthermore, in such a framework of software execution accelerated by discrete vel 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 lized 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 ent, De Bruijn or assembly graph construction, and other such ions, can be used in various higher level parent algorithms. For example, as described 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 analysis 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, ions.

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 hardware engines 13 described herein. In widespread practices of multi-threaded, multi-core, and multi-CPU software design, many software threads and processes communicate and cooperate seamlessly, without any central software modules, drivers, or threads to manage intercommunication. In such a format, this is practical because of the cache-coherent shared memory, which is visible to all threads in all cores in all of the CPUs; while physically, coherent memory g 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 onnect uration that incorporates a multiplicity ofthreads, e.g., 20a, b, c, and a multiplicity of processes running on one or the le 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 re 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 d 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 ith. 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 er 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 ment, data to be passed from one or more software threads 20 from one or more CPU cores 1000 to a hardware 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. onally, requests to process data in the shared memory 1014, or notification of results updated n, can be signaled between the re and/or hardware, such as over a suitably ured bus, e.g., DDR4 bus, such as in queues that may be implemented within the shared memory itself. rd 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 onnect, it becomes much more practical to package the FPGA 7 more compactly and natively within traditional CPU 1000 boards, 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 multi-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 e 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, ground, and CPU/FPGA interconnect, made within the e 2a. Inter-die connections may be made by die-to-die wire-bonding, or by connection to a common substrate 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 -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 CPU/FPGA onnect 3, e.g., by properly dedicated protocols, and to e, convert, and/or route memory access requests from internal FPGA engines 13 to the CPU/FPGA interconnect 3, via appropriate protocols, to the shared memory 1014a.

Additionally, some or all of this logic may be hardened 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 te 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 ption according to changing the CPU and/or the FPGA computation demands.

All ofthese packaging options share l advantages. The tightly-integrated CPU/FPGA platform becomes ible with standard 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 ocket board1002 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 chassis (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 .

Accordingly, in various instances, therefore, there may be no need for an expansion card to be installed so as to integrate the CPU and FPGA acceleration, because the FPGA 7 may be ated in to the CPU socket 1003. This implementation avoids the extra space and power ements of an expansion card, and avoids various additional failure points expansion cards sometimes have with respect to relatively low-reliability components. rmore, standard CPU cooling solutions (head sinks, heat pipes, and/or fans), which are efficient yet st since they are manufactured in high volumes, can be applied to FPGAs or CPU/FPGA packages in CPU sockets, whereas cooling for expansion cards can be expensive 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 c data processing applications, all these packaging options may facilitate easy installation of a y-integrated CPU+FPGA compute platform, such as within a DNA sequencer. For ce, typical modem "next-generation" DNA sequencers contain the sequencing apparatus (sample and reagent storage, fluidics tubing and control, 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 tightly-integrated CPU+FPGA platform, as herein described, may be achieved 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 alternatively by ling a new motherboard with both CPU(s) and FPGA(s), e.g., tightly coupled, as herein disclosed. Further, 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 servers with very high ility/availability.

Hence, in accordance with the teachings herein, there are many processing stages for data from DNA (or RNA) sequencing to g and aligning to g and/or de-duplicating to variant calling, which can vary ing 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 polyclonal clusters, detecting and trimming rs, key sequences, barcodes, and low y 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, cleanup, repair, coloring, annotation, comparison, transformation, splitting, splicing, analysis, subgraph selection, traversal, iteration, recursion, searching, ing, import, export, including mapping reads to a reference genome, aligning reads to candidate g locations in the reference , local assembly of reads mapped to a nce region, sorting reads by aligned position, marking and/or removing duplicate reads, including PCR or l duplicates, gnment of multiple overlapping reads for indel consistency, base y score recalibration, variant calling (single sample or joint), structural variant analysis, copy number variant analysis, somatic variant calling (e.g., tumor sample only, matched tumor/normal, or tumor/unmatched normal, etc.), RNA splice junction detection, RNA alternative splicing analysis, RNA transcript assembly, RNA transcript sion analysis, RNA differential expression analysis, RNA t calling, DNA/RNA difference analysis, DNA methylation analysis and calling, variant quality score recalibration, variant filtering, variant annotation from known variant databases, sample contamination detection and estimation, phenotype prediction, disease testing, treatment response prediction, custom treatment design, ancestry and mutation history analysis, population DNA analysis, genetic marker identification, ng genomic data into rd formats and/or compression files (e.g. PASTA, FASTQ, SAM, BAM, VCF, BCF), decoding genomic data from rd formats, querying, selecting or filtering genomic data subsets, general compression and decompression for genomic files (gzip, BAM compression), specialized compression and decompression for genomic data , genomic data encryption and decryption, statistics calculation, comparison, and presentation from genomic data, genomic result data companson, accuracy analysis and reporting, genomic file storage, al, retrieval, , 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 expensive when implemented on traditional e platforms. The sluggishness of such exclusively software implemented operations may be due in part to the xity 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 problems, in part due to the configuration of the various hardware smg engmes, acceleration by the various hardware implementations, and/or in part due to the CPU/FPGA tight coupling configurations. ingly, 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 ology, e.g. RTL), such as where the CPU software mostly serves the on of compiling large data packets for preprocessing via worker threads 20, such as aggregating the data into various jobs to be processed by one or more hardware implemented processing engines, and feeding the various data inputs, 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 t to , in s embodiments, a worker thread generates various packets ofjob data that may be compiled and/or streamed into larger job packets that may be queued up and/or further aggregated in preparation for er, e.g., via a DDR3 to the FPGA 7, such as over a high bandwidth, low latency, point to point interconnect protocol, e.g., QPI 3. In particular instances, the data may be ed in accordance with the particular data sets being erred to the FPGA. Once the ed 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 sing engines for processing y in accordance with one or more of the ne ions herein described.

Once processed, s data may then be sent back to the cluster and queued up for being sent back over the tight ng point to point interconnect to the CPU for post processing. In certain embodiments, the data may be sent to a regator thread prior to post sing. 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 disclosed above. Particularly, these functions are distinguishable by the facts that their algorithmic complexity (although having a very high net computational burden) are pretty limited, and they each may be configured so as to have a fairly uniform compute cost across their various sub-operations.

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 compute-intensive sub-functions in any given operation, and then implement that sub-function in custom FPGA logic (hardware acceleration), such as for the intensive sub-function(s), while implementing the balance of the operation, and ideally much or most of the algorithmic complexity, in software to run on CPUs/GPUs/QPUs, as described herein, such as with respect to .

Generally, it is l of many c data processing operations that a small percentage of the algorithmic complexity accounts for a large percentage of the l 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 ing 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 implement 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 , 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 compute load, in which case applying FPGA ration, 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 rated processing is desired, one or more ofthese functions may be med by a quantum processing unit. ] r, such a "piecemeal" 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 U+FPGA platform.

Particularly, in a loosely integrated platform, the n, 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 through certain low-bandwidth/high-latency cut-points, which divisions may not otherwise be desirable when optimizing the partitioning ofthe algorithmic complexity and computational loads. This may often result either in enlarging the boundaries of the hardware portion, encompassing an undesirably large portion of the algorithmic complexity in the red format, or in shrinking the boundaries of the hardware portion, undesirably excluding portions with dense compute load.

By contrast, on a tightly integrated CPU/GPU+FPGA platform, due to the cache-coherent shared memory and the high-bandwidth/low-latency CPU/GPU/FPGA onnect, the low-complexity/high-compute-load portions of a genomic data processing ion can be ed very precisely for implementation in custom FPGA logic (e.g., via the hardware engine(s) described 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. ularly, 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 ent small accesses over the tency interconnect 3' serviced by the U cache, y 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 in-place in CPU/GPU memory 1014, without streaming an entire copy of the data unit by DMA to CPU/GPU memory. Even if the desired software/hardware ry is not appropriate for a software thread 20 to make a atency, non-blocking queued handoff to the re 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 y being dramatically reduced 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, because the specific algorithms and requirements of signal/image processing and base calling vary from one sequencer technology to another, and because the ty of raw data from the sequencer's sensor is typically gargantuan (this being d to enormous after /image sing, 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 connected by a high dth transmission channel to the sequencer. However, DNA sequencers have been achieving increasingly high throughputs, at a rate of increase ing 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 increasingly inadequate to the task. Nevertheless, since a tightly integrated CPU + FPGA and/or a GPU + FPGA and/or a GPU/CPU + FPGA rm can be configured to be compact and easily instantiated within such a sequencer, e.g., as CPU and/or GPU and/or FPGA chip positioned on the sequencer's motherboard, or easily installed in a server adjacent to the sequencer, or a cloud-based server system accessible remotely from the cer, such a sequencer may be an ideal platform to offer the massive compute acceleration d 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 thereof 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; GPU/QPU; CPU and/or GPU and/or QPU + FPGA rm.

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 WO 14320 PCT/0S2017/036424 can also be configured so as to be performed by one or more quantum processing circuits such as to be implemented in a m processing platform.

Particularly, 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 exceeding the capabilities of simple general CPUs. Likewise, simple GPUs, have more able parallel arithmetic resources. However, GPUs often have awkward architectural limitations and programming restrictions that may prevent them from being fully utilized. Accordingly, these FPGA and/or m processing and/or GPU arithmetic resources can be wired up or otherwise configured by design to operate in exactly the designed manner with near 100% efficiency, such as for ming the calculations necessary to e the functions herein.

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

More particularly, in various instances, with t to Graphics Processing Units (GPUs), a GPU can be configured so as to ent one or more of the functions, as herein described, so as to accelerate the processing speed of the underlying calculations 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 g, aligning, sorting, licating, 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 n, so as to work in conjunction with a server's CPU and/or FPGA to accelerate the application and processing performance and shorten the computational cycles required for performing such functions. Cloud s, 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 compute-intensive tasks can also be offloaded to the cloud, such as to be performed by a quantum processing unit.

Accordingly, if a tightly integrated CPU+FPGA or GPU+FPGA and/or CPU/GPU/FPGA with shared memory platform is employed within a sequencer, or ed or cloud based server, such as for signal/image processing, base calling, mapping, aligning, sorting, licating, 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 mming function for base calling, mapping, aligning, sorting, de-duplicating, and/or variant calling may be implemented in one or more FPGA s, where as other work may be done in the CPU and/or GPU expansion cards.

However, the tight U/FPGA integration and shared memory model, herein presented, may be further configured, 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 implemented as processing engines, and various of their functions may be offloaded for execution 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 sing 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 nce genome, as well as sorting, de-duplicating, and/or variant calling may be benefited from such GPU and/or FPGA and/or QPU acceleration. Specifically, mapping and alignment and/or variant 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 t 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 mance of sorting and/or variant calling. This type of FPGA acceleration works on a loosely-integrated CPU/GPU+FPGA platform, and in the configurations described herein may be extremely fast. Nevertheless, there are some additional advantages that may be gained by moving to a tightly-integrated CPU/GPU/QPU + FPGA platform.

Accordingly, with respect to mappmg and aligning and t calling, in some embodiments, a shared advantage of a tightly-integrated U+FPGA and/or quantum processing platform, as described herein, is that the map/align/variant calling acceleration, e.g., re acceleration, can be efficiently split into several discrete compute-intensive ions, such as seed generation and/or mapping, seed chain formation, paired end rescue scans, s alignment, and gapped alignment -Waterman or Needleman-Wunsch), De Bruijn graph formation, performing a HMM computation, and the like, such as where the CPU and/or GPU and/or quantum ing software performs lighter (but not necessarily less x) tasks, and may make acceleration calls to te 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 latencies, but may be more ent in a tightly-integrated GA, GPU+ FPGA, and/or quantum computing platform with cache-coherent shared , high-bandwidth/low-latency interconnect, and distributed software/hardware coordination model. Additionally, such as with respect to variant calling, both Hidden Markov model (HMM) and/or dynamic programming (DP) algorithms, ing 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 sequence explaining the observed sensor measurements, in a configuration so as to be well suited to the parallel cellular layout ofFPGAs and quantum circuits described herein.

Specifically, an efficient utilization ofhardware and/or software resources in a distributed sing configuration can result from reducing hardware and/or quantum computing acceleration to discrete compute-intensive functions. In such instances, 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 complex, and therefore may consume large quantities of physical FPGA resources p-tables, flipflops , block-RAMs, etc.). In such instances, moving a n or all of various discrete 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 compute 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 m computing platform.

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

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

Additionally, in various instances, ility within the map/align/variant calling algorithms and features thereof may be improved by reducing hardware and/or quantum ration 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, although hardware algorithms can be modified and reconfigured in FPGAs, generally such s to the hardware designs, e.g., via re, may require several times as much design effort as similar changes to software code. In such ces, the computeintensive portions of mapping and alignment and sorting and de-duplicating, and/or variant calling, such as seed g, 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 optimized 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 quantum circuits ofa quantum processing machine. ingly, 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, ype-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 candidate 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 ofobserving the read given the truth ofthe haplotype.

Such an operation 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 sequencing 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 ation 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 der of the parent haplotype-based variant calling algorithm, either in a looselyintegrated or tightly-integrated CPU+FPGA, or GPU+FPGA or CPU and/or GA and/or QPU rm. For ce, in a loose integration, software threads may construct and prepare 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 ate haplotypes from the graph, and queue up haplotype-read pairs for DMA transfer to FPGA hardware engines, such as for pair-HMM or PD-HMM comparison. The same or other software threads can then receive the pair-HMM s queued and DMA-transferred back from the FPGA into the CPU/GPU , and perform genotyping and Bayesian probability ations 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 instance, as can be seen with respect to , the U 1000 may e one or more, e.g., a plurality, 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 WO 14320 PCT/0S2017/036424 performance of one or more of the operations herein described, such as for mapping, aligning, sorting, and/or variant calling. Because of the high bandwidth, tight coupling onnect 3, data ning to the data structures and/or related to the processing results may be shared substantially seamlessly n 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 ured 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 urations 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. Particularly, in various embodiments, when processing data through a genomics ne, 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 operations may involve both software and hardware processing components.

] Consequently, data may need to be shared and/or otherwise communicated, between the software ent running on the CPU and/or GPU and/or the QPU and the hardware component ed in the chip, e.g., an FPGA or ASIC. Accordingly, one or more of the various steps in the processing 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 icably 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 sharing of information for the performance of such tasks, the CPU and/or GPU and/or QPU may be loosely or tightly 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 is platform is provided.

For instance, the platform may include a motherboard, a memory, and plurality of integrated circuits, such as forming one or more of a CPU/GPU/QPU, a mapping module, an alignment module, a sorting , and/or a t call module. Specifically, in particular embodiments, the platform may include a first integrated circuit, such as an integrated circuit g a central sing 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 genomics analysis functions, as bed herein, such as where the CPU/GPU includes a first set of physical electronic interconnects to t with the motherboard. In other embodiments, a m processing unit is provided, wherein the QPU includes one or more quantum circuits that are configured for performing one or more of the functions sed herein. In various instances, a memory is provided where the memory may also be attached to the motherboard and may further be electronically 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 sequences, and/or an index, e.g., such as a hash table, of the one or more genetic reference Additionally, the platform may e 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 structured ASIC) 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, sorting, de-duplicating, variant calling, e.g., an HMM function, etc. Particularly, the hardwired digital logic ts of the FPGA may be arranged as a set of processing s to perform one or more pre-configured steps in a sequence analysis pipeline of the genomics analysis rm, such as where the set(s) of processing engines include one or more of a mapping and/or aligning and/or sorting and/or de-duplicating and/or variant calling module, which s may be formed ofthe separate or the same subsets ofprocessing engines.

For instance, with respect to variant calling, a pair-HMM or PD-HMM calculation is one of the most compute-intensive steps of a haplotype-based variant calling WO 14320 PCT/0S2017/036424 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 benefit in accelerating other compute-intensive steps in additional FPGA and/or QP engines, to e a greater up of variant calling, or a portion 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 quantum processing s include: callable-region detection, where reference genome regions d by adequate depth and/or quality of d reads are selected for processing; active-region ion, where reference genome loci with vial evidence of possible variants are identified, and windows of sufficient context around these loci are selected as active regions for further 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, repairing 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 candidate haplotypes from the ly 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 ented by each haplotype, and onize 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 rm. r, calling such a variety of hardware acceleration functions from many integration points in the variant calling software may become inefficient on a loosely-coupled CPU/GPU/QPU+FPGA platform, and ore a tightly-integrated CPU/GPU/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. onally, 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.

WO 14320 PCT/0S2017/036424 Hence, an ideal model for such graph processmg, employing a tightlyintegrated CPU/GPU/QPU and/or FPGA platform, is to retain such graphs in cache-coherent shared memory for alternating processing by CPU and/or GPU and/or QPU software and FPGA hardware functions. In such an instance, a re thread processing a given graph may iteratively command various compute-intensive graph processing steps by a hardware engine, and then the software could inspect the s and determine the next steps between the hardware calls, such as exemplified in the s of . This processing model, may be controlled by a suitably configured workflow management system, and/or may be configured 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 s, 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 addition 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 joining them into longer sequences, s, 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 nce 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 ed, such as where overlapping reads may agree on a single sequence in one segment, but branch into multiple sequences in an adjacent segment, 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 considered to adjoin contiguously to the sequences in connected edges or nodes. In ular instances, such an ly 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 overlapping by k-1 tides. In various methods there may be one or more transformations med n one or more sequence graphs and k-mer graphs.

Although assembly graphs are ed in haplotype-based variant calling, 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 ce ne, de novo ly graphs tend to be more unstructured and complex, with cycles, dangling paths, and other anomalies not only permitted, but subjected to special analysis. De novo assembly graph coloring is sometimes employed, assigning "colors" to nodes and edges signifying, for example, which biological sample they came from, or matching a reference sequence. Hence, a wider variety of graph analysis and processing functions need to be employed for de novo assembly graphs, often iteratively or recursively, and especially due to the size and complexity of de novo assembly graphs, processing functions tend to be extremely compute 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 processing a given graph may iteratively command various compute-intensive graph processing steps to be performed by a hardware engine, and then inspect the s to thereby determine the next steps to be performed by the re, such as by making appropriate re calls. Like above, this sing model, is y benefitted by implementation on a tightly-integrated CPU+FPGA platform, with cache-coherent shared memory and high-bandwidth/low-latency CPU/FPGA onnect.

Additionally, as described herein below, tertiary is includes genomic processing that may follow graph ly and/or t calling, which in clinical applications may include variant annotation, phenotype prediction, disease testing, and/or treatment response prediction, as described herein. 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 ration of primary and/or secondary processing, which are very e intensive, and it is ideal to continue with tertiary analysis on the same rm, for convenience and reduced turnaround time, and to minimize transmission and copying of large genomic data files. Hence, either a loosely or tightly-integrated CPU/GPU/QPU+FPGA platform 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 WO 14320 PCT/0S2017/036424 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 genomic data iteratively, leveraging the U/QPU and/or FPGA acceleration of secondary processmg, in response to partial or intermediate tertiary results, which may benefit additionally from the tight integration configuration. For example, after ry analysis detects 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 nce regions impacting the detection, thus improving the accuracy and confidence of relevant variant calls, and in tum improving the confidence in the detection call. Additionally, if tertiary analysis determines information about the ancestry or ural 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 cy of variant calls and ing the accuracy of further tertiary analysis steps.

