WO2023122363A1 - Résumés dynamiques d'état graphique pour séquençage de nucléotides - Google Patents

Résumés dynamiques d'état graphique pour séquençage de nucléotides Download PDF

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Publication number
WO2023122363A1
WO2023122363A1 PCT/US2022/077422 US2022077422W WO2023122363A1 WO 2023122363 A1 WO2023122363 A1 WO 2023122363A1 US 2022077422 W US2022077422 W US 2022077422W WO 2023122363 A1 WO2023122363 A1 WO 2023122363A1
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Prior art keywords
sequencing
status
data
analysis
nucleotide
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PCT/US2022/077422
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English (en)
Inventor
Jeremy Ward
Rodger Constandse
Michael SMOOT
Michelle BEORIS
Adam French
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Illumina Software, Inc.
Illumina Cambridge Limited
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Priority claimed from US17/936,722 external-priority patent/US20230207063A1/en
Application filed by Illumina Software, Inc., Illumina Cambridge Limited filed Critical Illumina Software, Inc.
Priority to CN202280084191.7A priority Critical patent/CN118451509A/zh
Publication of WO2023122363A1 publication Critical patent/WO2023122363A1/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B45/00—ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
    • G16B50/30—Data warehousing; Computing architectures
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search

Definitions

  • sequencing machines that determine nucleotide-fragment reads for a sample genome (or other nucleic-acid polymer) and sequencing-data-analysis software that analyze the base calls for such nucleotide-fragment reads.
  • some existing sequencing machines include on-machine software that cause the sequencing machines to display interactive graphical options to start and stop sequencings runs. After sequencing runs finish, such on-machine software can likewise cause the sequencing machines to display options to view detailed sequencing metrics for respective sequencing runs.
  • existing sequencing-data-analysis software In addition to on-machine software, existing sequencing-data-analysis software often run on separate computing devices to align base-call data for nucleotide-fragment reads with a reference genome and determine (and depict) data for variants of nucleotide bases that differ from the reference genome. While such on-machine software and sequencing-data-analysis software (together “existing sequencing management systems”) provide useful options to start and stop sequencing runs or view results of sequencing-data analysis, existing sequencing management systems (i) generate limited or misleading graphic grids depicting separate sequencing machine processes and separate sequencing-data-analysis processes and (ii) limit functions and control of an end-to- end sequencing process for a sample across the sequencing machine and sequencing-data analysis.
  • existing sequencing management systems provide misleading graphics that omit critical information concerning the end-to-end sequencing process for genome samples or hide such critical information behind layers of different graphical user interfaces. For instance, in some existing graphic grids, existing systems provide generalized graphics concerning a sequencing run and a corresponding secondary-data analysis for variants. But conventional sequencing graphics fail to indicate when an error or breakdown has occurred at certain points after the sequencing run or after the corresponding sequencing-data analysis. By failing to indicate such errors, existing systems often offer no visibility into a sequencing process with multiple potential failure points.
  • the disclosed system can query the status of various stages in an end-to-end sequencing process and generate a graphical status summary for the sequencing process that depicts icons indicating statuses of the various stages.
  • the disclosed systems can generate a graphical status summary for a nucleotide sequencing taskset that includes icons depicting statuses of a sequencing run, a data transfer of base-call data to a device for variant analysis, and the variant analysis — each part of the same nucleotide sequencing taskset.
  • the disclosed system can quickly provide a graphical status summary of an end-to-end sequencing process marked by various tasks within a nucleotide sequencing taskset. From a graphical user interface depicting graphical status summaries for active nucleotide sequencing tasksets, the disclosed system can also cause computing devices to display selectable options for viewing more detailed summaries of individual stages or for intervening at particular stages of a given nucleotide sequencing taskset.
  • FIG. 1 illustrates an environment in which a sequencing status system can operate in accordance with one or more embodiments of the present disclosure.
  • FIG. 2 illustrates a schematic diagram of the sequencing status system querying the status of various nucleotide sequencing tasks part of a nucleotide sequencing taskset and generating a graphical status summary for the nucleotide sequencing taskset in accordance with one or more embodiments of the present disclosure.
  • FIG. 3 A illustrates a graphical user interface for planned nucleotide sequencing tasksets for respective nucleotide sequencing tasksets in accordance with one or more embodiments of the present disclosure.
  • FIGS. 3B-3F illustrate graphical user interfaces comprising graphical status summaries for respective nucleotide sequencing tasksets in accordance with one or more embodiments of the present disclosure.
  • FIG. 3G illustrates a graphical user interface for completed nucleotide sequencing tasksets in accordance with one or more embodiments of the present disclosure.
  • FIGS. 4A-4B illustrates graphical user interfaces comprising detailed summaries for respective nucleotide sequencing tasksets in accordance with one or more embodiments of the present disclosure.
  • FIG. 5 illustrates an example architecture diagram of the sequencing status system in relation to an overall sequencing environment in accordance with one or more embodiments.
  • FIGS. 6A-6B illustrate series of acts for querying the status of various tasks of a nucleotide sequencing taskset and generating a graphical status summary for the nucleotide sequencing taskset in accordance with one or more embodiments of the present disclosure.
  • FIG. 7 illustrates a block diagram of an example computing device in accordance with one or more embodiments of the present disclosure.
  • FIG. 8 illustrates a block diagram of an example optical system for image-based genomic sequencing in accordance with one or more embodiments.
  • FIG. 9 illustrates an example imager for image-based genomic sequencing in accordance with one or more embodiments.
  • FIG. 10 illustrates an example diagram for performing image-based genomic sequencing in accordance with one or more embodiments.
  • This disclosure describes one or more embodiments of a sequencing status system that queries the status of various nucleotide sequencing tasks within a taskset and generates a graphical status summary that efficiently depicts the status of individual tasks from the nucleotide sequencing taskset.
  • the sequencing status system can send or receive data with a sequencing device concerning a sequencing run status and also determine statuses (e.g., from other computing devices) for various other nucleotide sequencing tasks for variant analysis following the sequencing run.
  • the sequencing status system Based on real time status queries, the sequencing status system generates a graphical status summary for a nucleotide sequencing taskset that includes a grouped series of dynamic status icons depicting statuses of various tasks, such as a sequencing run, a data transfer of base-call data to a device for variant analysis, and the variant analysis.
  • a sequencing run a data transfer of base-call data to a device for variant analysis
  • the variant analysis a graphical status summary for a nucleotide sequencing taskset that includes a grouped series of dynamic status icons depicting statuses of various tasks, such as a sequencing run, a data transfer of base-call data to a device for variant analysis, and the variant analysis.
  • the sequencing status system can detect a corresponding status and change the representative icon to efficiently communicate the status of both critical nucleotide sequencing tasks and an overall progress of an active nucleotide sequencing taskset.
  • the sequencing status system receives a status query from a computing device concerning a nucleotide sequencing taskset for determining sample genomes’ composition using a sequencing device and a server for variant analysis.
  • the sequencing status system determines statuses of various nucleotide sequencing tasks, including a sequencing run, a data- analysis transfer of base-call data generated during the sequencing run, and a variant analysis of the base-call data.
  • the sequencing status system Based on the determined statuses, the sequencing status system provides a graphical status summary for the nucleotide sequencing taskset — including a run status icon indicating a status of the sequencing run, a data-transfer-status icon indicating a status of the data- analysis transfer, and a variant-analysis-status icon indicating a status of the variant analysis.
  • the sequencing status system can generate different graphical status summaries for different nucleotide sequencing tasksets to display in a single, integrated graphical user interface — that is, an active sequencing interface.
  • the sequencing status system is executed by a local server corresponding to a sequencing device.
  • the sequencing status system can efficiently detect the status of sequencing runs from a nearby and connected sequencing device.
  • the sequencing status system can also quickly determine the status of (i) a data-analysis transfer of base-call data generated by a completed sequencing run and transferred to the local server and (ii) a variant analysis of the base-call data by complimentary software executed by the local server.
  • the sequencing status system can query the sequencing device (or other computing devices) to determine the status of external-data transfers of the base-call data or results data from the variant analysis to various external storages.
  • the sequencing status system can generate a group of dynamic status icons that together form a graphical status summary for a nucleotide sequencing taskset.
  • the sequencing status system can change (or cause to change) the dynamic status icons that are ordered in positions representing different tasks from the nucleotide sequencing taskset.
  • Such dynamic status icons can include, for instance, different icons in different ordered positions representing statuses for one or more of a sequencing run, an extemal-call-data transfer of base-call data generated during the sequencing run to an external storage, a data-analysis transfer of the base-call data to a computing device (e.g., local server) for a variant analysis, the variant analysis, or an extemal-analysis-data transfer of data generated during the variant analysis to an external storage.