However, if tertiary analysis is done on a CPU-only platform after primary and secondary processing (possibly accelerated on a te platform), then re-analysis with secondary processing tools is likely to be too slow to be useful on the tertiary is platform itself, and the alternative is transmission to a faster platform, which is also prohibitively slow. Thus, in the absence of any form ware or quantum acceleration on the tertiary analysis platform, primary and secondary processing must generally be completed before tertiary analysis begins, t the possibility of easy re-analysis or ive secondary analysis and/or pipelining of analytic functions. But on an FPGA and/or quantumaccelerated rm, and especially a tightly-integrated CPU and/or GPU and/or QPU and/or FPGA platform where secondary processing is maximally efficient, ive is 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 functioning may take place at a faster speed, with greater cy, 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 s may be distributed such that various of the functions may be configured so as to be implemented by the host CPU and/or GPU and/or QPU software, whereas in other instances, s 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 Structured ASIC may be tightly coupled, 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 ons 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 directly in accordance with their operational ters, 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 al memory. Of course, 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 el processing capabilities, y resulting in even faster processing . For instance, in certain embodiments, a chip of the disclosure may e 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, instead of a hash table and hash function, which may in various instances, rely on al, e.g., host memory. In such instances, the running of a portion or an entire pipeline can be accomplished in 6 or 10 or 12 or 15 or 20 s or less, such as from start to finish.

As indicated above, there are various ent 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., red into the chip, its function may be performed by the hardware, however, 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 on, such as a , e.g., quantum server, accessible via the cloud, so that the particular module's functionality may be engaged for further processing of the data, in accordance 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 s 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 recalibration, and/or variant calling may take place on the cloud.

Particularly, once the genetic data has been generated and/or processed, e.g., in one or more primary and/or secondary processing protocols, such as by being , aligned, and/or sorted, 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 ons on the generated and/or processed genetic data such as for further, e.g., tertiary, processing, as ed in FIGS. 40. For example, the system may be configured for further processing of the ted and/or secondarily processed data, such as by running it through one or more tertiary processing pipelines 700, such as one or more of a array analysis pipeline, a genome, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, epigenome analysis ne, 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 nes, as well as RNA sequencing pipelines and other genetic analyses pipelines. onally, in various instances, an additional layer of processing 800 may be provided, such as for disease diagnostics, therapeutic treatment, and/or prophylactic prevention, such as including NIPT, NICU, Cancer, LDT, AgBio, and other such disease diagnostics, laxis, 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 nes, metagenome pipelines, genotyping pipelines, variants, e.g., GATK/MuTect2 pipelines, and other such pipelines.

Hence, the devices and methods herein disclosed may be used to generate genetic ce data, which data may then be used to generate one or more variant call files and/or other associated data that may r be t to the execution of other tertiary processing pipelines in accordance with the s and methods disclosed herein, such as for particular WO 14320 PCT/0S2017/036424 and/or general disease diagnostics as well as for prophylactic and/or therapeutic treatment 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 c sequence data. Such data may be generated or otherwise acquired from any le 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 ary processing protocols 600. The secondary processing protocols may include one or more of mapping, aligning, and sorting of the generated genetic sequence data, such as to produce one or more t call files, for example, so as to ine how the genetic sequence data from a subject differs from one or more reference ces or genomes. A further aspect of the disclosure may be directed to performing one or more other analytical functions on the generated and/or sed genetic data, e.g., secondary result data, such as for onal processing, e.g., ry processing 700/800, which processing may be med 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, acquisition, 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 f may then be directly processed, such as by a local computing resource 100, or may be itted to a remote location, such as to a remote computing resource 300, for further processing, e.g. for secondary and/or tertiary processing, see FIGS. 42. For instance, the generated genetic sequence data may be processed locally, and directly, such as where the sequencing and secondary processing functionalities are housed on the same chipset and/or within the same device on-site 10. Likewise, 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 communicably connected, such as via a local network 10. In a further instance, the genetic 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 tion 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 k connection 10/30. The local computing resource 100 may include or otherwise be associated with one or more of a data generation 110 and/or a data acquisition 120 mechanism(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 genetic sequences of a subject or group of subjects, such as where the genetic sequence data is in a BCL or FASTQ file .

For example, such a data generating mechanism 110 may be a pnmary processor such as a sequencer, such as a NGS, a sequencer on a chip, or other like mechanism for generating genetic ce information. Further, such data acquisition mechanisms 120 may be any mechanism configured for receiving data, such as ted genetic ce information; and/or together with the data generator 110 and/or ing resource 100 is capable of ting the same to one or more ary processing protocols, such as a secondary processing 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 networked 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 processing 300 and/or storage 400. In s ments, one or more of these ents 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 er 130 ured 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 performing primary, secondary, and/or tertiary processing. For instance, any suitable compiler 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 ent and coherent transfer of data between system components, but may further be achieved by implementing a n-time (JIT) computer language er uration. Further, in certain instances, 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 various stages ofthe platform nes.

Specifically, as used herein just-in-time (JIT) refers to a device, system, and/or method for converting ed and/or generated file formats from one form to another. 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 various code from one form into another. For instance, in one implementation, a JIT er may function to convert bytecode, or other program code that contains instructions that must be interpreted, into instructions that can be sent ly to an associated processor 140 for near immediate execution, such as without the need for interpretation of the instructions by the ular machine ge. Particularly, after a coding program, e.g., a Java program, has been written, the source ge statements may be compiled by the compiler, e.g., Java compiler, into bytecode, rather than ed into code that contains instructions that match any given particular hardware platform'sprocessing language. This bytecode compiling action, ore, is platform-independent code that can be sent to any rm and run on that platform regardless of its underlying processor.

Hence, a suitable er may be a compiler that is configured so as to compile the bytecode into platform-specific executable 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 suitably 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 specific computer platform on which it was to be implemented. In the present compiling , 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 specific 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 r, 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 genetic 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 s instances, the system herein disclosed may include a first and/or a second compiler 130a and 130b, such as a virtual compiling machine, that handles one or a plurality ofbytecode instruction conversions at a time. For ce, using a Java type just-in-time compiler, or other ly configured second compiler, within the present system platform, will allow for the compiling of instructions into de 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 sor 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 step(s) of translation to machine code or translation into another format, which may then be executed directly, thereby allowing for one or more of ahead-of-time compilation (AOT) and/or interpretation.

More ularly, as implemented within the present system, a typical genome sequencing dataflow generally es 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 ce, a typical DNA sequencer 110, e.g., an NGS, produces raw signals representing 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. Likewise, the reads of the generated BCL/FASTQ files may then be further processed within the , as herein described, so as to produce g and/or alignment data, which ed data, e.g., of the mapped and aligned reads, may be in a SAM or BAM file format, or alternatively a CRAM file . Further, the SAM or BAM file may then be processed, such as through a variant calling 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 ted, just-in-time (JIT) or other dual compiling or dynamic translation compilation analysis, may be configured and deployed herein so as to reduce such high storage costs. For ce, 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 re and/or quantum acceleration platforms, e.g., such as for mapping, aligning, sorting, and/or variant calling (or ent functions thereof, such as de-duplicating, HMM and Smith-Waterman, compression and decompression, and the like), in hardware s on an integrated circuit, such as an FPGA, or by a quantum processor. Hence, by implementing JIT or similar analysis along with such acceleration, the c data can be sed in a manner so as to generate desired file formats on the fly, at speeds comparable to normal file access. Thus, considerable storage savings may be ed by JIT-like processing with little or no loss of access speed.

Particularly, two general options are useful for the underlying storage of the genomic data produced herein so as to be accessible for JIT-like processing, these include the storage of unaligned 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 e compressed BAM or CRAM files). However, since the accelerated processing disclosed herein allows any of the referenced file formats to be derived rapidly, e.g., on the fly, the underlying file format for e 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 un-aligned, read data, there is an advantage to storing unaligned reads so that the data fields are minimized. Likewise, there is an advantage to storing the processed and compressed data, such as in a CRAM file format.

More ularly, 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, e this ation may be y 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 aligning 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 s may be ssed prior to storage, be decompressed after storage, and processed rapidly, such as on the fly, so as to convert one file format from r.

An advantage to storing aligned reads is that much or all of each read's ce content can be omitted. Specifically, system efficiency can be enhanced and storage space saved by only storing the differences between the read sequences and the selected reference genome, such as at indicated variant alignment positions of the read. More specifically, since differences from the reference are usually sparse, the aligned on and list of differences can often be more compactly stored than the original read sequence.

Therefore, in various instances, the e of an aligned read , e.g., when storing data related to the differences of aligned reads, may be preferable to the storage ofunaligned read data. In such an instance, if an aligned read and/or t 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 y of other data, such as metadata derived from the various computations determined herein, may also be stored. Such ated data may include read mapped, alignment and/or uent sing data, such as alignment scores, mapping ence, 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 another file format. Hence, the metadata file allows the system to create a bit-compatible version of any other file type. For instance, proceeding forward from an ating 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 greater 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 particularly useful when there is a plurality of mapping/aligning/sorting/variant calling systems 100/300, such as with one on either side of the cloud 50 interface facilitating the tic uploading and processing of the data, which can be further processed such as using the JIT technology herein bed.

For instance, an underlying storage format for JIT compiling and/or processing may n only minimal data fields, 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 instance, the underlying storage for JIT analysis may be in a local file system 200, such as on hard disk drives and solid state drives, or a network e ce 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 e. Additional data, such as metadata and/or other information (which may be small) ary to uce 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 formats to be reproduced, data processing commands to reproduce each format, unique ID (e.g., URL or MD5/SHA hash) of reference genome, various parameter settings, such as for mapping, ent, sorting, variant calling, and/or any other processing, as described herein, randomization seeds for processing steps, e.g., utilizing pseudo-randomization, to deterministically reproduce the same results, user Interface, and the like.

] In various ces, the data to be stored and/or retrieved in a JIT or r 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 , such as for storage and/or retrieval as a SAM, BAM, CRAM, or other custom file format, and provide user tools to rapidly convert the JIT object into the desired format (e.g., in a local temporary storage 200) using the accelerated processing disclosed herein. Another option is to present the appearance of multiple file formats, such as BCL, FASTQ, SAM, BAM, CRAM, VCF, etc. to the user, and the user applications, in such a manner that the file system access to various file formats utilizes a JIT ure, thus only one file type needs be saved, and from these file type, all other files can be generated 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. ] ingly, 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. Additionally, JIT s useful even ifonly a single file format is to be accessed, because compression is still ed relative to storing the accessed format directly. In such an instance, the underlying file storage format may be ent 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 sed above, the file is compressed prior to storage, and decompressed 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 consumed 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 ation network node, such as where the underlying format may be chosen ily for st compressed file size, and/or where all d file formats may be generated at the ation node by or for c 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 described, may be utilized in (or by) both the source network node, to generate and/or compress the underlying format for transmission, and the destination network node, to decompress and/or te other desired file formats by accelerated genomic data processing. Nevertheless, 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 sing.

For example, a data server that sends large amounts of genomic data may utilize hardware and/or quantum accelerated genomic data sing so as to generate the compressed ying format for transmission to various 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 corresponding hardware and/or quantum accelerated genomic data processing apparatus.

Further, in another e, a data server that es uploads of large amounts of genomic data, e.g., from various sources, may utilize hardware and/or quantum rated genomic data processing and/or storage, while the various source nodes may use slower software run on a CPU/GPU to generate the compressed ying file format for transmission. Alternatively, hardware and/or quantum accelerated genomic data processing may be utilized by one or more intermediate network nodes, such as a gateway server, n the source and destination nodes, to it and/or receive genomic data in a compressed underlying file format, according to the JIT or other c translation analysis methods, thus gaining the benefits of reduced transmission time and network utilization without overburdening the said intermediate network nodes with ive 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 compiler, and may further include a ssor unit 150 that is configured for compressing data, such as ted and/or acquired primary and/or secondary processed data (or tertiary data), which data may be compressed, such as prior to er 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 decompressed subsequent to transfer and/or prior to use.

As bed 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 g ion, so as to generate one or more ofmapped, aligned, , deduplicated , and/or variant 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 mance of one or more genomics and/or bioinformatics pipeline analyses, such as for ry 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 ne processing ure. Specifically, secondary processing of genomics data may be performed by a first hardware and/or quantum accelerated processor 100 so as to produce results data, and tertiary processing may be performed on that results data, such as where the r processing is med 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 performing tertiary processing of the genomics variation data produced by the first circuit 100. Accordingly, the results data derived from the first integrated server acts as an analysis engine driving the further processing steps described herein with reference 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, ing 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 , 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 processing protocol as described herein. The ability to receive and process primary sequence data ly 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 converted by the JIT system, 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, deduped, and/or t 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 l genomics 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 d, 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 t calling protocol, a VCF file is produced, which VCF also typically needs to be stored. In such an instance, in order to adequately 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 ented herein, along with the JIT compilation, compression, and storage, y ameliorates these inefficiencies, resource costs, and expenses. For instance, in view of the methods implemented and the sing speeds achieved by the present accelerated 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 sion 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 s may be generated rapidly, e.g., on the fly, in accordance with the methods disclosed herein, such as in a just in time, or JIT, compiling format.

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 processing and e 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. However, once produced, the BAM file itself must then be stored, requiring further time and resources. Likewise, the BAM or CRAM file may be sed 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 , thereby incurring further resource costs and expenses. More particularly, in a typical ce, the FASTQ file for a human genome consumes about 90 GB ofstorage, per file. Likewise, a typical human genome BAM file may consume 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 intensive to go from one step to another, e.g., from one file format to another, 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 e a large amount of computing resources and time to re-do the processes ed to rate 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, typical 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 protocols employed herein make use of the accelerated processing speeds achieved by the present hardware and/or quantum accelerators, so as to realize enhanced efficiency, at reduced time and costs both for processing as well as for storage. Instead of storing 2, 3, or more copies of the same general data in ent 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 rated sing 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 s herein disclosed, only a single file format need be stored, while the other file formats may easily and rapidly be generated rom. 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 processing steps desired by the user. uently, the system may be configured for ease of use such that if a user simply cts with a graphical user interface, such as presented at an associated display of the device, e.g., the user clicks on the FASTQ, BAM, VCF, etc. button presented 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 necessary for regenerating the requested file in the ted 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 le that includes all of the configurations of how the system was run to create the compressed and/or stored file.

Such metafile data details how the particular file , 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 backwards using the configuration stored in the metafile. This can be about an 80% or more reduction in storage and economic cost ifthe computing function is d 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 , as disclosed herein, a user may connect with a storage device, such as for the storage of input data. For e, 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 , 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 download output, e.g., results data, from the platform. ically, the system may e an API, e.g., an S3 or "S3-like" object that allows access to one or more memories of the system, for the storage 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 . Accordingly, the system may be configured so as to allow a user to have access to remotely stored files, e.g., via an S3 or S3-like API, such as by accessing the API via a cloud based ace on a personal ing device.

] Such an API therefore may be ured for allowing access to the cloud 50 to thereby t the user with one or more ofthe cloud based servers 300 disclosed herein, such as to upload and/or ad 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 r 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 val, analyses, and storage ofgenomics data.

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

This expense is increased when storing the large s 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 attractive. The present solutions 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 ented 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, instead 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 service that employs a compute instance 300, which may be launched by an alternative API, so as to compresses data before storage 400, and/or decompress data upon val. In such an ce, what is stored, therefore, is not the actual file, but rather what is stored is a compressed version ofthe original file.

Specifically, in such instance, 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 ressed, 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 ssed 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 .

Hence, because of the rapid and efficient computing and/or compressmg functionalities achieved by the present systems, the user need not even know that the file is being compressed before storage, and subsequently decompressed post storage and presented 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 t 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 , wherein the storage services can be d at lower costs, by ing a e and/or compress instance along with a storage functionality. In such an instance, the l 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 ured as a rapid and efficient storage and retrieval system that allows for the rapid compressed 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 genomics data storage 400, and is compatible with the Just In Time processing functionalities disclosed herein, above. Therefore, in accordance with the devices, systems, and methods disclosed herein is an object storage service that may be provided, n the storage service implements a rapid compression functionality, such as genomics specific compression so as to store genomics processing results data.

More particularly, as can be seen with respect to A, in one exemplary implementation, the BioIT systems provided herein may be configured such that a ne server system 300, e.g., a portion 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 d metadata. se, once the original CPI files are decompressed and processed, the resulting results data (F2) files may then be ssed and stored as a CF2 file. Accordingly, 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 e 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 compute instance(s), 4) the e instance(s) performs the requisite compression on the FI file, and generates 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 processed 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 feature of this system is that several different users 100 may be allowed to access the stored data 400 substantially simultaneously. For instance, the compression s and methods disclosed herein are useful in conjunction with the BioT rms sed 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 others, with the right permissions.

With respect to performing genomic analysis, 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 format, and thereby request the performance of one or genomics operations, such as a mapping, ng, sorting, de-duplicating, variant calling, and/or other operations. The system 300 receives the request at a ow manager API, the workflow manager system then assesses the incoming requests, indexes the jobs, forms a queue, allocates the resources, e.g., ce allocation, and generates the pipeline flow. Accordingly, when a request comes in and is cessed and queued, an ce allocator, e.g., API, will then spin up the various job specific instances, described in greater detail herein below, in accordance with the work ts.

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

Additionally, where a plurality of jobs are ted 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 compressed and stored, such as in an appropriate memory instance, e.g., a first data base, such as an elastic or flexible storage , so as to wait while the further pipeline instance(s) is spun up and retrieves the results data for further processing, such as in accordance with the systems and methods sed herein above. r, as new requests come in and/or t 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 ses ofthe system running efficiently.

] Likewise, the system 300 may constantly be taking the results data and storing the data 0, 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 s 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 disclosed herein, the system may have a common backbone to which other services may be coupled and/or additional ces, e.g., instances, may be brought online so as to make sure all of the pipeline ions run smoothly and efficiently. Likewise, 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 presentation to the requester. It is noted that in various instances, the specified compute and compress instance, as well as the ied compute and decompress instance, may be a single or multiple instances, and may be ented as a CPU, FPGA, or a tightly coupled CPU/FPGA, y 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 sures herein, in one aspect, a device for performing one or more of a multiplicity of ons in performing genomics sequence analysis operations is provided. 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 ed for further sing, e.g., for ary analysis, the results thereof may then be transmitted back to the local user 100, e.g., after being ssed, stored 400, and/or subjected to onal processing, e.g., tertiary processing by the system server 300.

In certain ces, the ary 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 ce 200 includes a core of CPUs, such as from about 4 to about 14 to about 24 or more CPU cores, and may further include one or more FPGAs. The local box-top 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 k 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 g processing jobs data to, e.g., genomics jobs to be processed, and reading processed 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 sequencer, 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 operations 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 ing 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 processing on genetics data. For instance, as disclosed herein, the ing ce 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. Particularly, the components of the computing device 100 including the processing units, associated memories, and/or associated PCie card(s), having one or more FPGA/ASIC chipsets therein, may be in communication with one another, all ofwhich may be provided within a housing, such as in a box set manner that is typical within the art. More particularly, 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 ated 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 computing 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 ce, 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 s data thereof may be read back to the associated CPU via the SSD . uently, in this embodiment, the local computing system 100 may be configured to offload various high-compute functionalities to an associated FPGA, thereby enhancing speed, accuracy, and efficiency of bioinformatics sing. 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 plurality ofusers that may be located remotely from the box Particularly, in various instances, a cloud-based server solution 50 may be provided, such as where the server 300 may be accessible remotely. ingly, in particular instances, one or more of the integrated circuits (CPU, FPGA, QPU) disclosed WO 14320 PCT/0S2017/036424 herein may be provided and configured for being accessed via a cloud 50 based interface.

Hence, in particular instances, a work bench box set computing ce, as described above, may be provided where the box set configuration is adapted so as to be portable to the cloud and accessible ly. However, such a configuration may not be sufficient for handling a large of amount of traffic from remote users. Accordingly, in other cases, one or more of the ated circuits 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 instance, in one embodiment, a computing resource, or local server 100, having one or more, e.g., a licity, of CPU and/or GPU and/or QPU cores, and associated memories, may be provided in conjunction with one or more of the 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 ible remotely. In this implementation, such as where a single FPGA is provided, the FPGA(s) may be d for being reconfigured, such as partially reconfigured, between one or more of the s steps ofthe genomics analysis pipeline.

However, in other instances, a server system is provided 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 ing the reconfiguration step.

Accordingly, in s instances, one or more FPGAs may be provided, such as where the FPGA(s) are adapted so as to be reconfigurable between various ne operations. However, in other instances, one or more of the FPGAs may be configured so as 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, gh, in some ces, a single FPGA may be provided and configured for being at least partially reconfigured between performing both a mapping and an alignment ion.

Additionally, other operations in the pipeline that may also be performed by reconfigurable or dedicated FPGAs may e performing a BCL sion/transposition operation, a Smith-Waterman operation, an HMM operation, a local realignment operation, and/or s other variant calling operations. Likewise, various of the ne operations may be configured for being performed by one or more of the associated CPUs/GPUs/QPUs of the system. Such operations may be one or more less computationally intensive operations of the ne, such as for preforming a sorting, deduplication, and other variant calling operations. Hence, the overarching system may be configured for performing a combination of operations part by CPU/GPU/QPU, and part by hardware, such as by an FPGA/ASIC of the .

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 ne of operations discloed herein. For instance, various U/QPU and/or hardwired 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 c analysis operations, such as an FPGA instance for performing mapping, another for performing aligning, another for performing 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 dedicated genomic analysis ions, such as CPU/GPU/QPU instance for performing signal processing, g, de-duplication, compression, s variant calling operations, and the like. In such instances, an ated memory or memories may be provided, such as between the various computation steps of the pipeline, for receiving results data as it is computed, ed, and sed throughout the system, such as n the various CPU and/or FPGA ces and/or layers thereof. Further, it is to be noted that the size of the various CPU and/or FPGA instances may vary dependent on the computational needs of the cloud based system, 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 configured for scheduling and directing the nt 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 ted 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 e g switched from one instance to another, such as a switchable elastic block storage memory. In yet other instances, the memory may be instance non-specific and therefore capable of being icably coupled to a plurality ofinstances, such as for elastic file storage.

Further, the workflow manager may be a dedicated ce 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 resources will be utilized in the mance 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 instance that may be run by a processor, e.g. a CPU/GPU/QPU core. In various ces, the workflow manager may have a database ted 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 managing how data flows throughout 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 s 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 performing the BioIT operations of the system herein described. ically, certain system configurations have been optimized so as to maximize the flow of data from various CPU/GPU/QPU instances to s 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 ically, the system architecture may be configured in such a manner that the GA hardware are sized and configured to run in an optimally efficient manner so as to keep both ce platforms busy during all run times, such as where the CPUs outnumber the FPGA(s) 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 processed 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 directs the flow of data to identified CPUs and/or FPGAs that are ured 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 U/QPU cores may be configured to feed data into a small, , large, extra-large FPGA, or a portion thereof. Specifically, in one embodiment, a CPU specific instance may be provided, such as for performing 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 ce 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 various 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 selected 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 optimize data flow, and thus, the system may be configured so as to be elastically scaled up or down as needs be, e.g., to minimize expense 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 flexible architecture of one or more instances, which may be directly d together, or capable of being coupled together, in a manner that is adapted such that the CPU/FPGA software/hardware are run efficiently so as to ensure the present CPUs/GPUs/QPUs optimally feed the available 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 ow 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 include a plurality of numbers and sizes of CPUs/GPUs/QPUs, and additionally, there may be cloud accessible ces 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 instances.

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 sing platforms disclosed .

Accordingly, herein presented, are systems and architectures, le combinations of the same, and/or methods for implementing them for the efficient formation and use of a bioinformatics and/or genomics processing rm of pipelines, such as is made accessible via the cloud 50.

] In such s, the number and configurations of the selected CPU(s)/GPUs/QPUs may be selected and configured to process the less computationally intensive operations, 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 storing of data, e.g., s data, n the various steps of the procedures and/or between the various different instance types, y avoiding ntial period of instance latency. Specifically, during mapping and ng, very little of the CPU/GPU is utilized, because of the intensive nature of the ations, these tasks are configured for being performed by the re implementations. Likewise, during variant calling, the tasks may be split in such a way as to be roughly fairly distributed between 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 manager 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 t online, and operationally coupled together, should be selected and ured in such a manner that the activated CPUs and FPGAs, as well as their attendant re/hardware, are kept optimally busy. ularly, 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 component parts running proficiently.

Hence, in this manner, the work flow management controller of the system may be configured for ing 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 directed to the number of CPUs necessary so as to optimally m 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 ary so as to optimally perform those operations. An elastic and/or an efficient memory may further be included for efficiently transmitting the results data e operations from one ce to another. In this manner, 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 med, and the optimal configuration and usage of the ces 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 offerings may cause inefficiencies 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 . For instance, the system may be configured such that there is a greater proportion of ble 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 selected FPGA instance(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 e 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 flexibly or elastically organized so that the most intensive, specific computing functions are med by the hardware instances or QPUs, and those functions that can be run through the CPUs, are directed to the appropriate U at the appropriate level for general processing purposes, and where ary 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 utilization. Specifically, the architecture may be configured to maximize ency and reduce latency by ing the various instances on various different virtual levels. Particularly, a plurality, e.g., a significant and/or all, of the Level 1 CPU/GPU ces can be configured to feed into the s Level 2 FPGA instances that have been specifically configured to perform ic functions, such as a mapping FPGA and an aligning FPGA. In a r level, one or more additional (or the same as Level I) CPUs may be provided, such as for performing a sorting and/or deduplicating operations and/or s variant calling operations. Further still, one or more additional layers of FPGAs may be configured for performing a Needleman-Wunsch, Smith- an, an HMM, variant calling operation, and the like. Hence, the first level CPUs can be engaged to form an initial level of a cs 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 ted FPGA instances, such as where the FPGA ce is configured for performing intensive computing functions.

In this , in a particular implementation, the CPU/GPU instances in the pipeline route their data, once prepared, to the one or two mapping and aligning Level 2 FPGA instances. Once the mapping has been med 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 genomics analysis function, such as for performing a sorting function.

Additionally, once the Level 3 CPUs have performed their processing, the resultant data may then be forwarded 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), Waterman (SW) processing function, e.g., at a NW or SW dedicated FPGA instance. 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 memories 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 ent instances, on one or more ent levels, so as to provide a ne platform for cs processing. Such a configuration may involve more than a scaling and/or combining instances, the instances may be configured so that they specialize in performing dedicated functions. In such an instance, the Mapping FPGA instance only performs mapping, and likewise the ng FPGA instance only ms aligning, and so on, rather than a single instance ming 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 ce, 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 process, such as when the FPGA completes the mapping operation, it may be reconfigured so as to perform one or more of aligning, variant calling, Smith-Waterman, HMM, and the like.

WO 14320 PCT/0S2017/036424 Hence, the pipeline manager, e.g., workflow management system, may on to manage the queue of c 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 instances for further processing, such as for mapping and/or aligning, 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 s 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 g ions, such as where the entire platform is elastically and/or efficiently sized and ented to minimize cost of the expensive FPGA instances, while max1m1zmg utilization, minimizing latency, and therefore optimizing operations. ingly, in this manner, less hardware instances are needed because of their pure processing capabilities and hardwired icity, 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 element that may be included, e.g., as an ment, to the pipeline architecture, disclosed herein, is one or more elastic and/or efficient memory modules, which may be configured to function for providing block storage of the data, e.g., s 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 storage s may be inserted between one or more of the processing levels, e.g., between the different instances and/or instance levels. In such an instance, the storage device may be configured such that as data gets processed and results ed, 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 . The same storage device may be employed between all instances, or instance levels, or a multiplicity of storage devices may be employed between the various instances and/or instance levels, such as for storing and/or compiling and/or for queuing of results data. Accordingly, 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 memories (memories capable of being coupled to a plurality of instances sequentially) and/or efficient memories (memories capable 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 instance, in one exemplary embodiment with respect to configurations employing such elastic memories, prior to sending data directly from one ce and/or one level of processing to r, the data may be routed to an EBDS, or other memory device or ure, e.g., an efficient memory block, for storage and thereafter routed to the appropriate hardwired-processing module. Specifically, 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 r node for routing the stored data to the next processing level. In this , one or more, e.g., multiple, BDS modules may be included in the pipeline and configured for being flipped from one node to r so as to participate in the transitioning ofdata throughout the pipeline.

] 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 t 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 oftheir specific tasks at that level.

] Accordingly, there are many steps in the processing pipeline, 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 ted, then the result data may be stored, e.g., within an EDS device, queued and prepared for the next stage of processing by being d 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.

WO 14320 PCT/0S2017/036424 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 ce): Mapping, 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: lication, temporary storage; CPU: variant calling 1, temporary e; 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. onally, a work flow management system may be included to control and/or direct the flow of data h 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 implemented by any suitably configured ce 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 med on one or more CPU instances and one or more FPGA instances on one or more tical 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 various instances, the WMS may have a database ionally coupled to it. In such an ce, the database includes the various ions or jobs to be queued, pending 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. Consequently, when a pending job is identified, the WMS initiates a new analysis protocol on the data and farms it out to the appropriate instance node(s). 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 processing nodes to access tive 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 passing 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 written to the memory to be stored, and once one job is completed, r node fires up, reads the next job, and performs the next iterative 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 instances. 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 instances may also include complimentary 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 cally control the usage ofthe nodes.

] Further, with respect to the use of memories, one or more EBDS, or other ly 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 s 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 storage devices, such as one storage device per instance or instance type that is switchable between instances, e.g., elastic block storage device. Accordingly, in a manner such as this, each level of processing instances and/or memory may be cally 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 s 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 bed, 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 licity of jobs being sed by a virtual matrix of processors that are coupled to suitably ured memory devices so as to tate the efficient processing and data from one ce to another. Further, as indicated, a single memory device may be provided, where the memory device is configured for being coupled to a plurality 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 ller may be included and configured so as to control the functioning of such memory s as they switch from one instance 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 , e.g., among all of the ces, such as by flowing the data through the system, allowing each level to be scaled independently and to bring processors online as needed to efficiently scale.

Additionally, the workflow management system algorithm may be configured so as to determine the number of jobs, the number of resources to process those jobs, the order of processing, and s 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 workflow. 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 cient and takes up too much time. Rather, by flipping the elastic storage from one set of instances to another, e.g., g it from one node and ing to a second node, can greatly enhance the efficiency of the . Further, in various instances, instead of employing EBSD, one or more elastic file storage devices, e.g., single memory devices capable of being coupled to a multiplicity of instances without needing to be flipped from one to another, may be ed, so as to further enhance the transmission of data n instances, making the system even more efficiency. 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 ly coupled together. And, as indicated above, the CPUs can be directly coupled to the FPGAs, such as where such ng is via a tight coupling ace as described above.

Accordingly, with respect to user storage and mg of the generated results data, from a system wide perspective, all of the generated results data need not be stored. For instance, the generated results data will typically be in a particular file format, 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 consume a lot of memory thereby incurring a lot of expense. Nevertheless, an advantage of the present devices, systems, and methods , all of these files need not be stored. Rather, given the rapid processing speeds and/or the rapid compression and decompression rates achievable by the ents and methods ofthe system, only a single file format, e.g., a ssed 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 , implementing the devices and methods of the system, all other file formats may be derived. And, e of the rapid compression and decompression rates achieved 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 cs 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 resource 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., s 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 ive.

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 performed on that data. The system 300 will then perform the requested jobs and store the results data in database 400. As noted, in ular instances, the system 300 will store the generated results data in a single file format, such as a CRAM file. Further, with the click of a button, the user can access the stored file, and with r click of a button, all of the other file formats may then be made ible. For instance, in accordance with the methods disclosed herein, given the systems rapid processing capabilities, which would then be sed and generated behind the scene, e.g., on the fly, thus cutting 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, because the various processing ions of g, aligning, sorting, deduplicating , and/or variant calling, may be implemented in a hardwired and/or quantum processing configuration, the production of results data, in one or more file formats, may be achieved rapidly. Additionally, because ofthe close coupling architectures 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 e of their seamless ation with the associated storage devices, the data that results from the processing operations ofthe system, which data is to be stored, may be both ently compressed prior to storage and decompressed subsequent to e.

Such efficiencies thereby lower storage costs and/or the penalties related to decompression of files before use. Accordingly, because 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 regeneration 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 reverse direction, such as to reproduce a VCF or FASTQ or BCL file, tively.

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 forward direction, a checksum of the file may be taken. Likewise, once result data is produced, when going backward, another checksum may be generated. These checksums may then be used to ensure that any r file formats to be generated and/or recreated by the system, in the d or reverse direction, match identically to one another and/or their compressed file formats. In a manner such as this it may be d that all of the necessary data is stored, in as efficient 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 nt 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 sed time it takes prior to processing to decompress the compressed stored file. However, the JIT system allows this to be done without ntial penalties.

More particularly, enting the devices and processes disclosed herein, not only can generated ce 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 rated hardware and/or quantum 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 retrieved, decompressed, and/or converted from one file format to another, and/or be analyzed, such as by the JIT engine(s) being loaded into the hardwired processor, or configured 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 ated 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 s, which engines may include one or more compression engines 150 that are configured for working in the background.

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

For instance, when genetic data is generated, it is usually produced in a raw data format, such as a BCL file, which then may get ted 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 ed or otherwise 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 corresponding BAM file may be generated. And likewise, from the BAM file a corresponding VCF may be ted. onally, SAM and CRAM files may also be ted during appropriate steps. Each one of these steps may be performed very rapidly, especially once the appropriate file format has once been generated. Hence, once the BCL file is received, e.g., straight from the cer, 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 complex. Accordingly, these lexed BCL files may then be de-multiplexed, and respective FASTQ files may be generated representing the genetic code for each individual subject. For instance, if in one sequencing run N BCL files are generated, 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 ted to BCL files, which may then be converted to a FASTQ file or used directly for g, aligning, and/or sorting, variant calling, and the like. This process may be automated 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 therefore 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. ingly, as the generated and/or processed genetic data comes off of the sequencer, the data may be processed and/or stored and made available to other users on other systems, such as in a x-like cache 400. In such an instance, the ted 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 , 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 generate a BAM, CRAM, SAM, and/or VCF, thereby automatically generating and outputting the various sing 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 s c files produced to be compressed, e.g., prior to data storage, and to be decompressed rapidly prior to usage. Hence, JIT processing can e and/or compress and/or store the data as it is coming off the sequencer, where such storage is in a secure genomic dropbox memory cache. This c x cache 400 may be a cloud 50 accessible memory cache that is configured for the storing of genomics data received 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 , 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 generate a g, 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 sed above, on the fly, JIT can be used to regenerate 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 ssed and/or stored and can be used to produce one or more of the other file formats. 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 formats, 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 remotely and made accessible in the cloud 50. This configuration, along with the devices and methods disclosed herein, is d to enable a user to rapidly m 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 ssed data may be a SAM, BAM, CRAM, and/or VCF file.

Specifically, the second ing resource 300 may be another work-bench solution, or it may be a server configured resource, such as where the computing resource is accessible via the cloud 50, and is configured for performing g and/or aligning and/or sorting 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 y process that data and generate one or more results data, which data may then be compressed and/or . Additionally, from the results data file one or more BCL, FASTQ, SAM, BAM, VCF, or other file formats may be generated, e.g., on the fly, using JIT sing. This configuration y alleviates the l transfer speed bottleneck.

Hence, in various embodiments, the system 1 may e, 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, ssion 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 , 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 ting apparatus, 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 ly by the first mapping and/or aligning and/or sorting and/or variant calling instrument 100, as described herein, or may be accessed indirectly by the server resource 300, which may then process the sequence 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 accessible, with the appropriate sions, 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 configured se, where the database is configured as a genomics dropbox cache 400, such as where s results data may be stored in a SAM, BAM, CRAM and/or VCF file, which may be accessible remotely.

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 , 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. Further, in various instance, a second mapping and/or aligning and/or sorting and/or variant calling ment 300, e.g., a cloud based instrument, with the proper ities, may also be ted 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 immediately uploaded into the cloud 50 and stored in a genomics dropbox 400, or it may be transmitted to a BioIT processing system 300 for further sing and/or compression 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 s data from secondary processing may be decompressed and used such as for tertiary 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 compressed, e.g., by a local computing resource 100, prior to er and/or storage 200, or the data may be transferred directly into the genomics dropbox folder for storage 400. Once received 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, ng, g, and/or variant calling to produce secondarily sed results data that may then be re-compressed for storage. Afterward, the ssed 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 processing ures. As the data may be compressed when stored and substantially immediately decompressed when retrieved, it is ble for use by many different s and in many different bioanalytical protocols at different times, simply by accessing the dropbox storage cache 400. ore, in such manners as these, the BioIT platform pipelines presented herein may be ured so as to offer incredible flexibility of data generation and/or analysis, and are adapted to handle the input of particular 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 methods for performing genetic sequencing analysis, which may include 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 genetic ce file format, such as in a ssed 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 e, once received, 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 ng 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 ission 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 ed by the system may be streamed or otherwise transferred to the system ly from the sequencing apparatus, e.g., NGS 110, and as such the transferred file may be in a BCL file format. 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 ly. For instance, the platform pipeline processors can be configured to receive BCL data that is streamed directly from the sequencer, as described with respect to or it may receive data in a FASTQ file format. However, receiving the ce data directly as it is streamed off of the sequencer is useful because it enables the data to go ly from raw sequencing data to being directly sed, 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 received, e.g., by a computing resource 100 and/or 300, it may be mapped and/or aligned by the computing resource, which mapping and/or aligning 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 s 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 position sorted, such as through binning by reference range and/or sorting of the bins by nce position. Further, the sequence 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 report, 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 BAM/CRAM file, such as for storage and/or further processing. Furthermore, once the BAM/CRAM file has been retrieved, the mapped and/or aligned ce data may be forwarded to a variant g 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 t 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 d, the data may then be forwarded to one or more of the pipeline modules disclosed , 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 ded to a variant g module, so as to produce one or more VCF files. These steps may be repeated for r 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 itted 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. Consequently, 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 generated may be compressed and/or transferred from one system component to r, 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 ed, 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 itted to a remote resource 300 for such processing. Once mapped and/or aligned, the resulting ce data, e.g., in a SAM file format, may be processed further such as by being compressed 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 results 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 adapted so as to process BCL data directly, thereby eliminating a FASTQ file conversion step. Likewise, the BCL data may be fed directly to the pipeline to produce a unique output VCF file per sample. Intermediate SAM/BAM/CRAM files can also be generated on demand. The system, therefore, may be configured for receiving and/or transmitting one or more data files, such as a BCL or FASTQ data file ning sequence information, and processing the same so as to produce 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 ssed file and convert it to an original 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 . For instance, the results data from these procedures 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 nment 114d, and/or compression/decompression 114e.

The same or another 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 compressed 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 is.

For instance, if a further DNA or RNA analysis, or some other kind of analysis, is afterward desired, a user may want to run the data through another pipeline, and hence having access to the regenerated al data file is very useful. Likewise, 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 g and aligning to the new reference .