  • a computing device e.g., local server
  • the sequencing status system changes the icon representing an individual nucleotide sequencing task according to a detected status. Based on a change in detected status, for instance, the sequencing status system can cause the display of a different icon for the task in the corresponding position (as the status changes) to indicate the task having started, scheduled, progressed, completed, stopped, or an error. As the status of other nucleotide sequencing tasks change, the sequencing status system can likewise change to a different corresponding icon to represent the changed status of the task. At each position for a task in an ordered graphical status summary, therefore, the sequencing status system can indicate a not-started status, a scheduled status, or other status with a different and corresponding icon.
  • the sequencing status system can present snapshot sequencing metrics along with a graphical status summary in an active sequencing interface.
  • Such sequencing metrics may represent collective metrics giving a quality or statistical snapshot of a particular nucleotide sequencing taskset.
  • the sequencing status system can identify and surface a collective base-call-quality metric indicating an accuracy of base calls generated during a sequencing run within a nucleotide sequencing taskset.
  • the sequencing status system can identify and surface a collective pass filter metric indicating a subset of base calls generated during the sequencing run that satisfy a quality filter.
  • the sequencing status system provides options in an active sequencing interface to intervene in a nucleotide sequencing task for a given nucleotide sequencing taskset. For instance, the sequencing status system can intelligently surface a cancel option or a re-initiate option for a particular task or a particular nucleotide sequencing taskset. Based on the status indicated by a given dynamic status icon in a graphical status summary, therefore, the sequencing status system can provide intuitive selectable options for technicians to intervene (or otherwise manage) a nucleotide sequencing taskset.
  • the sequencing status system provides several technical benefits relative to existing sequencing management systems, such as by improving the efficiency and functionality of graphical user interfaces relative to existing sequencing management systems. For instance, in some embodiments, the sequencing status system generates and surfaces an efficient snapshot of a nucleotide sequencing taskset in the form of a graphical status summary. As indicated above, some existing sequencing management systems provide graphics that omit important stages of a sequencing process for genome samples, such as by including information about a sequencing run but omitting information about the data produced by the sequencing run or produced by a corresponding sequencing-data analysis for variants.
  • the sequencing status system queries and integrates status information for tasks in a nucleotide sequencing taskset into a visually efficient summary for the nucleotide sequencing taskset.
  • the disclosed graphical status summary can quickly communicate the status of individual tasks from a nucleotide sequencing taskset, such as a sequencing run, a data transfer of base-call data, a variant analysis, and/or other nucleotide sequencing tasks.
  • the disclosed active sequencing interface can efficiently visualize collective sequencing metrics for a nucleotide sequencing taskset alongside a corresponding graphical status summary.
  • the sequencing status system By surfacing one or both of a graphical status summary and collective sequencing metrics, the sequencing status system (i) obviates the cumbersome graphical interface navigation that hinders existing sequencing management systems and (ii) efficiently communicates status information for tasks that such existing systems surface only through multiple clicks or navigation steps.
  • the sequencing status system generates and surfaces dynamic status icons for individual tasks from a nucleotide sequencing taskset that efficiently communicate the status of the corresponding task.
  • some existing sequencing management systems omit information concerning important stages of a sequencing process in graphic grids or other selective status graphics. For instance, existing graphic grids give no indication of processes in between a sequencing run and a sequencing-data analysis or processes after the sequencing-data analysis.
  • the sequencing status system generates and integrates dynamic status icons for important tasks from a nucleotide sequencing taskset that existing systems omit.
  • a graphical status summary includes a data- transfer-status icon indicating a status of a data-analysis transfer of base-call data from a sequencing device to a computing device for variant analysis. Because such a data-analysis transfer is often necessary for a sequencing process to progress, unlike existing systems, the sequencing status system efficiently represents the status of such a data-analysis transfer in a compact visual summary. Similarly, unlike existing systems, the sequencing status system can generate and surface separate icons in a graphical status summary for (i) an extemal-call-data transfer of base-call data generated during the sequencing run to an external storage or (ii) an extemal-analysis-data transfer of data generated during the variant analysis to an external storage. By surfacing dynamic status icons in a graphical status summary, the sequencing status system obviates cumbersome interface-upon-interface navigation that hinders existing sequencing management systems.
  • the sequencing status system intelligently surfaces options — unavailable at the summary level for existing sequencing management systems — in an active sequencing interface to intervene in a nucleotide sequencing task.
  • existing systems often limit summary level options to cancel an ongoing sequencing run or another nucleotide sequencing task.
  • the sequencing status system intelligently surfaces selectable options based on (and relevant to) a particular graphical status summary. For instance, the sequencing status system can provide a selectable option to pause or re-initiate a particular nucleotide sequencing task in an active sequencing interface when a status of (and dynamic status icon indicates) the particular nucleotide sequencing task is experiencing an error or has stopped.
  • the disclosed sequencing status system can intelligently surface selectable options for a particular nucleotide sequencing task based on a determined status for the particular task.
  • nucleotide sequencing task refers to an operation or a process performed by a computing device as part of determining a sequence of nucleotide bases for one or more sample genomes (or other nucleotide polymers) or part of saving data from determining such a sequence or from a corresponding analysis.
  • a nucleotide sequencing task can include an operation or a process performed by a sequencing device that determines nucleotide-base sequences of fragments from a sample genome or performed by another computing device (e.g., server) to analyze data for the nucleotide-base sequences and determine variants within the nucleotide-base sequences with respect to a reference genome.
  • a nucleotide sequencing task can likewise include an operation or a process of preserving data generated from determining a nucleotide-sequence (e.g., base-call data) or an analysis thereof.
  • a nucleotide sequencing task can include, but is not limited to, a sequencing run, an extemal-call-data transfer of base-call data generated during the sequencing run to an external storage, a data-analysis transfer of the base-call data to a computing device (e.g., local server) for a variant analysis, the variant analysis, or an extemal-analysis-data transfer of data generated during the variant analysis to an external storage.
  • a computing device e.g., local server
  • nucleotide sequencing taskset refers to a group of tasks performed by one or more computing devices that, as a collective process, determine a sequence of nucleotide bases for one or more sample genomes (or other nucleotide polymers) or save data from determining such a sequence or from a corresponding analysis.
  • a nucleotide sequencing taskset can include a group of operations or processes (i) performed by a sequencing device to determine nucleotide-base sequences of fragments from a sample genome or save data related to the determined nucleotide-base sequences and (ii) performed by another computing device (e.g., server) to analyze data related to the determined nucleotide-base sequences, determine variants within the nucleotide-base sequences with respect to a reference genome, or save data resulting from the analyzed data.
  • a nucleotide sequencing taskset comprises tasks starting from a sequencing run that generates base-call data through completing (and storing a copy of) variant analysis of the base-call data.
  • the sequencing status system can generate or provide a graphical status summary for a nucleotide sequencing taskset.
  • graphical status summary refers to one or more graphics (or data for such graphics) depicting a stage or progress of a nucleotide sequencing taskset.
  • a graphical status summary includes a set of graphics that visually represent a current stage or progress of a set of nucleotide sequencing tasks that are part of a particular nucleotide sequencing taskset.
  • a graphical status summary includes individual status icons that depict a stage or progress of individual tasks from a nucleotide sequencing taskset.
  • a graphical status summary can include a group of status icons that together depict a status or progress of a nucleotide sequencing taskset.
  • the sequencing status system provides a textual status summary for a particular nucleotide sequencing taskset (e.g., a textual-taskset-status summary) or a textual status summary for a particular nucleotide sequencing task (e.g., a textual-task-status summary).
  • a “status icon” refers to a graphical-user-interface element or graphic that depicts a stage or progress of a nucleotide sequencing task.
  • a status icon can include a graphic, located at a designated position within a graphical status summary, that visually represents a determined status of a nucleotide sequencing task.
  • a status icon can be dynamic and change to represent a changed status of a nucleotide sequencing task.
  • the sequencing status system can change a dynamic status icon to represent that a nucleotide sequencing task has started, been scheduled, progressed, completed, stopped, or encountered an error.
  • a dynamic status icon may also be depicted in a particular color (e.g., green for completed, blue for in progress, grey for not started or scheduled, red for stopped or in error).
  • a status icon can also represent other suitable statuses.
  • sequencing run refers to an iterative process on a sequencing device to determine a primary structure of nucleotide fragments from a sample (e.g., genomic sample).
  • a sequencing run includes cycles of sequencing chemistry and imaging performed by a sequencing device that incorporate nucleotide bases into growing oligonucleotides to determine nucleotide-fragment reads from nucleotide fragments extracted from a sample and seeded throughout a nucleotide-sample slide.
  • a sequencing run includes replicating nucleotide fragments from one or more genome samples seeded in clusters throughout a nucleotide-sample slide (e.g., a flow cell).
  • a sequencing run can be a Sequencing By Synthesis (SBS) run.
  • SBS Sequencing By Synthesis
  • a sequencing device can generate base-call data in a fde.