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 s data. In such an instance, it is not the data that agrees with the reference that is important, 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 ng agreement and disagreement with the reference), or a sub-portion of it (showing only agreement or disagreement with the nce), may be configured for being compressed and stored. It may be seen, therefore, that as only the ences and/or variations between the reference and the genome being examined are the most useful to e, in various ments, only these differences need be stored, as anything that is the same as the reference 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 accessible system 1, provided herein, is a workflow management controller 151, which may be used to te the system flow. Such system animation may include utilizing the s 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 pipelines disclosed herein. Accordingly, the workflow management ller 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 , as well as the results data that is generated by the processing of that data, so as to identify and determine s relationships between that data and/or with any other data that may be entered into the system. More ularly, the A/I module may be configured for analyzing various genomic data in correspondence with a plurality of other factors, so as to determine any relationship, e.g., effect based relationships, between the s 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 ersa.

Specifically, as described in greater detail below, the A/I module may be configured to correlate the genomics data of a subject generated by the system with any electronic medical records, for that subject or others, so as to ine any relationships between them and/or any other nt factors and/or data. Accordingly, such other data that may be used by the system in ining any relevant effects and/or relationships that these s may have on a subject and/or their genomic data and/or health include: NIPT data, NICU data, Cancer related data, LDT data, nmental 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., ic or mate read data, germline variants data, allele data, RNA data, and other such data related to a subject's c 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. ularly, 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 implemented in an FPGA and/or Quantum Processing Unit. ically, 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 process the various data flowing through the system.

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 specifically, the A/I module may be configured so as to implement a machine ng protocol with respect 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 subject's whose genomic DNA has been sed, may be obtained, and may likewise be stored in the database. As described in r detail below, the processing engine(s) may be configured to analyze the subjects genomic data, as well as their EMR data, so as to ine any correlations between the two. These ations will then be explored, ed relationships strengthened, and the results thereof may be used to more effectively and more efficiently perform the various functions 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 predictive correlations 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 duals. Particularly, by ining 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 ations, 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 t's genome in comparison to known or determined genetic disease s, and/or by determining ce 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 respect to the present systems, a pileup of reads is ed. The pileup may overlap regions known to have a higher probability of a significant variance. Accordingly, the system on one hand will analyze the pileup to determine the presence of variance, while at the same time, based on its previous findings, will y know the likelihood that a variance should or should not be there, e.g., it will have an l tion 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 ating those variants with one or more e states or other health conditions.

For example, in an exemplary learning protocol, the A/I analysis may include taking an electronic image of a pileup of one or more s 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 e states. This may be done again and again with the system learning to process the information, make the appropriate associations, and make the correct calls quicker and quicker, and with greater accuracy. 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 , 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 , e.g., the variant caller, so as to make more accurate calls, sooner, and with greater efficiency, and/or to allow the tertiary processing module to better identify unhealthy conditions.

Accordingly, the system receives many inputs with known answers, performs the analysis and computes the answer, and thereby learns from the process, e.g., s an image of a pileup, with respect 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 n variations and various conditions, e.g., in a plurality 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 building of a correlation n one or more of the EMR (including phenotypic data), the pileup image, and/or known variants (genotypic data) and/or disease states or conditions, e.g., from which the predictions may be made. In various instances, the system may include a transcription function, so as to be able to transcribe any of the al 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 subject as well as for sensing the environmental and/or physiological conditions of the user at that on. Other sensed data may also be collected. For instance, the mobile computing device may include a GPS tracker, and/or its location may be ined 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, logical status, and other sensed data of the subject owner of the mobile er 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., rly and periodically, to one or more of the system databases herein, wherein the collected ZNA data may be correlated with the subject's patient history, e.g., EMR records, and/or their genomic data, as ined by the system herein.

Likewise, in s instances, one or more of these data may be forwarded from the ZNA collection and analysis platform, to a l repository, e.g., at a government ty, so as to be analyzed on a greater, e.g., nationwide, scale, such as in accordance with the Artificial Intelligence disclosed herein. For ce, the database, e.g., governmental 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 ually 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 ller 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 instances, the workflow ment ller 151 not only keeps track of all ofthe incoming data, but it also efficiently organizes and tates the secondary and/or ry processing of the received data. Accordingly, the workflow management controller 151 allows the system 1 to ssly connect to both small and large sequencing centers, where all kinds of genetic material may be coming through one or more sequencing instruments 110 at the same time, all ofwhich may be transferred 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 licity of samples may be received within the system 1, and hence the system 1 may be configured for receiving and efficiently processing the samples, either sequentially or in el, such as in a multi sample sing regime. Accordingly, to streamline and/or automate multi sample processing, the system may be controlled by a comprehensive Workflow Management System (WMS) or LIMS (laboratory information management system) 151. The WMS 151 enables users to easily schedule multiple workflow runs for any pipeline, as well as to adjust or accelerate NGS analysis algorithms, platform nes, and their attendant applications.

In such an instance, 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 processing 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 se 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 workflow management system 151, such as LIMS (laboratory ation management system). LIMS, in this instance, may be a standard tool that is employed for the management of laboratories, or it may be a specifically designed tool used for managing process flow.

In any instance, the workflow management controller 151 tracks a bar-coded sample from when it arrives in a given site, e.g., for storage and/or processing, until the results are sent out to the user. Particularly, the workflow management ller 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 ated with the sample is read, and based on that reading the system determines what the ted work flows are, and prepares the sample for sing. 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 together. In one particular model the ted or received data may be run h the system to produce processed data, the processed data may then be run through a GATK lent module, the results may be compared, 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 sing nes. 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 workflow is set up, it can be saved, and then a modified bar code gets assigned to that workflow, and the automated process takes place in ance 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 analysis. 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 reconfigure the system for analyzing the new sample set. With the technology sed herein the system can be ly automated. The present system, particularly, is ured so as to automatically receive multiple samples, map them to multiple different workflows and pipelines, and run them on the same or multiple different system cards.

Accordingly, the workflow management system 151 reads the job requirements of the bar codes, allocates resources for performing the jobs, e.g., regardless of on, s the sample e, and directs the s to the ted resources, e.g., processing units, for processing. Hence, it is the workflow manager 151 that determines the secondary 600 and/or tertiary 700 analyses protocols that will be run on the received s.

These processing units are resources that are available for delineating and performing the operations allocated to each data set. Particularly, the work flow controller 151 controls the various operations associated with receiving and reading the sample, determining jobs, allocating ces for the performance ofthose jobs, e.g., secondary 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 l system from start to finish, e.g., from sample receipt to VCF generation, and/or through to tertiary processing, see B.

In additional 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 ed, such as for disease diagnostics, therapeutic ent, 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 ted by one or more ofthe present primary and/or secondary and/or tertiary pipelines. ingly, 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, ed, and managed completely offsite by securely connecting to a remote ing resource 300 via a private cloud interface 30. ularly, in a general implementation of a bioinformatics analysis platform, the local computing 140 and/or storage 200 functions are maintained locally on site 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 ely off site 400. Further, where there is a need for stored data 400 to be made ble to other remote users, such data may be transferred and made available via a global cloud 50 interface for remote storage 400 y, but for global access. In such an instance, where the computing resources 140 required for performance of the computing functions are minimal, but the storage requirements extensive, the computing function 140 may be maintained locally 100, while the storage function 400 may be maintained remotely, e.g., for either private or global access, with the fully processed data being transferred 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 processed data, such as by employing the JIT protocols disclosed 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 generated and stored, e.g., 400, the data may then be recalled, 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 on remote from the storage facility 400, e.g., locally 100. In such an instance, the local storage resource 200 serves merely as a storage cache where data is placed while g er to or from the cloud 30/50, such as to or from the remote storage facility 400.

Likewise, where the computing on 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 storing the processed data 200 are vely l, as compared to the computing resources 300 required to s the data, the data to be processed 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 ing ces, e.g., one or more super computing resources. In such an instance, once the data has been processed by the cloud based er 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 analysis and/or diagnostics. Ofcourse, the remotely ted data 300 may also be stored remotely 400.

This may further be ified with respect to a l secondary 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 further processed thereby, e.g., in a secondary 600 or tertiary 700 processing function, to obtain processed results 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 tes sequenced read data using a local data generating 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 functions on that data, such as a Burrows-Wheeler transform or Needlemen- Wunsch and/or Smith-Waterman alignment 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 presented herein. In such an instance, the system can be configured such that local 100 and remote 300 computing ces are configured so as to run seamlessly together, such that data to be sed thereby can be allocated real time to either the local 200 or the remote 300 computing ce t paying an extensive penalty due to transfer rate and/or in operational ency. This may be the case, for instance, where the software and/or hardware and/or quantum sing 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 generating or for receiving ted 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 red 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 configured for one or more of generating data, such as sequencing data 111. In various embodiments, 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 generating 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 associated nucleotides. In such an instance, the data generating mechanism 110, e.g., sequencer 111, may further be configured for performing preliminarily processing on the generated data so as for signal processing 111 b or to m 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 ce, the produced data 111 may be generated locally and directly, such as by a local data generating 110 and/or computing resource 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 computing resource 100 such as for secondary processing 140 and/or e thereby in a local storage resource 200, such as while awaiting further local processing 140. In such an instance, where the data generation resource is remote from the local sing 100 and/or storage 200 resources, the corresponding ces may be configured such that the remote and/or local storage, remote and local processing, and/or communicating protocols employed by each resource may be adapted to smoothly and/or seamlessly integrate with one another, e.g., by g the same, similar, and/or equivalent software and/or by having the same, similar, and/or equivalent hardware urations, and/or employing the same communications and/or transfer ols, which, in some instances, may have been implemented at the time ofmanufacture or later thereto.

Specifically, in one implementation, these functions may be implemented in a hardwired uration such as where the sequencing on and the ary processing function are ined upon the same or associated chip or chipset, e.g., such as where the sequencer and secondary sor are directly interconnected on a chip, as herein described.

In other implementations, these functions may be implemented on two or more separate 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 entations, a combination of optimized hardware and software implementations for performing the recited functions may also be employed.

More specifically, the same configurations may be implemented with respect to the performance of the mapping, aligning, sorting, 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 include software and/or hardware configured for performing one or more secondary 600 tiers ofprocessing functions 5, and/or or tertiary tiers 700/800 of processing functions, on locally and/or remotely generated data, such as genetic sequence data, in a manner that the processing and results 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 configured for generating and/or receiving primary data, such as genetic sequence data, e.g., in a BCL and/or a FASTQ file format, and running one or more secondary 600 and/or tertiary 700 processing protocols on that generated and/or acquired data. In such an instance, one or more of these protocols may be ented in a software, hardware, or combinational format, such as run on a m processor, a CPU, and/or a GPU. For instance, 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 operation 113, variant calling 115, or other related function 114 on the acquired or ted data in software and/or in re. ingly, in various embodiments, the data generating ce, such as the sequencer 111, e.g., NGS or sequencer on a chip, whether ented in software and/or in re, or a combination of the same, may further be configured to e an initial tier of sors 500 such as a scheduler, various ics, comparers, rs, 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 re, or a combination ofthe same, may further be configured 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 ming a Burrows-Wheeler transform and/or other algorithms for building a hash table and/or g a hash function 112a on said data, such as for hash seed mapping, so as to generate mapped sequence data. r still, the local computing 100 resource whether implemented in re and/or in hardware, or a combination of the same, may further be configured to include an initial tier of processors 600 such as may also include an alignment engine 113, as herein described, or may otherwise include mming 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 man-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 resource 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 sing functions on the genetic sequence data, such as on the mapped and/or aligned ce data. Thus, the one or more other modules may include a suitably configured engine 114, or otherwise include programming, for running the one or more other processing functions such as a sorting 114a, de-duplication 114b, recalibration 114c, local realignment 114d, duplicate marking 114f, Base Quality Score bration 114g function(s) and/or a compression function (such as to produce a SAM, Reduced BAM, and/or a CRAM compression and/or ression file) 114e, in accordance with the methods herein described. In various instances, one or more of these processing ons may be configured as one or more pipelines 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 produce a t 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 on 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 ing function 100 that may be configured for employing a computer processing resource 150 for performing one or more further processing functions 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 computing ce 300 so as to te 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 further processed. This may be useful, for instance, where the remote ing 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 sequence data; and where r processing is desired, such as to determine how the ted 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., tertiary, 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 sing resource 300 ated therewith, in a manner so as to allow the user to m one or more quantitative and/or qualitative processing functions 152 on the generated and/or acquired data. For ce, in one configuration, the system 1 may include, e.g., in addition to y 500 and/or secondary 600 sing 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, aligned 113, sorted 114a, and/or further 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 ured for performing such requested secondary 600 and/or tertiary processing 0 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 sorting 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 processing function 0 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 pipeline processing functions 700 such as to te genome, e.g., whole genome, data 122a, epigenome data 122b, nome 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 ne, microbiome analysis pipeline, RNA sequencing pipelines, and other genetic analyses pipelines. Further, the system 1 may be configured for performing an additional tier of processing 800 on the generated and/or sed data, such as ing 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) ations 123e, or other such health care related 123fprocessing on. See C.

Hence, in various embodiments, where a primary user may access and/or configure the system 1 and its various components directly, such as h direct access therewith, such as through the local computing resource 100, as presented herein, the system 1 may also be adapted 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 ure and run the system 1 within the local environment. Additionally, 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 m interface (API), accessible as through one or more graphical user interface (GUI) components. Such a GUI may be configured to allow the third-party user to access the system 1, and using the API to configure the various ents of the system, the modules, ated pipelines, and other associated data generating and/or processing onalities 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.

Accordingly, in various instances, the system 1 as herein presented may be adapted so as to be urable by a primary, secondary, or tertiary user of the system. In such an instance, the system 1 may be adapted 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 ns of the system necessary or useful for running the analytics ted 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 graphical user ace (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 e the system and its components as desired.

In such an ce, 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, mapping 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 desired 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 ne 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, ing structural variants pipelines, as well as other tertiary analyses pipelines, such as a micro-array analysis pipeline, exome is pipeline, microbiome analysis ne, RNA cing pipelines, and other genetic es pipelines. Furthermore, where desired the GUI menu and system function calls may direct the user selectable operations of one or more of a third tier of operations 800 ing: 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 d 123fprocessing functions.

Accordingly, the menu and system function calls may include one or more primary, secondary, and/or tertiary processing functions, so as to allow the system and/or its component parts to be configured such as with respect to performing one or more data analysis pipelines as selected and configured by the user. In such an instance, the local computing resource 100 may be configured to correspond to and/or mirror the remote WO 14320 PCT/0S2017/036424 computing resource 300, and/or likewise the local storage ce 200 may be configured to correspond and/or mirror the remote storage ce 400 so that the various ents 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 ible to third parties, for running etary analysis protocols 121a on the generated and/or processed data, such as by running through an artificial intelligence interface ed to find correlations there between.

The system 1 may be configured so as to perform any form of tertiary sing 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 h 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 appropriately ured 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. Further, 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 selected 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 ure 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 party and/or requested or desired to be run thereby.

With respect to C, one or more ofthe above demarcated modules, and their tive functions and/or associated 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 ol over a global cloud based internet connection 50, such as via a suitably configured data acquisition mechanism 120.

Accordingly, in such an instance, a local computing resource 100 may include a data acquisition mechanism 120, such as configured 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 ss and steady manner, such as over a cloud based network 50 where the processing functions are buted both locally 100 and/or ly 300. Likewise, where one or more ofthe results ofsuch processing may be stored locally 200 and/or remotely 400, such that the system seamlessly allocates to which local or remote resource a given job is to be sent for processing and/or storage regardless of where the resource is physically positioned. Such distributed processing, erring, and acquisition may include one or more of sequencing 111, mapping 112, aligning 113, sorting 114a, duplicate marking 114c, deduplication, recalibration 114d, local realignment 114e, Base Quality Score bration 114f function(s) and/or a compression on 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 processing prior to nsmission and/or rage.

Specifically, the system 1 may be configured for producing and/or acquiring c sequence data 111, may be configured for taking that genetic sequence data and sing it locally 140, or transferring the data over a suitably ured cloud 30 or hybrid cloud 50 network such as to a remote processing facility for remote processing 300.

Further, once processed the system 1 may be configured for storing the processed data remotely 400 or transferring it back for local storage 200. ingly, 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 first tier of primary and/or secondary processing functions 600, which tier may include one or more of: sequencing 111, mapping 112, aligning 113, and/or sorting 114a so as to produce one or more variant 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 ons 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 ne 122d, variants, e.g., GATK 122e and/or MuTect2, analysis 122fpipeline, as well as other tertiary analyses pipelines, such as a micro-array analysis pipeline, a microbiome analysis pipeline, an exome analysis pipeline, as well as RNA sequencing pipelines and other c 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 e one or more of ting and/or acquiring data related to and including: non-invasive prenatal testing (NIPT) 123a, NIP ICU 123b, cancer d 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.

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 information to or from the associated local processing 100 and/or remote 300 processing resources as well as to store information either locally 200 or remotely 400 in a manner that allows the user to choose what information 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 med on ted and/or acquired data, but also how those ces get deployed, and/or where the results of such processing gets stored. For instance, in one configuration, the user may select whether data is ted either locally or remotely, or a combination thereof, whether it is subjected to secondary processing, and if so, which modules ofsecondary sing it is subjected to, and/or which resource runs which ofthose processes, and further may determine whether the then generated or acquired data is further subjected to tertiary processing, and if so, which modules and/or which tiers of tertiary processing it is subjected to, and/or which resource runs which of those processes, and likewise, where the results ofthose processes are stored for each step ofthe operations. ularly, in one ment, the user may configure the system 1 of A so that the generating of genetic sequence 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 functions, such as mapping 112, aligning 113, sorting 111, and/or producing a VCF 116, from a menu of available sing options. The user may then select whether the locally sed data is subjected to ry processing, and if so which s 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 s options for the various tiers of tertiary processing 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 ure 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 sing on generated and/or acquired data. Specifically, the user or second and/or third party may ure the system 1 for producing and/or acquiring genetic sequence data, either locally 100 or remotely 200.

Additionally, the user may configure the system 1 for taking that genetic sequence data and mapping, aligning, and/or g it, either locally or remotely, so as to produce one or more variant call files . Additionally, the user may configure the system for ming a tertiary 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 sing on the generated and/or acquired data, and where that sing 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 directly such as by directly accessing the local computing function 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 , the modules, associated pipelines, and other associated data ting 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 provide the requested processing, and where the results data will be stored.

Accordingly, in s instances, the system 1 may be configurable by a primary, secondary, or tertiary 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 directly generates, causes to be generated by the system 1, or causes to be transferred to the system 1, such as over a network associated 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 ary 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 Administration (FDA) 70b, or allow primary users and/or third parties to collate, compile, and/or access a data base of genetic information derived or otherwise acquired and/or compiled 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 y user or third party, and therefore, one or more of the computer resources 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 results data therefrom. The GUI or other interface may be ured for allowing the user to manage the system components, e.g., via a suitably configured web portal, and to track sample processing progress, less of whether the computing ces 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, aligning 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 ces. Hence, in an instance such as this, each dual user may build thereon a unique, or may use a ermined, analysis workflow, such as by ng 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 plurality 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 s icons into a workflow interface, so as to build a desired ow process, which once built may be saved and used to ish the control instructions for the sample set barcodes. Once the desired work projects have been selected, the work flow management controller 151 may configure the desired workflow processes (e.g., secondary analysis), and then identify and select the resources for ming the selected analysis.

] Once the workflow analysis process begins, the dashboard may be viewed so as to track progress through the system. For example, the dashboard may indicate how much data is running through the system, what processes 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 ts to be run, and which runs have been completed. Essentially, full access to everything that's running on the system, or a subportion 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 selected allows a user to select control functions that are capable of being implemented locally. Another tab may be ured for accessing "cloud resources 300," which when selected allows a user to select other control functions that are capable of being ented remotely. Accordingly, in cting with the ard, a user can select which resources 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 complexity 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, instructing 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 controlled by the workflow manager ller 151, a user of the system can still set the control parameters, and when needed can bring cloud based ces 300 on line.

Accordingly, the controller 151 can expand to the cloud 50/300 as needed to bring on line onal processing and/or e resources 400.

In various instances, the desktop interface may be configured as a mobile application or "app" that is ible 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 research projects, so as to form an electronic cohort market place that is accessible via the dashboard app, e.g., a web based browser ace. 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 l ofthe system by a user.

Accordingly, in various ments, as can be seen with t 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 computing 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 ce, the local and distal ces may be configured for communicating with one another in a manner so as to share information, such as l data, seamlessly between the two. Particularly, the local ces 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 configured such that a generating and/or analyzing function 152 is configured for being med locally 100 by a local computing 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 ces 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. Further, where such processed data is meant to be transferred, such as to a remote computing 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 sing may be of a global nature and may include receiving data from a plurality of local computing resources 100, ing such ities of data, annotating the data, and comparing the same, such as to interpret the data, determine trends thereof, analyzing the same for various biomarkers, and aiding in the development of diagnostics, therapeutics, and/or prophylactics. ingly, in various instances, the remote computing resource 300 may be configured as a data processing hub, such as where data from a variety of sources may be transferred, processed, and/or stored while waiting to be transformed and/or erred, such as by being accessed by the local computing 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 ces 100 so as to allow for oration amongst researchers and/or resources 100. Such oration may include various data sharing protocols, and may additionally include ing the data to be transferred, such as by ng 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 ed for er.

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 generating 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 generating genetic cing 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 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 intranet 10 connection. In some instances, the sequencer 121 may be housed in a separate facility than that of the computing 100 and/or e 200 resource and thus may be connected over an internet 30 or hybrid cloud connection 50.

In such ces, 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 transmitted directly 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 processed 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 transformer 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 intranet 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 WO 14320 PCT/0S2017/036424 and/or storage 200 resource and thus may be connected over an et 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 ing the generated sequence data with t to determining its genomic and/or exomic characteristics 152a, its epigenomic es 152b, any various DNA and/or RNA s 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 ry processes as described herein.

As indicated, the generated and/or processed data may be transformed, such as by a suitably configured transformer such as prior to ission throughout the system 1 from one component thereof 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, including 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 another; indexing 151 b, such as including ing and/or collating the ted 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 creating a lockable and unlockable, password protected dataset, such as prior to transmission over an internet 30 and/or hybrid cloud 50.

Hence, as can be seen with respect 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 , 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, eutic and/or prophylactic discovery and/or development needs of the user. Particularly, the hybrid cloud 50 and/or the various components of the system 1 may be operably connected with compatible and/or corresponding API interfaces that are adapted to allow a user to remotely 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 icate in a secured, encryptable environment.

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 analytic functions on generated data, such as annotation 311, interpretation 312, diagnostics 313, and/or a detection and/or an analysis function for determining the ce of one or more biomarkers, such as in the genetic data.

The interpreter 313 may be part ofor separate from the local computing ce 100, such as where the interpreter 310 is coupled to the computing resource 100 via a cloud ace, such as a hybrid cloud 50.

Further an additional 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 y of data to be transmitted. For instance, the collaborator may be ured for securing the data sharing process 321, for ensuring the y of transmission 322, g control ters 323, and/or for initiating a security protocol 324. The collaborator 313 is configured for allowing for the sharing of data, such as for facilitating the collaboration of processing, as such the collaborator 320 may be part of or te from the local computing resource 100, such as where the collaborator 320 is coupled to the computing resource 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 computing resource 300, such as for enhancing system efficiency by offloading ing 300 and/or storage 400 functions 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 tertiary processing stage. ularly, presented herein are s 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 rm may be provided and configured as a multiplicity of integrated circuits, which integrated circuits may be adapted as, or otherwise be included within, one or more of a central or graphics processing unit, such as a general purpose CPU and/or GPU, a hardwired implementation, and/or a quantum processing unit. Particularly, in various embodiments, one or more pipelines of the genomics processing rm 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 optimized re and/or hardware and/or quantum processing implementations for the performance of the various c sequencing and/or secondary and/or tertiary processing ons, herein disclosed, which may be run on one or more integrated circuits. Such integrated circuits may be ssly coupled together and may further be ssly 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 software run on a CPU, GPU, and/or QPU, and/or may be ented as a firmware configured integrated t, 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 processing 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 positioned within the same housing and/or otherwise coupled together, or they may be oned on separate boards or pCIE cards that are capable of communicating 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 sing units and/or integrated 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 integrated 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 nes of analyses where the various data generated may be fed into and out of, back and forth between, 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 n the multiplicity of integrated circuit, and more particularly to expedite the analyses herein.

For instance, in some instances, the various s for use in accordance with the methods disclosed herein may include, or otherwise be associated with, one or more cing devices, for ming 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 ty, such as made accessible via a cloud based interface. In other ces, the sequencing may be med 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 cing is performed by use of a nductor technology that delivers benchtop next gen sequencing, and/or by an integrated circuit ured as, or to otherwise include, a field effect transistor employing a graphene channel layer. In such instances, where the sequencing is performed by one or more integrated circuits configured as, or to include, a semiconducting sequencing microchip, the ) may be positioned remotely from the one or more other processing units and/or integrated circuits disclosed herein, which may be configured for performing secondary and/or tertiary analytics on the sequenced data. Alternatively, the chips and/or processing units may be positioned relatively 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 facility.

In this and other such instances, a sequencing and/or BioIT ics 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 is, 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 various lytic 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 sequence analysis may involve the comparing of a generated or received nucleic acid or protein sequence to one or more ses of known sequences, such as for performing ary analysis on the received data, and/or in some instances, for performing disease diagnostics, such as where the database of known sequences for performing the comparison may be a se containing morphologically distinct and/or abhorrent sequence data, that 1s data of genetic samples pertaining to or believed to pertain to one or more diseased states.

Accordingly, in various ces, once isolated and sequenced, the c, 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, aligning, g, 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 difference 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 lactic results. More particularly, after variant calling, the mapper/aligner/variant caller may output a standard VCF file that is ready for and may be icated to an onal integrated circuit for performing tertiary analysis, such as analyses related to , e.g., whole genome, analysis, genotyping, e.g., joint genotyping, analysis, micro-array analysis, exome analysis, microbiome analysis, an epigenome analysis, a metagenome analysis, a joint genotyping analysis, a variance analysis, e.g., a GATK analysis, ural variants analysis, somatic variants analysis, and the like, as well as an RNA-sequencing or other genomics analysis.

Hence, the bioanalytic, e.g., the BioIT, rm herein presented may include highly optimized algorithms for mapping, ng, sorting, duplicate marking, haplotype variant g, compression and/or decompression, such as in a software, hardwired, and/or a quantum sing configuration. For example, although one or more ofthese functions may be configured to be performed entirely or partially in a hardwired configuration, in particular instances, the secondary and/or tertiary sing rm may be configured for running one or more software and/or quantum processing applications, such as one or more programs directed at ming 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 further processed by one or more other highly optimized algorithms for one or more of whole genome is, genotyping analysis, microarray analysis, exome analysis, microbiome analysis, epigenome analysis, metagenome analysis, joint genotyping, and/or a variant, e.g., GATK analysis, such as implemented by software being run on a general purpose CPU and/or GPU and/or QPU, albeit in certain instances one or ore ofthese functions may at least partially implemented in hardware.

Accordingly, as can be seen with reference to , in various embodiments, the multiplexed bioanalytical processmg platforms are configured for performing one or more of primary, secondary, and/or tertiary processing. For e, the primary processing stage produces genetic sequence data, such as in one or more BCL and/or FASTQ files for transfer into the system 1. Once within the system 1 the sequenced 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 ated metadata, and/or or other associated processed data, and in one or more tertiary processing stages, may m one or more other operations thereon, such as for the purposes ofperforming one or more diagnostics and/or prophylactic and/or therapeutic procedures there with.

Particularly, an is ofthe data may be ted, e.g., in response to a user request 120, e.g., made from a remote computing resource 100, and/or in se to data submitted by the third party 121, and/or data automatically retrieved from a local 200 and/or remote 400 storage facility. Such further 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 tertiary processing units may include a genomics sing platform that is configured to perform genome, epigenome, metagenome, genotyping, and/or s variant analysis, and/or other bioinformatics based analysis. Additionally, in a second tertiary processing tier, various disease diagnostic, research, and/or analysis protocols 800 may be med, 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 ses herein sed such as related to electronic l records (EMR) data, Federal Drug Administration testing and/or structuring data, data nt 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, stic, therapeutic, and/or prophylactic modalities ped through use of the system 1 and/or made accessible thereby.

Accordingly, in various instances, the devices, methods, and systems presented herein allow for the secure performance of genetic and bioanalytic analysis, as well as for the secure transfer of the results thereof, 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 e, the system may be configured 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 results thereof, in a forum that may be easily usable for ream processing.

Particularly, 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 d data, e.g., in a SAM or BAM file format, and/or for processing variant data, such as in a VCF format. For ce, the first tertiary processing platform may include 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 ne, structural variant pipeline, somatic variant calling pipeline, and in some ces, may include an RNA-sequencing analysis pipeline. One or more other c analysis nes may also be included.

More specifically, with reference to , in various instances, the multitiered and/or multiplexed bioanalytical processing platform includes a further layer of data generation and/or processing units. For instance, in certain instances, the bioanalytical smg platform incorporates one or more processing pipelines, in one or more of software and/or hardware implementations, that are directed to performing one or more tertiary processing protocols. For example, in particular instances, a platform of tertiary 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 pipeline, and/or other pipelines, such as an RNA pipeline. Additionally, a second layer of the tertiary processing analyses platform may include a number of sing pipelines, such as one or more of a micro-array analysis pipeline, a genome, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, ome analysis pipeline, metagenome analysis pipeline, microbiome analysis pipeline, genotyping analysis pipeline, including joint ping, ts analyses pipeline, including structural variants pipelines, c variants pipelines, and GATK and/or MuTect2 pipelines, as well as RNA sequencing pipelines and other genetic analyses pipelines.

WO 14320 PCT/0S2017/036424 Accordingly, m one embodiment, the multi-tiered lytical processing rm includes a metagenomics pipeline. For instance, a metagenomics pipeline may be included, such as for the performance of one or more nmental genomics processes.

Particularly, in various embodiments, the nomics 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 licity of living and/or dead organisms within it may be obtained, from which the A present may be ed, sequenced, and processed via, one or more of the sing platforms , 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 rganisms that are found in association with both y and diseased , including microorganisms found in the skin, blood, sputum, stool s) as well as external environmental agents.

There are a plurality of methods for deriving the sequenced genetic samples for performing metagenomic processing. A first method includes a targeted 16S ribosomal RNA cloning and/or gene sequencing protocol. 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 e of bio-diversity that is derived from naturally occurring biological samples, which may be used to inform the A/I or other databases of the system. However, 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 diversity of microscopic life. Specifically, in the shotgun sequencing method, an aggregate reference sequence may be ted, e.g., from many (e.g., tens of thousands) of reference genomes of different species. However, the aggregate size ofthis many reference genomes is huge. Hence, it is advantageous 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 several 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 aligned, 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 g 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 t in a sample, that species can be identified as being within that environment. Ideally, in a manner such as this, species not common to other species generally present in that environment may be identified.

Specifically, when coverage of all species is normalized for the obtained environmental samples, genetic diversity of all species present can be determined and can be compared against the entire coverage, such as by comparing a portion of a particular organism'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 observing the sequenced reads of a particular organism, ng a given species is or is not present. Bayesian ility 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 probability ofcancer.

Specifically, with the Bayesian probability interpretation the m expresses how a subjective degree ef can rationally change to account for the observed ce. Bayes' theorem is stated mathematically as the ing 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 t regard to each other. P(A I B), a conditional probability, is the ility 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 an 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 increasingly similar between organisms sharing lower taxonomic . 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 l clade (such as a genus or ) can be called present unambiguously, rather than a ic species or .

Nevertheless, the devices, systems, and methods of using the same disclosed herein can be employed to overcome these and other such difficulties.

Specifically, in one embodiment, a method for determining the presence of two or more species or clades oforganisms from a sample is provided. For instance, in a first step, reads of c 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 c reference sequences may be a whole genome, or may be a partial genome in order to reduce the amount of data required for each s, strain, or clade. However, using larger portions of a genome will increase the sensitivity of detection, and each reference sequence used should be selected to represent each species, , or clade that will be distinct from one another.

For this purpose, all or a portion of the genomic sequence from the 16S me of each species 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 sequence reads may be compared with each ofthe two or more s. Then it may be determined ifthe sample genomic sequence reads map to each ofthe indexes.

Likewise, the reads of the genomic sequence may also be aligned to the genomic reference sequence(s) to which they are mapped. This will generate an alignment score, in accordance with the methods herein, which may be used in ing the probability that a read indicates the presence or e 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 herein. 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 determine the likelihood that an organism having the genomic reference sequence is present in the sample. Likewise, a list of species, strains, 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 te 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 present, and a confidence . 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 exemplary implementation 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 material may be isolated 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 s 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 st, 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. Further, 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 ined ifthe genomic sample ofsequenced reads map to each ofthe two or more indexes.

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 ed c materials within the sample are mapped and/or aligned, at step 1060, the likelihood that a given sm having the reference sequence is present within the sample may be determined. And once sed a list of species, strains, and/or clades that are t in the sample may be identified and/or reported.

The tertiary processing platform disclosed herein may also include an omic pipeline. ularly, epigenetics studies the genetic effects not encoded in the DNA sequence of an organism. The term also refers to the changes themselves: functionally relevant changes to the genome that do not e a change in the nucleotide ce.

Nevertheless, epigenetic changes are stably heritable phenotypes that result from changes in a chromosome that does not alter the DNA sequence. These tions may or may not be heritable. Particularly, epigenetic changes modify the activation of n 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 underlying genome that is y 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, omics involves the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome ting of a record of the chemical s to the DNA and histone proteins of an organism. These changes can be passed down to an organism's ing 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 ellular organism to express only the genes that are ary for their own activity. Epigenetic changes are ved when cells divide. Particularly, most epigenetic changes only occur within the course of one individual organism's lifetime. 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 epigenetic inheritance systems may play a role in what has become known as cell memory. For instance, various covalent modifications of either DNA (e.g., cytosine methylation 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 phenotype of a cell or individual is affected by which of its genes are transcribed, heritable transcription states can give rise to epigenetic effects. Such effects on cellular and logical phenotypic traits may result from external or environmental factors that switch genes on and offand affect how cells express genes.

For instance, DNA damage can cause epigenetic changes. DNA damage 1s very frequent. These damages are largely repaired, but at the site of a DNA , epigenetic s 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 ing types of histone modifications (chromatin remodeling). Other examples of mechanisms that produce such changes are DNA methylation and histone modification, each of which alters how genes are sed without altering the ying DNA sequence.

Nucleosome remodeling has also been found to cause epigenetic silencing of DNA repair.

Further, DNA ng chemicals, can also cause considerable hypomethylation of DNA, such as through the activation 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 h 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; d, netic 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 , which in tum become fully differentiated cells. In other words, as a single fertilized egg cell - the zygote - continues to divide, the resulting daughter cells change into all the different cell types in an organism, including neurons, muscle cells, epithelium, endothelium 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 complex of DNA and the histone proteins with which it ates. If the way that DNA is wrapped around the histones changes, gene sion can change as well. A first way is post translational modification of the amino acids that make up e 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 carried 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 ng the shape of the es 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. 5-Methylcytosine performs much like a regular cytosine, pairing with a guanme m -stranded DNA. However, some areas of the genome are methylated more heavily than , and highly ated areas tend to be less transcriptionally active, through a mechanism 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 modifications occur throughout the entire ce, the unstructured N-termini of es (called histone tails) are particularly highly modified. These modifications include ation, methylation, ubiquitylation, phosphorylation, sumoylation, lation and citrullination.

Accordingly, DNA methylation is the presence 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 ing which es are methylated in a given sample genome. Bisulfite sequencing (MethylC-seq) is the most common method of detecting methylation using whole-genome sequencing, where un-methylated cytosine ('C') bases are chemically converted to uracil ('U')bases, which become thymine ('T')bases after PCR amplification.

Methylated 'C'bases resist conversion.

] Accordingly, in accordance with the s 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 reference sequence, prior to mapping/alignment. This allows for accurate mapping and alignment of the reads, which have bisulfite converted C's (now T's), thus revealing the sulfite converted (methylated) C's in the genomic sequence reads.

For reverse-complemented alignments, 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 mapper/aligner may be modified to perform these substitutions automatically for C-seq usage.

Alternatively, the /aligner may be modified to allow the forward alignment of read 'T'sto reference 'C's, and the reverse-complemented alignment 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 ous reverse-complemented alignment of read 'G'sto reference 'A's.

Additionally, provided herein are methods for determining the methylation state of cytosine bases in genomic sequence reads. For instance, in a first step, reads of genomic sequence from bisulfite-treated tide 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 cmg; methylated DNA immunoprecipitation sequencing, and methylation-sensitive restriction enzyme sequencing may be used to identify DNA ation across ns ofthe genome, at varying 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 genome. Further, RNA-sequencing and expression arrays may be used to identify expression levels or protein coding genes. Particularly, smRNA-sequencing may be used to identify expression of small noncoding RNA, ily miRNAs.

Consequently, once sequenced to produce reads, a genomic reference sequence may be built for comparison with the reads. CpG ons in the genomic reference sequence may then be . Further, the genomic reference ce may be preprocessed by ing 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 te 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 embodiments, 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 permitted with G to A replacements, in the read and genomic sequence reference.

These mapping and aligning procedures may be accomplished by the various re and/or hardware modules described herein. In some embodiments, the mapped and aligned data may then be communicated to a U/QPU for further is 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 analyzed over each marked reference CpG location. In such an instance, A thymine (T) that has replaced a cytosine (C) indicates a nonmethylated ne 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 e, a e (G) that has replaced an e (A) is marked as the e-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 nucleotide strand may be reported, and an associated confidence metric (e.g. p-value) in the methylation call may be made. In some embodiments, the ation status of the marked CpG locations may also be ted for each chromosome of a diploid pair of chromosomes.

With respect to histone modification, histone cation includes various naturally occurring chemical modifications of the histone proteins 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 isolate and sequence DNA that is tightly bound to histones (or other selected proteins). After ChIP-seq has been med, the sample may be ed, the DNA isolated and sequenced, and the sequenced DNA may then be mapped/aligned to a reference genome as disclosed herein, and the mapped coverage may be used to infer the level ofhistone binding at various loci in the genome. Additionally provided herein are methods of analyzing ChIP-derived nucleotide sequences, which is similar to the methods described below for analyzing structural variants.

Ofspecial note is that etics is useful in cancer research and diagnostics.

For instance, human tumors undergo a major disruption ofDNA methylation and histone modification patterns. In fact the aberrant epigenetic landscape of the cancer cell is characterized by a global genomic hypomethylation, CpG island er hypermethylation of tumor suppressor genes, an altered histone code for critical genes, and a global loss of monoacetylated and trimethylated e H4. Accordingly, the methods disclosed herein may be used for the purposes ofcancer research and/or stics.

] Further, the methods herein disclosed may be useful for generating one or more epigenomic ses and/or reference genomes. For example, the methods herein disclosed, e.g., employing an A/I learning protocol of the system, may be useful for ting a human reference of epigenomes, such as from , 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 factors that occur in various disease states, such as cancer, dementia, Alzheimer's disease, and other neurological conditions.