  • base-call data refers to data representing nucleotide- base calls for nucleotide-fragment reads and/or corresponding sequencing metrics.
  • base-call data includes textual data representing nucleotide-base calls for nucleotide-fragment reads as text, (e.g., A, C, G, T) along with corresponding base-call-quality metrics, depth metrics, and/or other sequencing metrics.
  • base-call data is formatted in a text file, such as a binary base call (BCL) sequence file or as a fast-all quality (FASTQ) file.
  • BCL binary base call
  • FASTQ fast-all quality
  • base-call data includes sequencing metrics.
  • sequencing metric refers to a quantitative measurement or score indicating a degree to which an individual nucleotide-base call (or a sequence of nucleotide-base calls) aligns, compares, or quantifies with respect to a genomic coordinate or genomic region of a reference genome, with respect to nucleotide-base calls from nucleotide-fragment reads, or with respect to external genomic sequencing or genomic structure.
  • a sequencing metric includes a quantitative measurement or score indicating a degree to which (i) individual nucleotide-base calls align, map, or cover a genomic coordinate or reference base of a reference genome; (ii) nucleotide- base calls compare to reference or alternative nucleotide-fragment reads in terms of mapping, mismatch, base-call quality, or other raw sequencing metrics; or (iii) genomic coordinates or regions corresponding to nucleotide-base calls demonstrate mappability, repetitive base-call content, DNA structure, or other generalized metrics.
  • nucleotide-sample slide refers to a plate or slide comprising oligonucleotides for sequencing nucleotide segments for sample genomes or other sample nucleic-acid polymers.
  • a nucleotide-sample slide can refer to a slide containing fluidic channels through which reagents and buffers can travel as part of sequencing.
  • a nucleotide-sample slide includes a flow cell (e.g., a patterned flow cell or non-pattemed flow cell) comprising small fluidic channels and short oligonucleotides complementary to adaptor sequences.
  • a nucleotide-sample slide can include wells (e.g., nanowells) comprising clusters of oligonucleotides.
  • a flow cell or other nucleotide-sample slide can (i) include a device having a lid extending over a reaction structure to form a flow channel therebetween that is in communication with a plurality of reaction sites of the reaction structure and (ii) include a detection device that is configured to detect designated reactions that occur at or proximate to the reaction sites.
  • a flow cell or other nucleotide-sample slide may include a solid-state light detection or imaging device, such as a Charge-Coupled Device (CCD) or Complementary Metal- Oxide Semiconductor (CMOS) (light) detection device.
  • CCD Charge-Coupled Device
  • CMOS Complementary Metal- Oxide Semiconductor
  • a flow cell may be configured to fluidically and electrically couple to a cartridge (having an integrated pump), which may be configured to fluidically and/or electrically couple to a bioassay system.
  • a cartridge and/or bioassay system may deliver a reaction solution to reaction sites of a flow cell according to a predetermined protocol (e.g., sequencing-by-synthesis), and perform a plurality of imaging events.
  • a cartridge and/or bioassay system may direct one or more reaction solutions through the flow channel of the flow cell, and thereby along the reaction sites. At least one of the reaction solutions may include four types of nucleotides having the same or different fluorescent labels.
  • the nucleotides may bind to the reaction sites of the flow cell, such as to corresponding oligonucleotides at the reaction sites.
  • the cartridge and/or bioassay system may then illuminate the reaction sites using an excitation light source (e.g., solid-state light sources, such as light-emitting diodes (LEDS)).
  • the excitation light may provide emission signals (e.g., light of a wavelength or wavelengths that differ from the excitation light and, potentially, each other) that may be detected by the light sensors of the flow cell.
  • nucleotide-fragment read refers to an inferred sequence of one or more nucleotide bases (or nucleobase pairs) from all or part of a sample nucleotide sequence.
  • a nucleotide-fragment read includes a determined or predicted sequence of nucleotide-base calls for a nucleotide fragment (or group of monoclonal nucleotide fragments) from a sequencing library corresponding to a genome sample.
  • a sequencing device determines a nucleotide-fragment read by generating nucleotide-base calls for nucleotide bases passed through a nanopore of a nucleotide-sample slide, determined via fluorescent tagging, or determined from a cluster in a flow cell.
  • variant analysis refers to a secondary and/or a tertiary analysis of base-call data performed by a computing device to align nucleotide-fragment reads with a reference genome, determine genetic variants based on the aligned nucleotide- fragment reads, and/or interpret the determined genetic variants.
  • a variant analysis can include a secondary analysis performed by a server executing variant-call software to align samples’ nucleotide-fragment reads with a reference genome, determine genetic variants of samples based on the aligned nucleotide-fragment reads with respect to the reference genome, and determine one or more of quality metrics, allele frequency metrics, or other sequencing metrics.
  • a variant analysis can include a tertiary analysis performed by a server executing bioinformatics software to determine potential genetic diseases (or genetic factors correlating with genetic diseases) based on determined genetic variants of a sample.
  • a reference genome refers to a digital nucleic-acid sequence assembled as a representative example (or representative examples) of genes for an organism. Regardless of the sequence length, in some cases, a reference genome represents an example set of genes or a set of nucleic-acid sequences in a digital nucleic-acid sequenced determined by scientists or statistical models as representative of an organism of a particular species.
  • a linear human reference genome may be GRCh38 or other versions of reference genomes from the Genome Reference Consortium.
  • a reference genome may include a reference graph genome that includes both a linear reference genome and paths representing nucleic-acid sequences from ancestral haplotypes, such as Illumina DRAGEN Graph Reference Genome hg!9.
  • nucleotide-base call refers to a determination or prediction of a particular nucleotide base (or nucleotide-base pair) for a genomic coordinate of a sample genome or for an oligonucleotide during a sequencing cycle.
  • a nucleotide-base call can indicate (i) a determination or prediction of the type of nucleotide base that has been incorporated within an oligonucleotide on a nucleotide-sample slide (e.g., read-based nucleotide-base calls) or (ii) a determination or prediction of the type of nucleotide base that is present at a genomic coordinate or region within a genome, including a variant call or a non-variant call in a digital output file.
  • a nucleotide-base call includes a determination or a prediction of a nucleotide base based on intensity values resulting from fluorescent-tagged nucleotides added to an oligonucleotide of a nucleotide-sample slide (e.g., in a cluster of a flow cell).
  • a nucleotide-base call includes a determination or a prediction of a nucleotide base from chromatogram peaks or electrical current changes resulting from nucleotides passing through a nanopore of a nucleotide- sample slide.
  • a nucleotide-base call can also include a final prediction of a nucleotide base at a genomic coordinate of a sample genome for a variant call file or other base- call-output file — based on nucleotide-fragment reads corresponding to the genomic coordinate.
  • a nucleotide-base call can include a base call corresponding to a genomic coordinate and a reference genome, such as an indication of a variant or a non-variant at a particular location corresponding to the reference genome.
  • a nucleotide-base call can refer to a variant call, including but not limited to, a single nucleotide variant (SNV), an insertion or a deletion (indel), or base call that is part of a structural variant.
  • a single nucleotide-base call can be an adenine (A) call, a cytosine (C) call, a guanine (G) call, or a thymine (T) call.
  • FIG. 1 illustrates a schematic diagram of a system environment (or “environment”) 100 in which a sequencing status system 106 operates in accordance with one or more embodiments.
  • the environment 100 includes a local server device 102 connected to one or more server device(s) 110, a sequencing device 108, and a client device 114 via a network 112. While FIG. 1 shows an embodiment of the sequencing status system 106, this disclosure describes alternative embodiments and configurations below.
  • the local server device 102, the sequencing device 108, the server device(s) 110, and the client device 114 can communicate with each other via the network 112.
  • the network 112 comprises any suitable network over which computing devices can communicate. Example networks are discussed in additional detail below with respect to FIG. 7.
  • the sequencing device 108 comprises a device for sequencing a genomic sample or other nucleic-acid polymer.
  • the sequencing device 108 analyzes nucleic-acid segments or oligonucleotides extracted from genomic samples to generate nucleotide-fragment reads or other data utilizing computer implemented methods and systems (described herein) either directly or indirectly on the sequencing device 108. More particularly, the sequencing device 108 receives nucleotide-sample slides (e.g., flow cells) comprising nucleotide fragments extracted from samples and then copies and determines the nucleotide-base sequence of such extracted nucleotide fragments.
  • nucleotide-sample slides e.g., flow cells
  • the sequencing device 108 utilizes SBS to sequence nucleic-acid polymers into nucleotide- fragment reads. In addition or in the alternative to communicating across the network 112, in some embodiments, the sequencing device 108 bypasses the network 112 and communicates directly with the local server device 102 or the client device 114.
  • the local server device 102 is located at or near a same physical location of the sequencing device 108. Indeed, in some embodiments, the local server device 102 and the sequencing device 108 are integrated into a same computing device, as indicated by dotted lines 122.