Accordingly, in various instances, an omics analysis may be performed, such as to identify one or more or the entire set of epigenetic modifications that have taken place on the genetic material of a cell. Particularly, employing the methods sed 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 e proteins of the cells of the organism. For example, an exemplary epigenomic 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 nucleotide sample. Likewise, at step 1010, a c 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 fied.

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 genomic sequence reads of the sample map to the index, such as by being mapped in accordance with the s 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 med. For ce, at step 1051, if greater context is desired, the base substitutions in the reads, as processed above, and/or the alignment orientation, and/or parameter restrictions may be adjusted, and the comparison steps 1030 - 1050 may be ed. This process itself may be repeated as desired until a ient level of context is achieved. Accordingly, once a sufficient 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 /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" s in the sequence, at step 1084, the "C" may be marked as a methylated "C". y, at step 1086, a determination 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 ions e 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 structurally 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 heterogeneity 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 includes ions, deletions, and duplications. There are several inversions known that are d to human disease. For instance, recurrent 400kb inversion in factor VIII gene is a common cause ofhaemophilia A, and smaller inversions affecting idunorate hatase will cause Hunter syndrome. More examples include an me and Sotos syndrome.

The most common type of complex structural variation are non-tandem duplications, where sequence is duplicated and inserted in inverted or direct orientation into another part of the genome. Other s of x structural variant include deletion-inversion-deletions, duplication-inversion-duplications, and tandem duplications with nested deletions. There are also cryptic 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 matic to detect include those having: large insertions and deletions (e.g., beyond the 50-lO0bp indel size); duplications, and other copy-number variations (CNVs); ions 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 mapping and an aligning operation as using the devices and methods disclosed herein. For instance, the reads of the genomic sample to be analyzed 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 indels, inversions, translocations) can be detected by analysis ofclipped and chimeric alignments.

Specifically, each structural variant involves one or more "break" ons, 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 ly overlap the structural variant breaks may be clipped at the break, and the reads substantially overlapping the structural variant breaks may be chimerically d, e.g., with two portions of a read mapped to different reference ons. However, read pairs overlapping ural variant breaks may be inconsistently aligned, with the two mate reads mapped to widely different reference locations, and/or with abnormal ve orientation ofmate reads. Such obstacles may be overcome by the s disclosed herein.

For instance, in n 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 ol. ularly, where a structural variant is known, its " and "outer" coordinates may be employed as a minimal and maximum range of sequence that may be affected by the structural ion. onally, known ion, loss, gain, inversion, LOH, everted, transchr and UPD ions may be classified and fed into the knowledge base ofthe present system.

In various ces, the determination of a structural t may be performed by a CPU/GPU/QPU g suitably configured software, such as employing previously determined sequencing data, and in other instances, structural variant analyses may be performed such as in the hardware sed herein. Accordingly, in particular instances, a method for analyzing genomic sequences for structural variants is provided. For instance, 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 mapped, and an alignment score may be determined.

As indicated above, the mapping 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 U/QPU for further is 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 possible structural ts may be determined based on any detected chimeric reads and/or l 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 variant haplotypes may be determined, such as by using HMM analysis of the chimeric reads and/or the unusual relative alignments. For example, pair HMM may be used for such a ination. The pair HMM may be accomplished using the hardware module.

Accordingly, in various instance, as can be seen with respect to D, a method for determining variations in the structure of an organism's chromosomes is presented. For instance, in accordance with the s disclosed herein, at step 1000, reads of genomic sequence data may be received. 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 reference sequence may be built so as to allow the received reads to be compared against the generated reference. More specifically, for these purposes, at step 1020 an index for the genomic reference sequence may be generated, for e, at step 1020 a hash table or prefix/suffix tree may be generated.

Hence, at step 1030, the reads of the sample genomic sequence may be compared with the ted index, such as in accordance with the re 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 ic 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 relative alignments. Furthermore, ior probabilities of each le structural variant may be calculated, and, optionally, at step 1096, structural variant haplotypes may be determined, such as by using HMM analysis, as described herein, of the chimeric reads and/or unusual relative alignments.

Further, the s, systems, and methods disclosed herein may be employed for the processing of RNA sequences. ularly, herein ted are methods for ing RNA-sequence reads, such as employing a spliced mapping and alignment protocol (e.g., with a suitably configured RNA /aligner). For instance, in one embodiment, a transcriptome pipeline may be provided, such as for ultra-rapid RNA-sequence data is.

Particularly, 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 , where mapping, aligning, and variant calling may be performed. However, in various instances, one or more gene annotations files (GTF) may be input into the , 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. uently, a 2-phase alignment may be performed, such as where in a first detection phase novel splice junctions 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 ted, configurable junction filters may be employed to give a single junction output. Position sorting may be performed, which may include binning by the reference range, and then the sorting of the bins by reference position, and ate marking may take place, such as based on the starting position and CIGAR string so as to achieve a high y ate report, whereby any duplicates may be removed. Haplotype variant calling may then be med, e.g., using a SW and HMM processing engine, and assembly may be performed. onally, the devices, systems, and methods disclosed herein may be employed for ming somatic variant g. For instance, a c variant calling ol 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 l" (non-tumor) sample may also obtained, 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 . The DNA/RNA form the sample(s) may be isolated and sequenced, 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 configured so as to be able to detect low variant 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 t genotype allele frequencies corresponding to the observed allele frequencies in the overlapping reads. For WO 14320 PCT/0S2017/036424 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 probability that a variant is present in the tumor sample but not the normal sample can be estimated.

Further, the somatic variant caller pipeline may be configured to provide information on tumor heterogeneity, e.g., that a series of distinct mutation events occurred, such as where one or more sections ofa tumor with different genotypes (a subclone) has been identified. Such ne information may be derived from a determination of variant allele frequencies and distributions thereof, and/or by explicitly calling variants differentially among multiple tumor samples. ingly, methods for detecting ce variants of cancer cells from a sample are ed. 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 ols 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 aligning may be accomplished by a software and/or hardware , as described herein. In some embodiments, the mapped and d data may then be communicated to a CPU/GPU/QPU for further is and processing. The reads may be sorted by mapped reference position, and any ate 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 ncies (e.g. 3% to %).

In some ments, germline ts may be detected in both noncancerous and ous samples, and somatic 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 e, 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 variant g procedure, as exemplified, in E, may be performed, such as to ate the probability that a variant is a cancer variant. For instance, at step 1000 reads ofgenomic sequence samples may be generated, e.g., via sequencing ofan NGS, and/or be ed, e.g., via transmission over a suitably 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 comparison of the reads, at step 1020 an index may be built from the c reference sequence, and at step 1030 the sample genomic sequence may be compared with the index, such as employing the software and/or hardware 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 respect 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 c variants n may optionally be detected. Likewise, at step 1094, posterior probabilities of each possible cancer variant may be calculated. Further, at step 1096, cancer variant haplotypes may optionally be determined, such as by implementing an HMM analysis in software and/or in hardware as disclosed herein. rmore, the devices, systems, and methods disclosed herein may be configured for performing a joint genotyping operation. Particularly, a joint ping operation may be employed so as to improve t calling accuracy, such as by jointly considering reads from a cohort of multiple ts. For ce, in various ces, genomic variations may be highly correlated in certain populations, e.g., where certain variants are common to a plurality of subjects. In such instances, the sensitivity and specificity of variant calling can be improved by jointly considering the ce 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 improved 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 t calls can be for any given subject.

Joint genotyping es 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 ces, joint genotyping 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 numbers of samples, because when dozens, hundreds, or thousands of s are involved, the total data size becomes impractical to rapidly access and manipulate.

Alternatively, joint genotyping 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 t using the joint information. This method uses additional output from each single-sample variant calling pass so as to better measure areas of weak evidence 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 ts from single-sample variant calling, a special gVCF format may be used to ent first-stage t (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 locations to yield improved joint genotype calls for each subject.

Accordingly, the joint genotyping pipeline may be configured to call variants from multiple samples faster and with r cy. Additionally, the joint genotyping pipeline may further be configured to ts pedigree as well as population variant calling from a cohort of samples. For instance, the pipeline 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 d to handle sample sizes of many thousands at once. Further, a combination of speed and hierarchical ng of multiple samples provides a computationally efficient analysis solution for joint genotyping. onally, the sequencing of the samples for joint genotyping may be performed within the same flow cell of a Next Gen sequencer thereby ng the system to simultaneously map/align multi-sample inputs thereby speeding up the overall process t 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 ing t 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 ed. A genomic reference sequence for comparison with the reads is built, and from the genomic reference sequence an index is generated. The c ce 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 duplicate 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 . This can enhance the variant calling accuracy (e.g., the ivity 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 lished cumulatively, after each of the s 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 reported.

Accordingly, in various embodiments, a somatic variant calling procedure, as exemplified, in F, may be performed, such as to calculate the ility that a t is a cancer variant. 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 suitably 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 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 hardware 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 c reference sequence to generate an ent 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.

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 probabilities of each le t 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 t 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 running 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 c data. The online "cohort" marketplace, therefore, presents a rapid and efficient way to deploy new c analytic applications, which ations 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 provides 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 .

] 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 er in a secure and protected manner, such as where the t's identity is restricted. Accordingly, 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 advance their research in a protected and secure environment, without risking the identity of their respective subject's genomes. Additionally, a user can enlist the help of other researchers in the analysis of their sample sets without identifying to whom those genomes belong.

For ce, a user can identify subjects having a ic genotype and/or phenotype, such as stage 3 breast , and/or having been treated with a particular drug. A cohort can be formed to see how these drugs affect ous cell growth on a genetic level.

Therefore, these characteristics, amongst , 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 manner, a given researcher need not be responsible for identifying and securing 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 identify the appropriate ion criteria for subjects, the genomic test(s) to be run, and the parameters by which the test is to be run. chers B and C, located remotely from cher A, may then sign up for the cohort, fy 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 analyzed using the same genetic analysis system employing the same parameters, the results data will be the same regardless of where and on what machine the test(s) are run.

Consequently, 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 suitably configured commerce, e.g., monetary exchange, program.

Anyone that s participation in the cohort can then ad the criteria and data file(s) and/or use genetic data of subjects they have already generated and/or stored in performing the ted 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 ion criteria. Specifically, this stored genetic and/or other data of the subject may be scanned so as to determine ility 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 ne analyses and used for inclusion within the .

] Accordingly, in s 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 genomes from various subjects, such as with respect to mapping, aligning, variant calling, and/or the like. Therefore, instead of having to gather subjects and collect sample sets individually, the cohort initiator can advertise the need for a prescribed 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 ing 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 workflow folder, and can thereby te 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 ry es.

In various embodiments, the cohort structured analyses may be directed to primary sing, e.g., of either DNA or RNA, such as with t 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, c, epigenomic, metagenomic, genotyping, variants, and/or other forms of tertiary analyses. Additionally, it is to be understood that although 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 ce, in certain instances, an analysis platform may be exclusively ed to performing tertiary analysis, such as on genetic data, or other forms of genomics and/or bioinformatics analyses.

For e, 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, variant calling, and the like, so as to produce results data that may WO 14320 PCT/0S2017/036424 be subjected to one or more other secondary and/or tertiary analyses ures, 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 mapping, aligning, sorting, variant calling, and/or others, but an ant parameter is that the workflow should be identical for each person that takes part of the cohort. Particularly, a unique e of the system is that the requester establishing the cohort sets forth the control parameters so as to ensure that the is to be performed are performed in the same , regardless 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 ted analysis on the data, based on the settings of the configuration file. Researches may sign up to be selected as a cohort participant, and if subscription is great a lottery or competition can be held to select the participants. In various instances, a g system could be initiated. The results data generated by the cohort participants 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 ance with the prescribed configuration, a device associated with the system will perform the requested 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 genetic 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 ssed 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 s are able as they do not set forth the identity of the subject. However, if the analyses need to be performed online, e.g., in the cloud, selected BCL and/or FASTQ files may be uploaded, the analyses med, and the results data may then be pushed back to the initial submitter, who can then upload the results data at the cohort interface. The original raw data may then be deleted from the online memory. 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 is" (JIT), is particularly useful in enhancing cohort efficiency. For instance, using typical procedures, the movement of data into and out ofthe cohort system is very expensive. Accordingly, gh in various configurations, raw and/or uncompressed data uploaded to the system may be stored there, in particular 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 , such as where the compression is effectuated in accordance with a JIT protocol. In this instance, storage ofsuch data, such as in a compressed form is less expensive, and therefore the cohort system is very cost efficient.

Additionally, in various instances, a plurality of cohorts may be ed within an online marketplace, and given the compression processes herein described, data may be transmitted from one cohort to another, so as to allow ches ofvarious different cohorts to share data between them, which without the compression methods disclosed herein could be prohibitively 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 instances, the system 1 may be configured for subjecting the generated and/or arily processed data to further processing, e.g., via a local 100 and/or a remote 300 computing resource, such as by running it through one or more tertiary processing pipelines, such as one or more of a micro-array analysis pipeline, a genome, e.g., whole genome analysis pipeline, genotyping analysis pipeline, exome analysis pipeline, epigenome is pipeline, metagenome analysis pipeline, microbiome is pipeline, genotyping analysis pipeline, ing joint genotyping, variants analyses pipeline, including structural variants nes, c variants pipelines, and GATK and/or MuTect2 pipelines, as well as RNA sequencing pipelines, and/or other tertiary processing pipeline. The results data from such processing may then be compressed and/or stored ly 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 locally, 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 nment 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 r 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 ined desires and protocols, 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 together, e.g., regardless of location and/or how connected, such as by being in a tightly coupled configuration and/or a seamless cloud based ng, 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 interfaces dispersed throughout the system, the employment of which allows the various users to configure the various components to run the s 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 ular component of the system 1, wherein the header describes the functionality and determines how to call a on, such as the parameters that are passed, the inputs received and outputs transmitted, and the manner in which this occurs, what comes in and how, what goes out and how, and what gets ed, and in what manner.

For example, in s 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 corresponding APis which communicate with one or more of the s drivers of the system, 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 ter 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 configured to run in corresponding fashion, such as at the kernel level ofeach component and the system 1 as a whole. In such an ce, one or more of the ed kernel's may have their own very low level, basic API that es access to the hardware and functions of the various components of the system 1 so as to access applicable registers and modules so as to configure and direct the processes and the s 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 s back, encodes, encrypts, and/or transmits the relevant data and further 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 s may be transmitted throughout the system either locally or globally. Additional APis may be configured and included as set forth in more detail above with respect to the secure storage ofdata.

Such configuring of the vanous APis, memones, and/or re of the system may include communicating with registers and also performing function calls. For e, as described herein above, one or more function calls ary 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 te the necessary processes and perform the required steps.

Specifically, because in certain embodiments one or more of these operations may be based on one or more structures, the various structures needed for enting these operations may need to be constructed. There will therefore be a function call that ms 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 contain 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 hardware 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 lines 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 system.

Additionally, the system may be configured to allow various components of the system to communicate with one another, such as to allow one or more chips of the system 1 to interface 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 ed therewith, so as to receive the FASTQ and/or other generated and/or processed genetic sequencing files directly from the sequencer or other sing component such as immediately once they have been generated and/or processed and then transfers that information to the configuration manager which then directs that information to the riate memory banks in the hardware and/or software that makes that information available to the pertinent s ofthe hardware, software, and/or system as a whole so that they can perform their designated functions on that ation 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 wherein the CLI may allow the user to call one or more of these ons directly.

In various ments, the CLI may be a software application, 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 accepts 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 ical 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 nes, which tiers, of which platforms will be used and the parameters of their use. For example, in operation, if instructed, the CLI will locate the reference, will determine if a hash table and/or index needs to be ted, 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 s at the GUI that the user can select the associated chip(s)/system 1 to perform.

Furthermore, a library may be ed wherein the y may include sting , le, 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 analyses, such as personal medical histories and ancestry analysis, 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 parameters 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. ingly, 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 ations 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 ancillary information necessary for running the system 1. Hence, the y may be ured for ing software for g 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 performed remotely, such as where the system component is adapted to run one or more comparative 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 conclusions about the data and/or determine vanous relationships with t 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 biomarkers 314. As indicated, one or more of these functions may be ed by the WMS, and/or performed by the A/I module disclosed herein.

Additionally, where a collaborative protocol is performed, the system 1 may be configured for ing an onic forum where data g 321 may occur, which data sharing ol may include 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 ces, the system 1 may be configured so as to allow a 3rd party analyzer 121 to run virtual simulations on the data. Further, one generated, the interpreted data and/or the data subjected to one or more collaborative analyses may be stored either remotely 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 secondary 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 genomic and/or bioinformatics generation and/or processing and/or analysis.

In such an ce, the genomic infrastructure may include a bioinformatics processing platform having one or more memories that are configured to store one or more configurable processing ures 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 e 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 genetic reference sequences and/or a list of splice ons pertaining thereto. The system may also e an input having a platform application programming interface (API) for selecting from a list of options one or more ofthe configurable sing structures, 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 sed genomic sequences of interest. A graphical user interface (GUI) may also be present, such as operably 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 ss 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 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 prophylactic discovery and/or development needs of the user. Particularly, the hybrid cloud 50 and/or the various components of the system 1 may be ly connected with compatible and/or corresponding API interfaces that are adapted to allow a user to remotely ure 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 ation 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 described above, the system may be implemented on one or more integrated circuits that may be formed ofone or more sets ofconfigurable, e.g., preconfigured and/or hardwired, digital logic circuits that may be interconnected by a plurality of physical electrical interconnects. In such an ce, the ated t may have an input, such as a memory ace, for receiving one or a plurality of the configurable structure protocols, e.g., from the memory, and may further be adapted for implementing the one or more ures on the integrated circuit in accordance 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 index(es).

In s ces, the digital logic circuits may be arranged as a set of processing engines that are each formed of a subset of the digital logic ts. 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 integrated 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 plurality of genomic processing pipelines, such as where each pipeline may have an input that is defined according to the platform application programming interface so as to receive the result data from the primary and/or secondary processing by the bioinformatics sing platform, and for performing one or more analytic processes thereon so as to produce result data. onally, the plurality of genomic processing pipelines may have a common pipeline API that defines a secondary and/or tertiary sing operation to be run on the result data from the primary and/or secondary 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 sing operations and to output result data ofthe secondary and/or tertiary processing according to the pipeline APL In such instances, 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 plurality of c analysis applications are accessible via an electronic medium by a computer such as for execution by a computer processor, so as to m a targeted analysis of the genomic pre- or post processed data from the result data of the primary, secondary, and/or tertiary processing, such as by one or more of the ity of genomic processing pipelines. In particular instances, each of the ity of genomic analysis ations may be defined by the API and may be configured for receiving the result data of the primary, 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 onally include, selecting, e.g., from the menu of the GUI, one or more genomic processing pipelines from a ity 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, secondary, and/or tertiary sing.

Additionally, in various ments, all of mapping, aligning, and sorting, and variant calling may take place on the chip, and local realignment, duplicate marking, base y score recalibration 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, tertiary, and/or other processing platform, such as including an interpretive and/or collaborative 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 on 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 modules disclosed herein can be implemented as either re, e.g., on the chip, or re, e.g., on the cloud, but in certain embodiments, all of the modules 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 s wherein some are positioned on one or more chips and some are positioned on the cloud. Further, as indicated, in various embodiments, the chip(s) itself may be configured so as to function in conjunction with, and in some embodiments, 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 disclosure may be a chip, such as a chip that is configured for processing cs data, such as by employing a pipeline of data analysis s. Accordingly, as can be seen with respect to , a genomics pipeline processor chip 100 is provided along with ated hardware of a genomics ne processor system 10. The chip 100 has one or more connections to external memory 102 (at "DDR3 Mem ller"), and a connection 104 (e.g., PCie or QPI Interface) to the outside world, such as a host computer 1000, for example. A crossbar 108 (e.g., switch) provides access to the memory interfaces to s tors. 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 ller 112 controls chip ions, especially coordinating the efforts of multiple processing 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 cluster 11/114 share one crossbar port, via an arbiter 115. The central controller 112 has tions to each ofthe engine clusters. Each engine cluster 11/114 has a number ofprocessing engines 13 for processing genomic data, including a mapper 120 (or mapping ), an aligner 122 (or aligning module), and a sorter 124 (or sorting module), one or more processing engines for the performance of other functions, such as variant calling, may also be provided. Hence, an engine cluster 11/114 can include other engines or modules, such as a variant caller module, as well.

In ance 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 central controller 112, which load-balances the data to the processing s 13. The sing engines return processed data to the central ller 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, communication with the host CPU may be through a vely loose or tight coupling, such as a low y, 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 mapping/aligning/variant calling in a hybrid software/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 cer, DSP al signal processor), CPU and/or GPU or U hybrid, accelerated integrated circuit, e.g., FPGA, ASIC (on network card), as well as other Smart Network Accelerators 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 ication 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 between the different platforms and/or with one or more associated accelerators, e.g., such as a hardwired FPGA ented accelerator, so as to enable ent geneous computing, and thereby icantly improve the computing ency of the system, which in various instances may be configured as a based server system. Hence, in certain instances, a high bandwidth, low latency, cache coherent interconnect protocol, such as a QPI, Coherent Processor Accelerator Interface (CAPI), /GPU, or other suitable interconnect ol may be employed so as to expedite various data transmissions between the various components 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 irrespective e the various data to be processed resides in the system. And, where such data is retained within an associated memory device, such as a RAM or DRAM, the transmission activities may further involve expedited and coherent search and in-memory se sing.

Particularly, in particular embodiments, such heterogeneous ing may involve a multiplicity of sing and/or acceleration 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 componentry within the system to address, read, and/or write to one or more associated memories in a single, consistent, and coherent manner. More ularly, a CCVI interconnect may be employed so as to connect s ofthe devices of the system, such as the CPU and/or GPU or CPU/GPU hybrid, FPGA, and/or ated memories, etc. one with the other, such as in a high bandwidth manner that is configured to increase transfer rates between the various components while cing 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 include QPI, CAPI, NVLink, or other interconnect that may be ured 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 alternative 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 commands to the processing engines 13, which ct the sing engines as to what data to process. Because of the tight coupling, the processing engines 13 access input data directly from the external memory 1014 or a cache associated therewith, process it, and write results back to the external memory 1014, such as over the tightly d interconnect 3, reporting status to the central controller 112. The l 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 itselfvia the DMA engines 110. rates a genomics pipeline processor and system 20, showing a full complement of processing engines 13 inside an engine cluster 11/214. The pipeline 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 processing engines 13 or sing engine types can include, without limitation, a mapper, an aligner, a sorter, a local realigner, a base quality recalibrater, a duplicate marker, a variant caller, a ssor and/or a decompressor. In some implementations, each engine cluster 11/214 has one of each sing engine type. Accordingly, all sing 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 . Processing engine types that require massive parallel processing or memory bandwidth, such as the mapper (and ed 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 satisfy their performance requirements or the performance requirements of the ne processor system 20. rates a genomics pipeline processor 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 variant calling, such as the building of a DBG or the performance ofan HMM. Such processing is accelerated 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 ened programmability. illustrates a processing flow for a genomics pipeline processor system and method. In some red implementations, there are three passes over the data. The first pass includes mapping 402 and alignment 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 , 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 bration (BQSR) 412, SAM output 414, reduced BAM output 416, and/or CRAM ssion 418). The steps and functions of the third pass may be done in any combination or subcombination, and in any order, in a single pass.

Hence, in this manner data is passed relatively seamlessly 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 ecture, such as described above, is used to stream reads from the host into circular buffers in memory, through one processing engine after another in sequence, and back out to the host. In some implementations, CRAM decompression can be a separate streaming 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 decompression.

In various instances, a hardware implementation of a sequence analysis 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 includes a number ofblocks or modules to do sequence analysis. The input to the hardware realization can be a FASTQ file, but is not d 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 modules may include the following blocks: Error Control, Mapping, Alignment, Sorting, Local Realignment, Duplicate g, Base Quality Recalibration, BAM and Side Information reduction and/or variant These blocks or s can be present inside, or ented by, the hardware, but some of these blocks may be omitted or other blocks added to achieve the purpose izing a sequence analysis ne. Blocks 2 and 3 describe two alternatives of the sequence analysis pipeline platform. The sequence analysis pipeline platform comprising WO 14320 PCT/0S2017/036424 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 e. Blocks 4-7 describe different interfaces that the sequence analysis pipeline can have. In Blocks 4 and 6 the ace can be a PCie and/or PI/CCVI/NVLink interface, but is not d 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 cer. Surrounding the hardware FPGA or ASIC or structured ASIC are a plurality of DDR3 memory elements and a PI/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 sequence analysis pipeline API that can be accessed by third party applications to perform tertiary analysis.

] FIGS. 50A and 50B depict an ion 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 transferring of data, such as data to be processed and result data, back and forth from the CPU/GPU to the FPGA 100.

B depicts the ion card ofA having a plurality, e.g., 3, slots containing a plurality, e.g., 3, processing chips ofthe disclosure.

Specifically, as depicted in FIGS. 50A and 50B, in various embodiments, an apparatus of the disclosure may include a computing architecture, such as embedded in a n field gate programmable array (FPGA) or application specific ated circuit (ASIC) 100. The FPGA 100 can be integrated into a printed circuit board (PCB) 104, such as a Peripheral Component ace - 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 surrounded by local memories 105, however, in various embodiments, as ed 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 various instances, the overall work flow of c sequencing involving the FPGA may include the following: Sample preparation, Alignment ding mapping and alignment), Variant is, ical Interpretation, and/or Specific Applications.

Hence, in various embodiments, an apparatus of the disclosure may include a ing architecture that achieves the high performance execution of thms, 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 e one genome to libraries of others, do gene function analysis, etc.

There are two other typical architectures 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 ecture, n instructions and data are fetched from a level 1 cache (LI cache) that is small but has ely 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 connected to DRAM. An advantage of this arrangement may be that applications in which programs 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 ing 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 related to the various operations described herein, such as via a high bandwidth, low latency onnect such as a QPI, CCVI, CAPI so as to further enhance performance as well as the speed and coherency of the data transferred throughout the system. In such an ce, cache coherency 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 numbers of small CPUs, each with their own small LI cache, wherein each CPU executes the same instructions on different s ofthe data. This is a so called SIMD (Single Instruction stream, Multiple 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 k.

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 rence. That leads to a high cache miss rate and processors that are idle while waiting for data to arrive from the external DRAM.

For instance, in s 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 ence 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 require 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 ement is that the charge decays over time, so it has to be refreshed periodically. The need to do this has led to architectures that organize the memory into independent blocks and access mechanisms that deliver multiple words of memory per request. This compensates 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 oriented rather than block ed. DRAMs work well in a caching architecture e 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 expansions cards of , such as for bioinformatics processing 10. The system includes a Bio IT processing chip 100 that is ured for performing one or more functions in a processing ne, such as base calling, error correction, mapping, ent, sorting, assembly, variant calling, and the like as described herein.

The system 500 further includes a configuration manager that is adapted for configuring the onboard functioning of the one or more processors 100. Specifically, in various embodiments, the configuration manager is adapted to icate ctions 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 uration manager may be adapted to configure the internal processing clusters 11 and/or engines 13 associated therewith so as to perform one or more desired operations, such as mapping, aligning, sorting, variant calling, and the like, in accordance with the ctions received. In such a manner only the clusters 11 containing the processing engines 13 for performing the requested processing operations on the data provided from the host system 1000 to the chip 100 may be d to process the data in accordance with the received ctions. onally, in various embodiments, the configuration manager may further be adapted so as to itselfbe adapted, e.g., ly, by a third party user, such as over an API tion, as described in greater detail herein above, such as by a user ace (GUI) presented by an App of the system 500. Additionally, the configuration manager 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 mapped, aligned, and/or sorted genomes or portions thereof. In s instances, the database may further include 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 ation generated from the Bio IT processor, e.g., results data, may be ted and stored as an electronic health , 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 information stored therein, so as to perform one or more statistical analyses on the data, such as to determine diseased states or trends or for the purposes of analyzing the effectiveness of one or more prophylactics or ents ning thereto. Such information along with the EMR data may then be further processed and/or stored in a further se 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 r 502 that includes a computing system. The computing system of the configuration manager 502 can e a personal computer or other computer workstation, or can be ented by a suite ofnetworked computers. The configuration 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 configuration for processing genomics data from a sequencer or other genomics data . The configuration manager 502 further es 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 ated 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 computers and servers. A system can include one or more sequencers ted (e.g. via Ethernet) to a server containing the chip, where c reads are generated by the multiple sequencers, transmitted 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 sequencing technology.

This primary analysis stage functions to translate al signals detected inside the sequencer into " of nucleotide sequences with associated quality (confidence) , e.g. FASTQ format files, or other formats containing sequence and usually quality information. Primary analysis, as mentioned above, is often quite ic in nature to the sequencing technology employed. In various sequencers, nucleotides are detected by sensing changes in fluorescence and/or ical charges, electrical currents, or radiated light. Some primary analysis pipelines often include: Signal processing to amplify, filter, separate, and measure sensor output; Data reduction, such as by quantization, decimation, averaging, transformation, etc.; Image processing or numerical processing to identify and enhance meaningful signals, and associate them with specific reads and nucleotides (e.g. image offset calculation, cluster identification); Algorithmic sing and tics to compensate for sequencing technology artifacts (e.g. phasing estimates, cross-talk matrices); Bayesian probability calculations; Hidden Markov models; Base calling (selecting the most likely nucleotide at each position in the sequence); Base call quality dence) estimation, and the like. As discussed herein above, one or more of these steps may be tted 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 , to determine the content of the sequenced sample DNA (or RNA etc.), such as by mapping and aligning reads to a reference genome, sorting, duplicate marking, base quality score recalibration, local re-alignment, and variant calling. Tertiary analysis may then follow, to extract medical or research implications from the determined DNA content.

Accordingly, given the sequential nature of the above processing ons, 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 cial because sequencers produce data that typically es both primary and secondary analysis so as to be useful and may further be used in s tertiary processing protocols, and integrating them in a single device is most efficient in terms of cost, space, power, and resource sharing. Hence, in one ular aspect, the disclosure is directed to a , 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 ity of reads of genomic data, where each of the plurality of reads of genomic data e a ce ofnucleotides. The system may include one or more of a memory, such as a memory storing one or more genetic reference sequences and/or an index of the one or more genetic nce sequences; and/or the system may include a chip, such as an ASIC, FPGA, or sASIC.

] One or more aspects or features of the subject matter described herein can be realized in digital onic circuitry, integrated circuitry, lly designed application ic integrated circuits (ASICs), field programmable gate arrays ), or structured ASIC computer hardware, firmware, software, and/or combinations thereof.

These various aspects or features can include implementation in one or more computer ms that are executable and/or interpretable on a programmable system including at least one programmable processor, which can be l or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input , and at least one output device. The programmable system or ing 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 ms, software, software applications, applications, components, or code, include machine instructions for a mmable processor, and can be implemented in a high-level procedural and/or object-oriented programming ge, and/or in assembly/machine language. As used herein, the term "machine-readable medium" refers to any computer program product, tus and/or device, such as for example magnetic discs, optical disks, memory, and mmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term "machine-readable signal" refers to any signal used to provide machine instructions and/or data to a programmable processor. The machine-readable medium can store such machine instructions nsitorily , such as for example as would a non-transient solid-state memory or a magnetic hard drive or any equivalent storage medium. The machine-readable medium can alternatively or additionally store such machine instructions 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 immense growth in data production and acquisition in the 21 st Century, a need has developed for sed processing power that is capable of handling the rowing 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 rms. However, although supercomputing is useful, it has proven to be insufficient for some of the very complex computing problems many of today's logy companies face. Particularly, since the sequencing of the human genome, the logical advancement in the ical arts has been exponential. Nevertheless, in view ofthe high rate and increased complexity of the raw data produced every day, there has evolved 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 approach to solving the worlds most nging computational needs.

Quantum computing has been known smce 1982. For instance, in the International Journal of Theoretical Physics, Richard n zed a system for performing quantum ing. 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 performed 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 avings in complex computations.

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 s, but can also be adapted for running specialized quantum algorithms. More ularly, complex ms can be modeled in the form of an equation, such as a onian, which may be represented in the quantum system in a manner that the behavior of the system provides information regarding the solution to the equation. 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, solving a model for the or of the m system may be configured so as to involve solving a differential equation related to the wave-mechanical description of a particle, e.g., Hamiltonian, of the quantum system.

In essence, 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 digital logic ers. 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 digital logic t an "on/off' state of a transistor serves as a basic unit of information, e.g., a bit. Particularly, a common digital er sor 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 information device that uses superpositions of entangled states, called quantum bits or qubits, to encode data.

The basis for performing such quantum computations 1s an information device, e.g., a unit, which forms the quantum bit. The qubit is analogous to the digital "bit" in ional l 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 m computation on both states simultaneously. In general, N qubits can be in a superposition of 2N states. Quantum algorithms, therefore, can make use of this superposition ty to speed up certain computations.

A qubit, therefore, is analogous to a bit in a ional l computer, and is a type of information device that exhibits coherence. Particularly, a m computing device is built up from a plurality 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, together in a controllable manner.

In such an instance, the m state of one information device affects the quantum state of each ofthe other information s 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 quantum-mechanical system. Specifically, for the qubit, the two quantum states involve entanglement, such as involving 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 cal bit is that multiple qubits t m 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., quantum bits, must fulfill several requirements. 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 performing computations. Second, the information devices must be capable of producing quantum effects like entanglement and superposition. Additionally, in certain instances, the information device may be configured for g information, e.g., m information, such as in a coherent form. In such instances, the coherent device may have a quantum state that persists without significant degradation for a long period of time, such as on the order ofmicroseconds or more. ularly, quantum lement is the physical 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 ndently of r, despite the space that separates 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 instances, the measurements of various physical properties, such as position, momentum, spin, and/or polarization, performed on entangled particles are ated. 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 ise 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 lement.

Hence, one particle of an entangled pair simply "knows" 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 classical bits that can only have one value at a time, entanglement allows multiple states to be acted on simultaneously. It is these unique entangled relationships 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 ormations 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, quantum dots, and the like. Specifically, a quantum gate is the basis for a quantum t 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 quantum circuits, like classical logic gates are for conventional l circuits. Specifically, a quantum circuit is a model for m ation where the computation is a sequence of quantum gates that are reversible transformations on a quantum ical analog of an n-bit register. Such analogous structures are referred to as n-qubit registers. Hence, unlike cal 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 transistors that act as electronic switches for performing logical ions, 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 Boolean logic, and therefore, all of the algorithms and mathematics that can be described with Boolean logic can be med 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 usually 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 specific quantum state is found by multiplying the vector that represents the state by the matrix representing the gate. Hence, given this uration quantum ational operations may be executed on a very small number of quantum bits. For instance, there are quantum algorithms that are configured for running much more complex computations faster than any possible probabilistic classical thm. Particularly, a quantum algorithm is an thm that runs on a quantum circuit model ofcomputation.

Where a classical algorithm is a finite sequence of y-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 computer. However, even though quantum algorithms exist, such as Shor's, Grovar's, and Simon's thms, all classical thms 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. Quantum 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 represented by either a "1" or a"O"; a m computer supports a sequence of qubits where a single qubit can represent a one, a zero, or any m superposition ofthose two qubit states. Consequently, a pair of qubits can be in any quantum superposition of 4 states, and three qubits can be in any superposition of 8 states. In general, a quantum computer with n qubits can be in an arbitrary superposition of up to 2n different states aneously, which compares to a normal er that can only be in one of these 2n states at any one time. Therefore, qubits can hold exponentially more information than their classical counterparts. In , a quantum computer 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 quantum algorithms. The calculation ends with a ement, 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 quantum 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. r, 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 ility 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 d from its ability to exist in a coherent superposition of basis states, and for the state of the qubit to have a phase.

A qubit will retain this y to exist as a coherent superposition ofbasis states as long as the qubit is iently isolated from sources ofdecoherence.

Consequently, to complete a ation using a qubit, the state of the qubit is measured. As indicated above, when a ement of the qubit is done, the quantum nature of the qubit may be temporarily 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 collapsed 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 several 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 m computing. t 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. Accordingly, the circuit model for quantum ing 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. Likewise, a computer processor may take the form of a quantum processor such as a onducting 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.

Accordingly, although in various embodiments, a computer processor may be configured as a aditional superconducting processor, in other embodiments, it the computer processor may be configured as a superconducting processor. For ce, 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 quantum tunneling, but may rather operate by emphasizing different principles, such as those principles that govern the ion of classical computer processors. In other embodiments, the computer processor may be ured as a traditional superconducting sor such as by being adapted to WO 14320 PCT/0S2017/036424 process through various quantum effects, such as superposition, entanglement, and/or quantum tunneling.

Accordingly, in various instances, there may be certain advantages to the implementation ofsuch superconducting sors. Particularly, due to their natural physical properties, superconducting processors in general may be capable ofhigher switching speeds and shorter computation times than non-superconducting processors, and therefore it may be more practical to solve n problems on superconducting processors. Further, detail and embodiments of ary quantum 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 various divisionals, continuations, and/or continuation 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 g complex computational ms are also presented. For instance, the quantum devices and systems herein disclosed may be employed for controlling the m state of one or more information devices and/or systems, in a coherent manner, so as to m 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, aligning, sorting, variant calling, and/or other genomics and/or bioinformatics pipeline. In particular embodiments, the one or more operations may include performing a burrowwheelers , smith-waterman, and/or an HMM operation.

Particularly, solving complex genomics 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 entation of the ational problem, encoding the logic circuit representation as a discrete optimization problem, and solving the discrete zation problem using the m sor. 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 ms, where each miniature optimization problem encodes a tive logic gate from the logic circuit entation. For instance, a mathematical circuit may employ binary entations of factors, and these binary representations may be osed to reduce the total number of variables required to represent the mathematical circuit. Accordingly, in accordance with the teachings herein, a computer sor may take the form of a digital and/or an analog processor, for instance, a quantum processor such as a superconducting quantum sor. A superconducting quantum processor may include a number of qubits and associated local bias devices, for ce two or more onducting qubits, which may be formed into one or more m logic circuit representations.

More particularly, in various embodiments, a superconducting integrated circuit may be provided. Specifically, in particular embodiments, such a superconducting integrated circuit may e 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 current 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 ed, 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 current 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 connection may be positioned between the first superconducting current 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 ed and positioned between the first onducting current path and the third superconducting t path, such as where the second superconducting connection may extend through both the first dielectric layer and the second dielectric layer. Additionally, at least a portion of the second superconducting current path may be encircled by an outer superconducting current path that may be formed by at least a portion of one or more of the first superconducting t path, at least a portion ofthe second superconducting current path, and/or the first and second superconducting connections. ingly, in such instances, the second WO 14320 PCT/0S2017/036424 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 thickness 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. r, in various ments, 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 mmable . In other embodiments, the second superconducting current path may be a portion of a superconducting mmable 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 sing device.