  • the local server device 102 may run a sequencing system 104 to generate, receive, analyze, store, and transmit digital data, such as by receiving base-call data or determining variant calls based on analyzing such base-call data.
  • the sequencing device 108 may send (and the local server device 102 may receive) base-call data generated during a sequencing run of the sequencing device 108.
  • the local server device 102 may align nucleotide-fragment reads with a reference genome and determine genetic variants based on the aligned nucleotide-fragment reads.
  • the local server device 102 may also communicate with the client device 114.
  • the local server device 102 can send data to the client device 114, including status information for nucleotide sequencing tasksets, a variant call fde (VCF), or other information indicating nucleotide- base calls, sequencing metrics, error data, or other metrics.
  • VCF variant call fde
  • the server device(s) 110 are located remotely from the local server device 102 and the sequencing device 108. Similar to the local server device 102, in some embodiments, the server device(s) 110 include a version of the sequencing system 104. Accordingly, the server device(s) 110 may generate, receive, analyze, store, and transmit digital data, such as data for determining nucleotide-base calls or sequencing nucleic-acid polymers. Similarly, the sequencing device 108 may send (and the server device(s) 110 may receive) basecall data from the sequencing device 108. The server device(s) 110 may also communicate with the client device 114. In particular, the server device(s) 110 can send data to the client device 114, including status information for nucleotide sequencing tasksets, VCFs, or other sequencing related information.
  • the server device(s) 110 comprise a distributed collection of servers where the server device(s) 110 include a number of server devices distributed across the network 112 and located in the same or different physical locations. Further, the server device(s) 110 can comprise a content server, an application server, a communication server, a web-hosting server, or another type of server. [0051] As further illustrated and indicated in FIG. 1, the client device 114 can generate, store, receive, and send digital data. In particular, the client device 114 can receive status data from the local server device 102 or receive sequencing metrics from the sequencing device 108.
  • the client device 114 may communicate with the local server device 102 or the server device(s) 110 to receive a VCF comprising nucleotide base calls and/or other metrics, such as a base-call-quality metrics or pass-filter metrics.
  • the client device 114 can accordingly present or display information pertaining to variant calls or other nucleotide-base calls within a graphical user interface to a user associated with the client device 114.
  • the client device 114 can present an active sequencing interface comprising a graphical status summary of a nucleotide sequencing taskset.
  • FIG. 1 depicts the client device 114 as a desktop or laptop computer
  • the client device 114 may comprise various types of client devices.
  • the client device 114 includes non-mobile devices, such as desktop computers or servers, or other types of client devices.
  • the client device 114 includes mobile devices, such as laptops, tablets, mobile telephones, or smartphones. Additional details regarding the client device 114 are discussed below with respect to FIG. 7.
  • the client device 114 includes a sequencing application 116.
  • the sequencing application 116 may be a web application or a native application stored and executed on the client device 114 (e.g., a mobile application, desktop application).
  • the sequencing application 116 can include instructions that (when executed) cause the client device 114 to receive data from the sequencing status system 106 and present, for display at the client device 114, data concerning a status of a nucleotide sequencing taskset or data from a VCF.
  • the sequencing application 116 can instruct the client device 114 to display graphical status summaries for multiple nucleotide sequencing tasksets.
  • a version of the sequencing status system 106 may be located on the client device 114 as part of the sequencing application 116 or on the server device(s) 110. Accordingly, in some embodiments, the sequencing status system 106 is implemented by (e.g., located entirely or in part) on the client device 114. In yet other embodiments, the sequencing status system 106 is implemented by one or more other components of the environment 100, such as the server device(s) 110. In particular, the sequencing status system 106 can be implemented in a variety of different ways across local server device 102, the sequencing device 108, the client device 114, and the server device(s) 110.
  • the sequencing status system 106 can be downloaded from the server device(s) 110 to the local server device 102 and/or the client device 114 where all or part of the functionality of the sequencing status system 106 is performed at each respective device within the environment 100.
  • the environment 100 includes a local database 118 and a remote database 120.
  • the local database 118 is located at or nearby a same location as the sequencing device 108, whereas the remote database 120 is location at a different and remote location.
  • the local database 118 and the remote database 120 can store information, such as base-call data, variant call files, data for sample nucleotide sequences, and sequencing metrics.
  • the local server device 102, the sequencing device 108, the server device(s) 110, and/or the client device 114 communicate with the local database 118 and the remote database 120 (e.g., via the network 112) to store and/or access information, such base-call data, variant call files, data for sample nucleotide sequences, and sequencing metrics.
  • the sequencing status system 106 generates a graphical status summary 124 of a nucleotide sequencing taskset for display on the client device 114. As executed by the local server device 102 or the server device(s) 110, for instance, the sequencing status system 106 receives a status query from the client device 114 concerning a nucleotide sequencing taskset.
  • the sequencing status system 106 determines statuses of various nucleotide sequencing tasks within the nucleotide sequencing taskset, including a status of a sequencing run from the sequencing device 108, a data-analysis transfer of base-call data from the sequencing device 108 to the local server device 102 or to the server device(s) 110, and a variant analysis of the base-call data by the local server device 102 or by the server device(s) 110.
  • the sequencing status system 106 provides the graphical status summary 124 for the nucleotide sequencing taskset — including data for a run status icon 126a indicating a status of the sequencing run, a data-transfer-status icon 126b indicating a status of the data-analysis transfer, and a variant-analysis-status icon 126c indicating a status of the variant analysis.
  • graphical status summaries can be displayed on other computing devices, such as the sequencing device 108, a local computing device connected to the local server device 102, or an external computing device connected to the server device(s) 110 or the local server device 102 through the network 112.
  • Graphical status summaries can take various different forms. While the graphical status summary 124, for instance, includes status icons at designated positions for three different nucleotide sequencing tasks, other graphical status summaries may include status icons for four, five, or another number of nucleotide sequencing tasks.
  • the sequencing status system 106 can exchange and orchestrate data across multiple computing devices to determine the status of nucleotide sequencing tasksets and generate graphical status summaries for such tasksets.
  • FIG. 2 illustrates an example of the sequencing status system 106 (i) querying other computing devices for the status of various nucleotide sequencing tasks that are part of a nucleotide sequencing taskset and (ii) generating a graphical status summary for the nucleotide sequencing taskset.
  • the client device 114 sends a status query 202a to the local server device 102 inquiring of a status of a nucleotide sequencing taskset.
  • the status query 202a includes data identifiers for each nucleotide sequencing task in a nucleotide sequencing taskset to prompt the sequencing status system 106 to determine a status for each nucleotide sequencing task.
  • the status query 202a includes a data identifier for the particular nucleotide sequencing taskset that prompts the sequencing status system 106 to determine a status for each nucleotide sequencing task.
  • the sequencing status system 106 determines a status of tasks that are part of the nucleotide sequencing taskset — based on receiving the status query 202a. For instance, as shown in FIG. 2, the sequencing status system 106 sends a status query 202b to the sequencing device 108 inquiring of statuses for one or more of a sequencing run, a data-analysis transfer of base-call data, or an extemal-call-data transfer of the base-call data to the local database 118 or other external storage.
  • the sequencing device 108 determines whether the sequencing run has started, been scheduled, progressed, completed, stopped, or encountered an error and, subsequently, sends status data 204a to the local server device 102 indicating a corresponding status. Similarly, also based on receiving the status query 202b, in some embodiments, the sequencing device 108 determines whether it has started, scheduled, progressed, completed, stopped, or encountered an error in sending base-call data 206a generated during the sequencing run to the local server device 102 and/or sending basecall data 206b to the local database 118.
  • the sequencing status system 106 In addition to communicating with the sequencing device 108 to determine a status of nucleotide sequencing tasks, as suggested by FIG. 2, the sequencing status system 106 also detects a status of tasks that are part of the nucleotide sequencing taskset from either the local server device 102 or the server device(s) 110. For instance, the sequencing status system 106 imports or determines, from one or more storage devices on the local server device 102, statuses for one or more of a data-analysis transfer of base-call data to the local server device 102, a variant analysis of the base-call data, or an extemal-analysis-data transfer of variant analysis data 208 generated during the variant analysis to the local database 118 or the remote database 120 or other external storage.
  • the sequencing status system 106 sends a status query to the server device(s) 110 to determine a status for the data-analysis transfer, the variant analysis, and/or the extemal-analysis-data transfer. Based on receiving the status query 202a from the client device 114, the sequencing status system 106 determines whether the data-analysis transfer, the variant analysis, and/or the extemal-analysis- data transfer has started, been scheduled, progressed, completed, stopped, or encountered an error. [0062] As further indicated by FIG.
  • the sequencing status system 106 sends status data 204b to the client device 114 — based on (or in response to) the status query 202a.
  • the status data 204b includes data indicators for a status of each nucleotide sequencing task within a taskset.
  • the status data 204b includes data indicators for an updated or changed status of one or more nucleotide sequencing tasks, but no data indicators for nucleotide sequencing tasks with an unchanged status from a previous status query.
  • the status data 204b from the local server device 102 can prompt the client device 114 to present a graphical status summary 210a.
  • the client device 114 presents the graphical status summary 210a for the queried nucleotide sequencing taskset.
  • the graphical status summary 210a includes a group of status icons that together indicate a status of the queried nucleotide sequencing taskset.
  • the graphical status summary 210a includes a dynamic status icon for each of a sequencing run, an extemal-call-data transfer of basecall data to the local database 118 or other external storage, a data-analysis transfer of the base-call data to the local server device 102 for a variant analysis, the variant analysis, and an external - analysis-data transfer of variant analysis data 208 generated during the variant analysis to the local database 118 or the remote database 120 or other external storage.
  • the client device 114 sends additional status queries as a data pull or receives additional status queries as a data push, the client device 114 can change the dynamic status icon for one or more tasks within the queried nucleotide sequencing taskset.
  • the sequencing status system 106 can facilitate status queries from different computing devices and provide a graphical status summary for display on different querying computing devices.
  • the sequencing device 108 sends a status query for a nucleotide sequencing taskset and receives status data in response to the status query. Based on the status data, the sequencing device 108 likewise displays a graphical status summary 214 within a graphical user interface. As shown, the graphical status summary 214 matches the graphical status summary 210a for the same nucleotide sequencing taskset.
  • the sequencing status system 106 can provide (and the client device 114 display) graphical status summaries for multiple queried nucleotide sequencing tasksets. Indeed, the client device 114 display a graphical status summary 210b for an additional queried nucleotide sequencing taskset. To determine a status of an additional nucleotide sequencing taskset, in some embodiments, the sequencing status system 106 sends or receives the same type of status queries or status data as described above for the graphical status summary 210a. Indeed, in some embodiments, the sequencing status system 106 can orchestrate in parallel the exchange of status queries and status data for different nucleotide sequencing tasksets to facilitate display of multiple corresponding graphical status summaries on a single computing device.
  • the sequencing status system 106 provides selectable options, for display in the client device 114, to intervene in a task for a given nucleotide sequencing taskset. As shown in FIG. 2, for instance, the sequencing status system 106 provides a selectable option 212 to intervene in a targeted nucleotide sequencing task corresponding to a particular status icon in the graphical status summary 210a.
  • the sequencing status system 106 identifies a status of a nucleotide sequencing task and identifies an operation corresponding to the status of the nucleotide sequencing task, such as by identifying a sequencing operation to re-initiate a nucleotide sequencing task based on a status indicating the task has stopped or encountered an error.
  • the sequencing status system 106 sends data (e.g., as part of the status data 204b) identifying or triggering an option for a re-initiate operation to the client device 114 based on determining that a variant analysis has encountered an error. Based on receiving the data identifying or triggering the option for the re-initiate operation, the client device 114 displays a re-initiate option as the selectable option 212 for display alongside, nearby, or within a same graphical user interface as the graphical status summary 210a.
  • data e.g., as part of the status data 204b
  • the client device 114 displays a re-initiate option as the selectable option 212 for display alongside, nearby, or within a same graphical user interface as the graphical status summary 210a.
  • the sequencing status system 106 gives tools to a computing device to manage a nucleotide sequencing taskset running across multiple devices.
  • the client device 114 sends a sequencing command 216a to the local server device 102 based on detecting a selection of the selectable option 212 to perform a sequencing operation for the targeted nucleotide sequencing task. For instance, the client device 114 sends the sequencing command 216a to the local server device 102 to re-initiate the variant analysis represented by a status icon in the graphical status summary 210a.
  • the local server device 102 re-initiates the variant analysis of base-call data from the corresponding sequencing run.
  • the sequencing status system 106 can intelligently determine and send data for display of options to intervene in a variety of nucleotide sequencing task for a given nucleotide sequencing taskset. For instance, in some cases, the client device 114 prompts the local server device 102 to send (or the client device 114 sends across the network 112) a sequencing command 216b to the sequencing device 108. As further shown in FIG. 2, based on receiving a sequencing command from the client device 114, the local server device 102 optionally sends the sequencing command 216b to the sequencing device 108 to perform a sequencing operation for a targeted nucleotide sequencing task, such as by re-initiating a sequencing run.
  • the sequencing status system 106 can orchestrate and manage different nucleotide sequencing tasksets at various stages. As shown in FIGS. 3A-3G and described below, the sequencing status system 106 can provide tools to plan, manage, intervene, and view summaries of different nucleotide sequencing tasksets starting from a sequencing run that generates base-call data through completing (and storing a copy of) variant analysis of the base-call data.
  • FIGS. 3A-3G illustrate the client device 114 presenting graphical user interfaces for managing planned tasksets, active tasksets, and completed tasksets from the perspective of the client device 114 implementing computer-executable instructions of the sequencing application 116.
  • FIGS. 4A-4B illustrate the client device 114 presenting graphical user interfaces comprising details for a nucleotide sequencing taskset. Rather than repeatedly refer to the computer-executable instructions causing a computing device to perform certain actions for the sequencing status system 106, this disclosure describes the client device 114 or the sequencing status system 106 performing those actions in the following paragraphs.
  • the client device 114 presents a planned sequencing interface 302 of the sequencing application 116 within a screen 301. Based on detecting a selection of a planned sequencing tab 304, for example, the client device 114 presents the planned sequencing interface 302.
  • the planned sequencing interface 302 includes planned taskset overviews 306 for nucleotide sequencing tasksets that have been created or scheduled to begin at a future time.
  • the planned taskset overviews 306 each in turn include high-level information concerning a planned nucleotide sequencing taskset, including a textual status summary (e.g., Planned, Locked), sequencing run name or nucleotide-sequencing-taskset name (e.g., Run A), secondary-analysis application type (e.g., DRAGEN Enrichment), owner, and date last modified.
  • a textual status summary e.g., Planned, Locked
  • sequencing run name or nucleotide-sequencing-taskset name e.g., Run A
  • secondary-analysis application type e.g., DRAGEN Enrichment
  • the planned sequencing interface 302 includes an import taskset option 308 and a create taskset option 310. Based on detecting a selection of the import taskset option 308, the client device 114 presents one or more graphical user interfaces to import a previously planned nucleotide sequencing taskset. By contrast, based on detecting a selection of the create taskset option 310, the client device 114 presents one or more graphical user interfaces to create a new nucleotide sequencing taskset.
  • the sequencing status system 106 also presents a snapshot of active nucleotide sequencing tasksets in which at least a sequencing run has commenced.
  • the client device 114 presents an active sequencing interface 314a of the sequencing application 116 within the screen 301. Based on detecting a selection of an active sequencing tab 312, for example, the client device 114 presents the active sequencing interface 314a. Indeed, in some cases, the client device 114 presents an error icon 313 alongside (or within proximity of) the active sequencing tab 312 to indicate an error or other issue with one or more active nucleotide sequencing tasksets can be viewed within the active sequencing interface 314a.
  • the active sequencing interface 314a includes active taskset overviews 316a, 316b, 316c, and 316d.
  • Each of the active taskset overviews 316a-316d include basic identifying information for a nucleotide sequencing taskset.
  • each of the active taskset overviews 316a-316d include a run name or nucleotide-sequencing-taskset name, a start date, a nucleotide-sample-slide-side indicator, a graphical status summary, a collective base- call-quality metric, and a collective pass filter metric for a corresponding nucleotide sequencing taskset.
  • the active taskset overviews 316a, 316b, 316c, and 316d respectively include graphical status summaries 324a, 324b, 324c, and 324d.
  • This disclosure describes each active taskset overview and graphical status summary in turn below.
  • the client device 114 presents a nucleotide-sequencing-taskset name 318, a start date 320, and a nucleotide-sample-slide-side indicator 322.
  • the nucleotide-sequencing-taskset name 318 includes a code, alphanumeric information, or other identifier for the nucleotide sequencing taskset.
  • the nucleotide- sequencing-taskset name 318 also includes an indicator for a type of nucleotide sequencing taskset, such as in vitro diagnostic (IVD) or research use only (RUO).
  • the start date 320 includes a data that a sequencing device (or another computing device) commenced a nucleotide sequencing taskset. In some cases, the start date 320 indicates a date and time at which a sequencing device began a sequencing run.
  • the nucleotide-sample-slide-side indicator 322 indicates a side of a nucleotide-sample slide comprising one or more samples corresponding to the relevant nucleotide sequencing taskset.
  • the active taskset overview 316a indicates side A or side B of a nucleotide- sample slide because, in some cases, a particular sequencing run for a nucleotide sequencing taskset may correspond to a single slide.
  • the client device 114 presents a graphical status summary 324a as part of the active taskset overview 316a.