Accordingly, provided herein are devices, systems, and methods for solving computational problems, especially problems d to resolving the genomics and/or bioinformatics bottleneck described herein above. In various ments, these devices, systems and methods introduce a que whereby a logic circuit representation of a computational problem may be solved directly and/or may be encoded as a discrete optimization problem, and the discrete optimization 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 bed herein may be implemented using any form of computer processor such as including traditional logic circuits and/or logic t entations, 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 optimization problems and as such may be particularly well-suited to be solved using the quantum processors, sed herein. In other instances, such computations may be resolved more generally by a computer processor that harnesses m effects to achieve such computation; and/or in other instances, such computations may be performed using a dedicated integrated t, such as an FPGA, ASIC, or ured ASIC, as described herein in detail. In some embodiments, the discrete optimization problem is cast as a problem by configuring the logic circuits, qubits, and/or rs in a quantum processor. In some embodiments, the quantum processor may be specifically adapted to facilitate g such discrete optimization problems.

As disclosed throughout this specification and the ed 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 l outputs, and a set oflogic gates (e.g., NAND gates, XOR gates, and the like) that transform the logical inputs to the logical outputs h a set of ediate logical inputs and intermediate logical outputs. A complete logic circuit may include a representation of the input(s) to the computational problem, a entation ofthe output(s) ofthe ational problem, and a representation ofthe sequence of intermediate steps in between the input(s) and the output(s).

] Thus, for various purposes of the present devices, systems, and methods, the computational problem may be defined by its input(s), its output(s), and the intermediate steps that transform the input(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 m as a discrete optimization problem, and the subsequent 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 layer coupling (e.g., coupling between the qubits of different layers) and intra-layer coupling (e.g., coupling between the qubits within a particular layer).

Accordingly, as indicated, in some embodiments, the structure of a logic circuit may be fied into layers. For example, the logical input(s) may represent a first layer, each sequential l (or arithmetic) operation may ent 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 ure of a logic circuit 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 (s) representing another layer), each layer may be embodied by a respective set of qubits in the quantum and/or superconducting processor. For example, in one embodiment of a quantum processor, one or more, e.g., each, row of qubits may be programmed to represent a respective layer of a quantum logic circuit. That is, particular qubits may be mmed to represent the inputs to a logic circuit, other qubits may be programmed to represent a first l operation ted by either one or a ity of logic gates), and r 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 representing various layers of the problem, it can be advantageous to enable independent c control of each respective set. Further, in various embodiments, s serial logic circuits may be mapped to the quantum processor, and the respective qubits mapped to facilitate the functional 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 problem.

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 problem, 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 circuit representation of the computational m 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 sed herein, the logic circuit representation of the computational problem may be encoded as a te optimization problem, or a set of optimization objectives, and in various embodiments, such as where a classical digital er processing gm is configured to solve the problem, the system may be ured 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 solution to the original computational problem.

Further, in other embodiments, the discrete zation problem may be solved using a computer processor, such as a quantum processor. In such an ce, solving the discrete optimization m may then involve, for e, evolving the m 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 discrete optimization problem may e three acts. First, the discrete optimization problem may be mapped to a computer processor. In some embodiments, the computer processor may e a quantum and/or super ting processor and g the discrete optimization problem to the computer sor may include programming the elements (e.g., qubits and couplers) of the quantum and/or superconducting sor.

Mapping the discrete optimization problem to the computer processor may include the discrete optimization problem in at least one computer or processor-readable storage , such as a computer-readable non-transitory storage medium or memory (e.g., volatile or nonvolatile Accordingly, in view of the above, in various instances, a device, system, and method for executing a sequence analysis pipeline, such as on genomics material, is provided.

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 nce 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 ments, the device may be a quantum computing device, such as formed of a set of quantum logic circuits, e.g., hardwired quantum logic circuits, for instance, where the logic circuits are interconnected with one another. 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 represented 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 engines, 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 sequence analysis pipeline on the reads of genomic data.

For ce, the set of processing s may be configured so as to include an image processing, base g, mapping, aligning, sorting, variant calling, and/or other genomics and/or bioinformatics processing module. For example, in various embodiments, a mapping , such as in a first hardwired configuration, may be included. Additionally, in r embodiments, an alignment module, such as in a second hardwired configuration, may be included. Further, a sorting module, such as in a third hardwired configuration, may be included. And, in additional embodiments, a t calling module, such as in a fourth hardwired configuration, may be included. Further still, in various ments, an image processing 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 m computing device and/or system may include a mapping module, where the mapping module comprises a set of quantum logic circuits that are arranged as a set ofprocessing engines, one or more h are configured for performing one or more steps ofa g procedure. For instance, one or more quantum processing engines may be configured to receive a read of c data, such as via one or more of a plurality of superconducting connections. Further, the one or more m processing engines may be configured 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 address in the index in the memory, so as to receive a record from the address, such as where the record represents on information in the genetic reference sequence. Further more, the one or more quantum processing engines may be configured to determine, 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 sing engines configured for ating 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 sequence of nucleotides. The sing 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 information in the genetic reference sequence. The processing engine may further be configured for determining, based on the position information, the one or more ng 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 ses a set of quantum logic circuits that are arranged as a set of processing engines, one or more of which are ured for performing one or more steps of an alignment procedure. For instance, one or more quantum processing engines may be configured to receive a plurality of mapped positions for the read from the , 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 t ofthe genetic reference sequence so as to generate a score for each alignment. Further, once one or more scores have been generated at least one best-scoring alignment of the read may be selected. In ular instances, the m ing device may include a set of quantum logic circuits that are arranged as a set of sing engines that are ured for performing a gapped or gapless alignment, such as a Smith Waterman alignment.

Further, in certain instances, a quantum computing device and/or system may include a variant calling module, where the t calling module comprises a set of quantum logic circuits that are arranged as a set of processing engines, one or more ofwhich are configured for performing one or more steps of a variant calling procedure. For instance, the quantum computing variant g 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 memory, where each read of c data and each candidate ype 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 receive the one or more candidate haplotypes, 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 candidate haplotypes, so as to determine a probability of each candidate haplotype representing a correct variant call. Additionally, one or more of the quantum processing engines may be configured to te an output based on the determined ility.

Additionally, in various instances, the set of quantum logic ts may be formed as one or more quantum sing engines that are configured to determine a probability of observing each read of the plurality of reads based on at least one candidate haplotype being a true sequence of nucleotides, e.g., of a source organism of the plurality of reads. In particular 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 onal ments, the one or more quantum processing engines may be configured for g the plurality of reads into one or more contiguous nucleotide sequences, and/or for generating the one or more candidate haplotypes from the one or more contiguous nucleotide sequences. For instance, in various embodiments, the g of the plurality of reads includes the one or more quantum processing engines constructing a De Bruijn graph.

Accordingly, in light of the above, a system for performing vanous computations in solving problems related to genomics and/or bioinformatics processing is provided. For instance, 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 ce analysis pipeline. Particularly, the Next Gen Sequencer may be ured for sequencing a ity 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.

However, in various instances, such as where the automated cer and/or an associated server is not ured 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 sequence data, such as in image, BCL, and/or in FASTQ form, and may further be configured for performing a primary, e.g., image processing, and/or a ary and/or tertiary sing analysis, such as a ce analysis pipeline, on the received data. For instance, the cloud accessible server may be one or more servers ing a CPU and/or a GPU one or both h may be associated with an integrated circuit, such as an FPGA or ASIC, as herein bed. Particularly, in certain 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 is 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 performed by another processing platform, such as an associated, e.g., tightly coupled, integrated circuit, such as an FPGA or ASIC, that is specifically configured for performing s of the steps in the ce analysis pipeline.

In such instances, where data and the results of analysis are to be transferred from one platform to another, 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 quantum computing platform that is configured herein to perform one or more steps in the ce analysis pipeline, as described herein, and may e the performance of one or more secondary and/or tertiary processing steps in accordance with one or more of the methods disclosed herein.

Further, with respect to quantum computing, detail and embodiments of exemplary m processors and the methods of their use that may be employed in conjunction with the t 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 orated by reference in their entireties.

Additionally, with respect to the artificial intelligence module set forth above, in one aspect, a cloud accessible cial intelligence module is provided, and is configured for being communicably and operably coupled to one or more ofthe other components ofthe BioIT pipeline disclosed herein. For instance, the A/I module may work closely with the WMS so as to efficiently direct and/or l the various processes of the system disclosed herein. Accordingly, in various embodiments, 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 ce, in various instance, the BioIT system may be configured for ing al data. In such an ce, the workflow manager system may be ured for ing the al data, and other such data, and implementing one or more deterministic rule systems, 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 ecture.

These constructions may be represented by one or more table structures. A series of tables, for instance, may then be employed by which ations may be made by the WMS in an iterative fashion. For example, in various use models a first correlation may be made with respect 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 command. The key may be any common identifier, such as a name, a number, 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. r, in other instances, the A/I module may be configured to provide a more comprehensive analysis on generated and/or provided data. For e, the A/I module may be configured so as to implement one or more machine learning protocols on the data of the system that are devised to teach the AI module to make correlations between the genomic data, e.g., ted 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 ed 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 s, so as to be able to draw ations more rapidly and tely 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 predict the outcome for the second kind of input data. ically, based on historical evidence, the A/I module may be ured 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 outcomes 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 ined genomic profile. Particularly, in combining these two datasets, the A/I module is configured for determining the various interrelationships between them. Accordingly, in a first step, a graph database or knowledge graph may be constructed. For example, in this ce, the knowledge graph may be composed of three typical elements, which basically include a subject, a predicate, and an object, these may form nodes, and the relationship between the nodes must be determined.

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 onships that can be determined.

For instance, relationships may be determined based on their effects, e.g., they are effect based; or they may be determined based on inferences, e.g., relationships that are n but determinable.

Accordingly, with t 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 sequence 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 tions, one or more allergies, one or more other conditions or phenotypic traits, e.g., blood pressure, cholesterol, etc. onally, in between these nodes are the relationships that may be determined.

Specifically, when building the dge graph, clinical data input into the system, such as from a medical records facility, e.g., electronic medical records, family history of medical conditions, etc. that may be encrypted and ly transferred electronically. Likewise, genomic data from the subject may be sequenced and generated in accordance with the secondary processing steps set forth . r, once these two nodes have been established one or more third nodes may be input into the system, from the presence ofwhich the onship(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 population 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, condition and/or characteristic; an allergy; phical region; diet, a food item and/or ient; an environmental condition; a geographical condition; powerlines, cellular 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 ed for multiple subjects having the same sis and/or condition. Hence, in a manner such as this the ation between the clinical and genomics worlds may be determined.

Accordingly, a step in building a clinical genom1cs graph is to define the anchor nodes, these represent the two ng elements n 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 effects caused by and/or the characteristics of one node or the other. These may form the known and/or observable 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 ine causal and/or predictable es the various ent relationships may be weighted, such as based on the degree of certainty, number of commonalities, number of instances sharing the node, number of common relationships, 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 platforms of the global system may be coupled together, so as to seamlessly transfer data between its various components. For instance, as indicated, the mapping, aligning, and/or variant calling pipelines may be configured for itting its data, e.g., results data, to the artificial intelligence module. Particularly, the A/I module may be ured for receiving inputs of data from one or more of the secondary processing platform components, and/or one or more of the other component of the system. More particularly, the A/I module is configured for receiving mapping, d, and/or variant called data from the mapper, aligner, and/or variant g processing engines, and for taking that data and using it to generate one or more nodes within the knowledge graph. Further, as ted, 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, mental, 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.

Additionally, 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 ure, 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 suitably 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 ate a relevant question to be answered by the A/I module. ingly, the user may review and/or select the bounding nodes, and then allow the system to te an appropriate knowledge map employing the selected WO 14320 PCT/0S2017/036424 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 ian who desires to know how a certain drug dosage is affecting a patient with respect to a given disease.

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 ted to secondary processing, and the s 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 history 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 system, e.g., all of the ined chromosomal properties may be uploaded, for instance, as a constellation of nodes, which nodes may be used to determine s relationships pertinent to the t, such as by querying the system and allowing it to generate the appropriate connections 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 entered into the system, any relationships between them may be determined by the A/I module, such as with t to common genotypes, phenotypes, conditions, environments, geographies, allergies, ethnic-cultural ounds, medications, and the like.

Further, relationships between two or more characteristics in a subject, or between ts, 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 historic systolic and diastolic 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 onships between the two, such that if a given systolic input is d into the system, the predicted diastolic output may be given, taking the predictive weights between the two into t. It is to be noted that although the preceding example was given with t to blood pressure, within a single subject, the same will apply to any to given nodes that are in a atical relationship to one another, such as with respect to a multiplicity of subjects and/or a variety ofconditions. onally, 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 ol. For instance, the A/I system may be adapted so as to s information in a layered or multi-staged fashion, such as for the purpose of deep learning. Accordingly, the system may be configured to te 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 relationships.

The more stages of learning that are initiated within the system the better the ing between junctions will be, and the deeper the ng. 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 modulate 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 features of the subject matter described herein can be implemented on a computer having a display device, such as for example a cathode ray tube (CRT), a liquid crystal y (LCD) or a light emitting diode (LED) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer. Other kinds of devices 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, including, but not limited to, acoustic, speech, or tactile input. Other possible input devices include, but are not limited to, touch s or other touch-sensitive devices such as single or point ive or capacitive trackpads, voice recognition re and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.

WO 14320 PCT/0S2017/036424 The t matter described herein can be embodied in systems, apparatus, methods, and/or es depending on the desired configuration. The implementations set forth in the foregoing description do not ent all implementations consistent with the subject matter bed herein. Instead, they are merely some examples consistent with aspects related to the described subject matter. Although a few variations have been described in detail above, other modifications or additions are possible. In particular, further features and/or variations 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 es and/or combinations 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 s. Other implementations may be within the scope ofthe following claims.

Claims

1. A method for improving the accuracy of a variant call by jointly evaluating reads that map to two or more s of a reference sequence that are homologous, the method sing: accessing, by one or more computers, a joint-pileup of a plurality of sequence reads, wherein the joint-pileup includes a first pileup of reads that have been aligned to a first region of the reference sequence and at least a second pileup of reads that have been aligned to a second region of the reference sequence, wherein the first region and the second region are homologous with each other; determining, by the one or more computers, a set of ate variants from the jointpileup defining, by the one or more computers, an order of processing of the candidate variants; evaluating, by the one or more computers, each of the candidate variants from the set of candidate variants based on the defined processing order; and generating, by the one or more computers and based on the tion of the candidate ts, a t call file that identifies one or more of the candidate variants.

2. The method of claim 1, the method further comprising: obtaining multiple homologous regions of a reference sequence from one or more memory devices.

3. The method of claim 1, wherein determining a set of candidate variants using the ileup comprises: using a De Brujin graph to extract candidate variants from the joint pileup.

4. The method of claim 3, wherein nodes in the graph ent the list of candidates, and wherein using the De Brujin graph includes generating the De Brujin graph using each region of the reference sequence as a backbone and aligning each candidate variant positions to universal coordinates.

5. The method of claim 1, wherein defining, by the one or more computers, an order of processing of the candidate variants comprises: defining, by the one or more computers, an order of sing of the candidate variants as a function of read length or insert size.

6. The method of claim 5, n defining an order of processing of the candidate variants as a function of read length or insert size comprises: generating a connection matrix that defines the order of processing of the candidate variants as a function of read length and insert size.

7. The method of claim 1, wherein evaluating, by the one or more computers each of the candidate variants from the set of candidate variants based on the defined processing order comprises: for each candidate variant of the set of candidate variants: generating candidate joint ypes, calculating an a posteriori probability of each of the joint ypes, computing a genotype matrix, pruning the candidate joint diplotypes, and including a next active position as evidence for a current position.

8. A system for improving the accuracy of a variant call by jointly evaluating reads that map to two or more regions of a reference sequence that are gous, the system comprising: one or more ers and one or more storage devices storing ctions that are operable, when executed by one or more computers, to cause the one or more computers to perform the operations comprising: accessing, by one or more computers, a joint-pileup of a plurality of sequence reads, wherein the pileup includes a first pileup of reads that have been aligned to a first region of the reference sequence and at least a second pileup of reads that have been aligned to a second region of the reference sequence, wherein the first region and the second region are homologous with each other; determining, by the one or more computers, a set of candidate variants from the joint-pileup; defining, by the one or more computers, an order of processing of the candidate variants; ting, by the one or more computers, each of the candidate variants from the set of candidate variants based on the defined processing order; and generating, by the one or more computers and based on the evaluation of the candidate variants, a variant call file that identifies one or more of the candidate variants.

9. The system of claim 8, the operations r comprising: obtaining multiple homologous s of a reference ce from one or more memory s.

10. The system of claim 8, wherein determining a set of candidate variants using the jointpileup comprises: using a De Brujin graph to extract candidate variants from the joint pileup.

11. The system of claim 10, wherein nodes in the graph represent the list of candidates, and wherein using the De Brujin graph includes generating the De Brujin graph using each region of the reference sequence as a backbone and aligning each ate variant positions to sal coordinates.

12. The system of claim 8, wherein defining, by the one or more computers, an order of processing of the candidate variants comprises: defining, by the one or more computers, an order of processing of the candidate variants as a function of read length or insert size.

13. The system of claim 10, wherein defining an order of processing of the ate variants as a function of read length or insert size comprises: generating a tion matrix that defines the order of processing of the candidate variants as a function of read length and insert size.

14. The system of claim 8, n evaluating, by the one or more computers each of the candidate variants from the set of candidate variants based on the defined processing order comprises: for each candidate variant of the set of candidate variants: generating candidate joint diplotypes, calculating an a iori probability of each of the joint ypes, computing a genotype matrix, pruning the candidate joint diplotypes, and including a next active position as evidence for a current position.

15. A computer-readable storage device having stored n instructions, which, when executed by a data processing apparatus, cause the data processing apparatus to perform operations for improving the accuracy of a variant call by jointly ting reads that map to two or more regions of a reference ce that are homologous, the operations comprising: ing, by one or more computers, a joint-pileup of a plurality of sequence reads, wherein the joint-pileup includes a first pileup of reads that have been aligned to a first region of the reference sequence and at least a second pileup of reads that have been aligned to a second region of the reference sequence, wherein the first region and the second region are homologous with each other; determining, by the one or more ers, a set of candidate variants from the jointpileup defining, by the one or more computers, an order of processing of the candidate variants; evaluating, by the one or more computers, each of the candidate variants from the set of candidate variants based on the defined processing order; and generating, by the one or more ers and based on the evaluation of the candidate variants, a variant call file that identifies one or more of the candidate variants.

16. The computer-readable storage device of claim 15, the ions further comprising: obtaining multiple homologous regions of a reference sequence from one or more memory devices.

17. The computer-readable storage device of claim 15, wherein determining a set of candidate variants using the joint-pileup comprises: using a De Brujin graph to extract candidate variants from the joint pileup.

18. The computer-readable storage device of claim 17, wherein nodes in the graph represent the list of ates, and wherein using the De Brujin graph includes generating the De Brujin graph using each region of the reference sequence as a backbone and aligning each candidate variant ons to universal coordinates.

19. The er-readable storage device of claim 15, wherein defining, by the one or more ers, an order of processing of the ate variants comprises: generating a connection matrix that defines the order of processing of the candidate variants as a function of read length and insert size.

20. The computer-readable storage device of claim 15, wherein evaluating, by the one or more computers each of the candidate variants from the set of candidate variants based on the defined processing order comprises: for each candidate variant of the set of candidate variants: generating candidate joint diplotypes, calculating an a posteriori probability of each of the joint diplotypes, computing a genotype , pruning the ate joint diplotypes, and including a next active position as evidence for a current position. WO 14320 W0 14320 PCT/USZOl