  • the graphical status summary 324a includes a set of status icons that together depict a stage or progress of an active nucleotide sequencing taskset. Indeed, the graphical status summary 324a orders the set of status icons in designated positions for different tasks within the active nucleotide sequencing taskset.
  • the sequencing status system 106 provides a graphical snapshot that efficiently communicates both an overall status of the active nucleotide sequencing taskset and a status of individual tasks. This disclosure describes the specific status icons and designated positions further below with respect to FIG. 3B.
  • the client device 114 intelligently presents a textual-taskset-status summary 326a within the active taskset overview 316a.
  • the sequencing status system 106 can determine a status of each nucleotide sequencing task within a nucleotide sequencing task. Based on the status of each nucleotide sequencing task — or an overall status of the nucleotide sequencing taskset — the sequencing status system 106 can determine and provide the textual -taskset-status summary 326a for display within the active sequencing interface 314a.
  • the textual-taskset-status summary 326a includes a textual description of the overall status of the active nucleotide sequencing taskset that compliments the graphical status summary 324a.
  • the textual -taskset-status summary 326a describes a current status of a last scheduled or a last commenced nucleotide sequencing task according to an order of the nucleotide sequencing taskset.
  • the graphical status summary 324a depicts status icons in designated positions according to the order of the nucleotide sequencing taskset.
  • the sequencing status system 106 can provide (and the client device 114 present) collective sequencing metrics for the nucleotide sequencing taskset as part of the active taskset overview 316a. As shown in FIG. 3B, for instance, the client device 114 presents a collective base- call-quality metric 328 for the nucleotide sequencing taskset. To surface this collective sequencing metric, in some cases, the sequencing status system 106 or the sequencing system 104 determines a percentage of base calls for nucleotide-fragment reads (generated during the sequencing run) that satisfy (e.g., equal or exceed) a threshold base-call-quality metric.
  • Such a threshold base-call- quality metric may be a quality metric of 30 according to a Phred algorithm (e.g., Phred quality score of Q30).
  • the sequencing status system 106 or the sequencing system 104 determines a percentage of variant calls in a variant call file that satisfy a threshold base-call-quality metric.
  • the collective base-call-quality metric 328 indicates an accuracy of base calls generated during a sequencing run (or an accuracy of variant calls in a VCF) that is part of the nucleotide sequencing taskset. Based on determining or receiving the collective base-call-quality metric 328, the sequencing status system 106 provides the collective base-call-quality metric 328 for display proximate to the graphical status summary 324a within the active sequencing interface 314a.
  • the client device 114 presents a collective pass filter metric 330 for the nucleotide sequencing taskset.
  • the sequencing status system 106 or the sequencing system 104 determines a percentage of nucleotide-fragment reads that pass a quality fdter.
  • the quality filter may be a chastity metric for nucleotide fragment reads.
  • a nucleotide- fragment read for a given cluster of oligonucleotides passes a chastity filter when no more than one base call has a chastity value below 0.6 in the initial twenty-five cycles of a sequencing run.
  • the collective pass filter metric 330 indicates a subset of base calls generated during the sequencing run that satisfy a quality filter. Based on determining or receiving the collective pass filter metric 330, the sequencing status system 106 provides the collective pass filter metric 330 for display proximate to the graphical status summary 324a within the active sequencing interface 314a.
  • each of the active taskset overviews 316b-316d include a same type of information as just described for the active taskset overview 316a. Accordingly, each of the active taskset overviews 316b-316d include a sequencing run name or nucleoti de-sequencingtaskset name, start date, nucleotide-sample-slide-side indicator, a graphical status summary, and collective sequencing metrics — but for different active nucleotide sequencing tasksets.
  • This disclosure describes certain differences among the active taskset overviews 316a-316d and graphical status summaries 324a-324d below.
  • the sequencing status system 106 can provide an expanded graphical status summary comprising additional detail concerning an active nucleotide sequencing taskset.
  • the client device 114 detects a selection of an expand option 332a, the client device 114 presents an expanded active taskset overview 340a within an active sequencing interface 314b.
  • the expanded active taskset overview 340a includes an expanded graphical status summary 336a with a textual -task-status summary for each dynamic status icon.
  • the graphical status summary 324a includes individual status icons arranged in a particular order with designated positions.
  • the graphical status summary 324a includes a run status icon 334a located at a sequencing run position (or first designated position), an external -transfer-status icon 334b located at an extemal-call-data-transfer position (or second designated position), a data-transfer-status icon 334c located at data-analysis-transfer position (or third designated position), a variant-analysis- status icon 334d located at variant-analysis position (or fourth designated position), and an extemal- transfer-status icon 334e located at an extemal-analysis-data-transfer position (or fifth designated position).
  • an individual status icon at each designated position in the graphical status summary 324a can take the form different graphics to indicate different statuses of a nucleotide sequencing task.
  • the run status icon 334a indicates a status of the sequencing run.
  • the run status icon 334a is a circular check-mark icon representing a completed status.
  • the external - transfer-status icon 334b indicates a status of an extemal-call-data transfer of the base-call data (generated during the sequencing run) to an external storage.
  • the extemal-transfer- status icon 334b is a blue clock icon or a darker shaded clock icon representing an in-progress status.
  • the data- transfer-status icon 334c indicates a status of a data-analysis transfer of base-call data (generated during the sequencing run) to a server for variant analysis.
  • the data-transfer-status icon 334c is a circular check-mark icon representing a completed status.
  • the variant-analysis-status icon 334d indicates a status of the variant analysis of the base-call data, which can be performed by a server (e.g., a local server device).
  • the variant-analysis-status icon 334d is a circular stop-sign icon representing a stopped status.
  • the extemal-transfer- status icon 334e indicates a status of an extemal-analysis-data transfer data generated during the variant analysis to an external storage.
  • the extemal-transfer-status icon 334e is a grey-filled-circle icon representing a not-started status.
  • the client device 114 presents a textual -task-status summary for each status icon within the expanded graphical status summary 336a.
  • the active sequencing interface 314b comprises textual task summaries 338a, 338b, 338c, 338d, and 338e that describe in words a status of the nucleotide sequencing task represented by the run status icon 334a, the extemal-transfer-status icon 334b, the data-transfer-status icon 334c, the variant-analysis-status icon 334d, and the extemal-transfer-status icon 334e, respectively.
  • the client device 114 vertically orders the run status icon 334a, the extemal-transfer-status icon 334b, the data-transfer-status icon 334c, the variant-analysis- status icon 334d, and the extemal-transfer-status icon 334e from top to bottom and presents the textual task summaries 338a, 338b, 338c, 338d, and 338e, respectively alongside the corresponding status icon.
  • the sequencing status system 106 provides more detailed information concerning each nucleotide sequencing task from a taskset for display within the expanded active taskset overview 340a. As shown in FIG. 3C, for instance, the client device 114 presents (i) a start date and time for each nucleotide sequencing task that has been started and (ii) a completed date and time for each nucleotide sequencing task that has been completed.
  • the sequencing status system 106 can intelligently provide a selectable intervention option for a particular nucleotide sequencing task — based on a determined status of the particular nucleotide sequencing task. As shown in FIG. 3C, for instance, the sequencing status system 106 determines a stopped status for a particular nucleotide sequencing task, here, the variant analysis. Based on the determined stopped status, the sequencing status system 106 provides (and the client device 114 presents) a re-initiate option 342a to re-initiate the particular nucleotide sequencing task.
  • the sequencing status system 106 Based on receiving an indication of a selection of the re-initiate option 342a from the client device 114, in some cases, the sequencing status system 106 re-initiates the particular nucleotide sequencing task from a point at which the task stopped (e.g., by requeuing the variant analysis to continue). By contrast, in certain embodiments, based on receiving an indication of a selection of the re-initiate option 342a from the client device 114, the sequencing status system 106 re-initiates the particular nucleotide sequencing task from the beginning (e.g., by restarting the variant analysis to continue).
  • the sequencing status system 106 can customize a graphical status summary for a nucleotide sequencing taskset.
  • FIG. 3D depicts the sequencing status system 106 generating a customized graphical status summary for a nucleotide sequencing taskset based on the queried status of individual tasks within the nucleotide sequencing taskset.
  • the client device 114 detects a selection of an expand option 332b
  • the client device 114 presents an expanded active taskset overview 340b within an active sequencing interface 314c.
  • the active sequencing interface 314c includes both the graphical status summary 324b and an expanded graphical status summary 336b for a same nucleotide sequencing taskset.
  • the client device 114 presents the graphical status summary 324b with a textual-taskset-status summary 326b as part of the active taskset overview 316b. Because the status of the nucleotide sequencing taskset represented by the graphical status summary 324b differs from the nucleotide sequencing taskset represented by the graphical status summary 324a, the sequencing status system 106 customizes the textual -taskset-status summary 326b to represent the differing status of a corresponding nucleotide sequencing taskset.
  • the textual-taskset-status summary 326b describes a current status of a last scheduled or last commenced nucleotide sequencing task — here, an extemal-analysis-data transfer of data (generated during a different variant analysis) to an external storage.
  • the textual- taskset-status summary 326a indicates a server cannot connect to an external storage.
  • the client device 114 presents the graphical status summary 324b with a set of status icons ordered horizontally with respect to the active sequencing interface 314a or 314c.
  • the graphical status summary 324b comprises a run status icon, an extemal-transfer-status icon, a data-transfer- status icon, a variant-analysis-status icon, and another extemal-transfer-status icon.
  • the sequencing status system 106 customizes the set of status icons for the graphical status summary 324b to indicate a status of a different nucleotide sequencing taskset — with a different sequencing run, different variant analysis, and other different tasks.
  • an extemal-transfer-status icon 344 indicates a different status of the extemal-analysis- data transfer of data resulting from a variant analysis to an external storage.
  • the client device 114 presents an error icon or warning sign icon to represent an error status for the extemal-analysis-data transfer.
  • the expanded active taskset overview 340b includes the expanded graphical status summary 336b with textual task summaries for status icons differing from the textual task summaries for the expanded graphical status summary 336a depicted in FIG. 3C.
  • the client device 114 presents a textual -task-status summary 346 that describes the nucleotide sequencing task represented by the extemal-transfer-status icon 344 with a warning message or an error message.
  • the textual -task-status summary 346 indicates that the extemal-analysis-data transfer for the nucleotide sequencing taskset has encountered an error and is unable to complete.
  • the sequencing status system 106 can likewise customize a selectable intervention option for different nucleotide sequencing tasks — based on a determined status of a particular nucleotide sequencing task. As shown in FIG. 3D, for instance, the sequencing status system 106 determines an error status for the extemal-analysis-data transfer of data generated during the variant analysis to an external storage. Based on the determined error status, the sequencing status system 106 provides (and the client device 114 presents) a re-initiate option 342b to re-initiate the extemal-analysis-data transfer.
  • the sequencing status system 106 Based on receiving an indication of a selection of the re-initiate option 342b from the client device 114, the sequencing status system 106 re-initiates (or sends a command to re-initiate) the extemal-analysis-data transfer either from a point at which the transfer encountered an error or from the beginning.
  • the sequencing status system 106 can customize one or both of a graphical status summary for a nucleotide sequencing taskset and a selectable intervention option based on a determined status of the nucleotide sequencing taskset or an individual nucleotide sequencing task.
  • FIG. 3E depicts the sequencing status system 106 generating a customized graphical status summary for a different nucleotide sequencing taskset based on the queried status of individual tasks within the nucleotide sequencing taskset. As shown by a transition from FIG. 3B to FIG. 3E — or a transition from FIG. 3D to FIG.
  • the client device 114 when the client device 114 detects a selection of an expand option 332c, the client device 114 presents, within an active sequencing interface 314d, the graphical status summary 324c as part of the active taskset overview 316c and an expanded graphical status summary 336c as part of an expanded active taskset overview 340c. Both the graphical status summary 324c and the expanded graphical status summary 336c represent a same nucleotide sequencing taskset.
  • the client device 114 presents the graphical status summary 324c with a textual-taskset-status summary 326c as part of the active taskset overview 316c.
  • the sequencing status system 106 customizes the textual-taskset-status summary 326c to represent the differing and current status of its corresponding nucleotide sequencing taskset.
  • the textual-taskset-status summary 326c describes as queued for beginning a variant analysis of base-call data from a sequencing run.
  • the client device 114 presents the graphical status summary 324c comprising a run status icon, an extemal-transfer-status icon, a data-transfer-status icon, a variant-analysis-status icon, and another extemal-transfer-status icon — each at corresponding designated positions.
  • a variant-analysis position (or fourth designated position) within the graphical status summary 324c, a variant-analysis-status icon 350 indicates a different status of the variant analysis from other graphical status summaries described above.
  • the client device 114 presents a light grey clock icon or a lighter shaded clock icon representing a queued status (or a scheduled status) for the variant analysis.
  • a server or other computing device has yet to commence the variant analysis as part of a corresponding nucleotide sequencing taskset.
  • the expanded graphical status summary 336c likewise includes textual task summaries for status icons differing from other textual task summaries for other expanded graphical status summaries described above.
  • the client device 114 presents a textual -task-status summary 352 that describes the variant analysis represented by the variant-analysis-status icon 350 to indicate that the variant analysis for the nucleotide sequencing taskset is queued or scheduled.
  • the textual-task-status summary 352 therefore, matches the textual -taskset-status summary 326c.
  • a textual-task-status summary for an individual nucleotide sequencing task is the same or similar to a corresponding textual-taskset-status summary for a corresponding nucleotide sequencing taskset.
  • the sequencing status system 106 customizes a selectable intervention option for a particular nucleotide sequencing task.
  • the sequencing status system 106 determines a queued status or a scheduled status for the variant analysis. Based on the determined error status, the sequencing status system 106 provides (and the client device 114 presents) a cancel option 354 to cancel the variant analysis. Based on receiving an indication of a selection of the cancel option 354 from the client device 114, the sequencing status system 106 cancels the variant analysis or sends a command to cancel the variant analysis.
  • the sequencing status system 106 provides a textual-taskset-status summary for a nucleotide sequencing taskset that corresponds to a particular nucleotide sequencing task. Indeed, in certain cases, the sequencing status system 106 can customize a textual-taskset-status summary to indicate a subtask for a more particularized status indicator.
  • FIG. 3F depicts an example of a more particularized textual-taskset-status summary. As shown by a transition from FIG. 3B to FIG. 3F — or a transition from FIG. 3E to FIG.
  • the client device 114 when the client device 114 detects a selection of an expand option 332d, the client device 114 presents, within an active sequencing interface 314e, the graphical status summary 324d as part of the active taskset overview 316d and an expanded graphical status summary 336d as part of an expanded active taskset overview 340d.
  • the client device 114 presents the graphical status summary 324d for a nucleotide sequencing taskset comprising a set of status icons indicating a sequencing run is in progress.
  • the graphical status summary 324d comprises a run status icon, an extemal-transfer-status icon, and a data-transfer-status icon each indicating an inprogress status.
  • the status icon depicts a blue clock icon or a darker shaded clock icon representing an in-progress status.
  • a computing device can initiate and perform in parallel multiple nucleotide sequencing tasks within a nucleotide sequencing taskset.
  • the sequencing status system 106 can likewise detect statuses and represent such parallel nucleotide sequencing tasks with corresponding status icons in real time.
  • the sequencing status system 106 customizes a textual -taskset-status summary 326d as part of the active taskset overview 316d to include more granular detail.
  • a sequencing device sends status data for a sequencing cycle to the sequencing status system 106 indicating a particular sequencing cycle and a particular nucleotide-fragment-read type of the sequencing cycle (e.g., most recently detected sequencing cycle and most recently detected nucleotide-fragment-read type).
  • the sequencing status system 106 provides (and the client device 114 presents) the textual- taskset-status summary 326d to indicate the particular sequencing cycle and the particular nucleotide-fragment-read type.
  • the textual -taskset-status summary 326d states, “Sequencing: Read 2, Cycle 102/151,” thereby indicating a specific nucleotide-fragment-read type and a specific cycle number.
  • the expanded graphical status summary 336d includes textual task summaries that generally indicate statuses of corresponding nucleotide sequencing tasksets.
  • the expanded graphical status summary 336d includes a textual -task-status summary for a run status icon, a textual-task-status summary for an extemal-transfer-status icon, and a textual-task-status summary for a data-transfer-status icon that respectively indicate that a sequencing run, an extemal-call-data transfer, and a data-analysis transfer are in progress.
  • the sequencing status system 106 can provide textual task summaries in an expanded graphical status summary that include more granular detail.
  • the sequencing status system 106 also provides a completed sequencing interface with summaries of completed nucleotide sequencing tasksets — with, for example, variant analysis completed and data from the variant analysis transferred to an external storage.
  • the client device 114 presents a completed sequencing interface 356 of the sequencing application 116 within the screen 301. Based on detecting a selection of a completed sequencing tab 358, for example, the client device 114 presents the completed sequencing interface 356.
  • the completed sequencing interface 356 includes completed taskset overviews 360 for nucleotide sequencing tasksets that have finished.
  • the completed taskset overviews 360 each in turn include high-level information concerning a completed nucleotide sequencing taskset, including a textual status summary (e.g., Completed), sequencing run name or nucleotide-sequencing-taskset name (e.g., Run 1234), secondary-analysis application type (e.g., DRAGEN Enrichment), collective sequencing metrics, and date completed.
  • a textual status summary e.g., Completed
  • sequencing run name or nucleotide-sequencing-taskset name e.g., Run 1234
  • secondary-analysis application type e.g., DRAGEN Enrichment
  • the sequencing status system 106 provides graphical user interfaces comprises details concerning nucleotide sequencing tasksets.
  • FIGS. 4A-4B illustrate the client device 114 presenting graphical user interfaces comprising details for a selected nucleotide sequencing taskset. Based on detecting a selection of a sequencing run name or a nucleotide- sequencing-taskset name in one of the graphical user interfaces shown in FIGS. 3A-3G, for example, the client device 114 presents a sequencing details interface 404a within a screen 402, as shown in FIG. 4A.
  • the client device 114 provides taskset materials details 406, sequencing run details 408, and sample sequencing metrics 410 for display within the sequencing details interface 404a.
  • the sequencing status system 106 provides, for display in the sequencing details interface 404a, a sequencing run name or nucleotide-sequencing-taskset name (e.g., Run 234), a sequencing run description or nucleotide- sequencing-taskset description, a secondary-analysis application type (e.g., DRAGEN Enrichment) and version (e.g., 3.5.2), a sequencing software application version (e.g., 2.0.5), an owner, and a textual-taskset-status summary (e.g., Completed).
  • a sequencing run name or nucleotide-sequencing-taskset name e.g., Run 234
  • a sequencing run description or nucleotide- sequencing-taskset description e.g., 3.5.2
  • a sequencing software application version e.g., 2.0.5
  • an owner e
  • the sequencing status system 106 provides, for display in the sequencing details interface 404a, a sequencing device name (e.g., Sequencer 2021), a technician name, a sample-nucleotide-slide type (e.g., S4 for a flow cell), a nucleotide-fragment read type for the sequencing run (e.g., Paired End), and nucleotide-fragment read lengths (e.g., Read 1: 151, index 1: 10).
  • a sequencing device name e.g., Sequencer 2021
  • a technician name e.g., a sample-nucleotide-slide type
  • a nucleotide-fragment read type for the sequencing run e.g., Paired End
  • nucleotide-fragment read lengths e.g., Read 1: 151, index 1: 10
  • the sequencing status system 106 provides, for display in the sequencing details interface 404a, collective sequencing metrics specific to a sample.
  • the sequencing status system 106 provides a selectable status option 412 that, when selected, causes the client device 114 to show a graphical status summary for the relevant nucleotide sequencing taskset detailed in the sequencing details interface 404a.
  • the client device 114 presents an expanded graphical status summary 414 of the relevant nucleotide sequencing taskset within a sequencing details interface 404b.
  • the client device 114 also presents textual -taskstatus summaries, start dates, and completed dates alongside (or in line with) status icons within the expanded graphical status summary 414.
  • the sequencing system 104 utilizes containers and pods to execute external workflows associated with nucleotide reads and base calls of a sample sequence.
  • the sequencing system 104 can analyze sequencing data via a diagnostic workflow to identify genetic markers or hereditary traits indicated within a genomic sample.
  • the sequencing status system 106 can send or receive data indicating a status of sequencing tasks performed by such containers or pods.
  • FIG. 5 illustrates an example diagram of components, applications, devices, and containers of a system architecture (e.g., installed on a local server device) involved in implementing an external diagnostic workflow in accordance with one or more embodiments.
  • the sequencing system 104 utilizes a sequencing device (e.g., the sequencing device 108) to communicate with various components or systems and to perform sequencing operations used by, and/or as instructed by, a version of a variant analysis model on a local server (e.g., a version of DRAGEN on the local server device 102).
  • a sequencing device e.g., the sequencing device 108
  • the sequencing system 104 communicates with a BaseSpace Sequencing Hub (“BSSH’) or cloudbased interface for research use only (“RUO”) and a lab information management system (“LIMS”) to generate base calls and other sequencing data for nucleotide bases of a genomic sample.
  • BSSH BaseSpace Sequencing Hub
  • RUO research use only
  • LIMS lab information management system
  • the sequencing system 104 Based on information from the BSSH RUO and/or the LIMS, the sequencing system 104 performs a real-time analysis (“RTA”) of a sample. More specifically, the sequencing system 104 performs RTA to determine base calls, variant calls, and/or various metrics from nucleotide bases of a genomic sample according to a sequencing plan. Based on the RTA, the sequencing system 104 generates a binary base call (“BCL”) file that includes raw data generated and output by one or more sequencing runs (e.g., via the RTA). Indeed, the BCL file can indicate base calls, variant calls, and/or other sequencing information for interpretation by a variant analysis model and/or some other system.
  • RTA real-time analysis
  • the sequencing system 104 provides control software (e.g., including a user interface) for planning or scheduling a sequencing run on a particular sample.
  • control software e.g., including a user interface
  • the sequencing system 104 provides control software and a user interface for planning one or more sequencing runs to, for example, test a genomic sample for a particular genetic marker according to plan parameters.
  • the control software enables a user to specify parameters for a sequencing run and/or to test for specific markers.
  • the sequencing system 104 can integrate the control software for the sequencing device with a user interface web portal (which includes a standalone web browser and control software integration) to interface with the sequencing device for planning a sequencing run.
  • the sequencing system 104 facilitates local planning for a sequencing run, where the planning software (e.g., the control software) is hosted by a local server device, such as a local edge server.
  • the sequencing system 104 facilitates cloud planning for a sequencing run, where the planning software (e.g., the control software) is hosted on a cloud server rather than a local server.
  • the execution of a variant analysis model can be local or cloud-based as well, depending on whether the server hosting the variant analysis model is a local server (e.g., the local server device 102).
  • the sequencing status system 106 can be executed either locally on a local server device located at or near the sequencing device 108 or remotely on a cloud-based server device in combination with (ii) a variant analysis model (e.g., DRAGEN) executed either locally on a local server device located at or near the sequencing device 108 or remotely on a cloud-based server device.
  • a variant analysis model e.g., DRAGEN
  • the system architecture 500 of the sequencing system 104 includes, or communicates with, containers or systems associated with one or more core services. Indeed, as shown, the sequencing system 104 includes the services of the system architecture 500.
  • the system architecture 500 includes a container orchestration engine 501 (e.g., K3S or Kubemetes) to manage and implement various pods and containers associated with performing genomic analyses via diagnostic workflows.
  • the sequencing system 104 utilizes a container orchestration engine 501 to orchestrate or coordinate a diagnostic workflow to analyze a genomic sequence for base calling, variant calling (e.g., as indicated by an application of a third-party system).
  • the container orchestration engine 501 includes pods and containers to perform other functions as well, including user management, application management, run management, variant analysis model management, instrument management, data copying, and audit logging.
  • the system architecture 500 includes a user management service 502 that includes a set of one or more user management pods or containers.
  • the user management service 502 performs various processes or functions for providing a single sign-on (“SSO”) experience system wide.
  • the user management service 502 can include one or more containers or pods that include or access user information for a third-party system to, for example, determine a diagnostic workflow (e.g., from one of the third-party systems) for analyzing a genomic sequence, including user settings or preferences for executing the diagnostic workflow. Based on the determination of the diagnostic workflow and/or the user settings, the user management service 502 can communicate with other services of the system architecture 500 to initiate performance of the diagnostic workflow to analyze a genomic sequence accordingly.
  • a diagnostic workflow e.g., from one of the third-party systems
  • the system architecture 500 includes or utilizes an application management service 504 in communication with the container orchestration engine 501.
  • the application management service 504 manages application package installation for diagnostic workflows.
  • the application management service 504 further includes a resource manager.
  • the resource manager can access or utilize a genomic analysis device resource as specified by an application specification and/or as part of a diagnostic workflow.
  • the resource manager identifies a resource label to access a designated resource, such as an FPGA or a CPU, as a schedulable resource for access via the container orchestration engine.
  • the application management service 504 includes (or receives from a third-party system) an application specification that indicates an FPGA or a CPU or some other genomic analysis device for executing a diagnostic workflow of a genomic analysis application (or a particular workflow pod), and the resource manager therefore accesses or communicates with the specified device (or other resource) for facilitating execution of the genomic analysis application (or the particular workflow pod).
  • the system architecture 500 includes or utilizes a run management and orchestration service 506.
  • the run management and orchestration service 506 includes one or more containers or pods for facilitating and executing genomic analysis via a diagnostic workflow, such as a sequencing run, a primary analysis, a secondary analysis, or a tertiary analysis.
  • the run management and orchestration service 506 includes computer code or instructions for executing a sequencing run (and/or further analysis) according to an installed version of a variant analysis model.
  • the run management and orchestration service 506 communicates with the workflow engine 514 to execute a custom diagnostic workflow for an application, such as an application associated with a third-party system (e.g., an oncology assay application, such as TS0500 application; a QC application; or another application).
  • the run management and orchestration service 506 further includes code for communicating with the data copy service 512 to copy input and output sequencing data (e.g., from a BCL fde generated by a sequencing device) for performing a genomic analysis and/or for storing in a database, such as a local network attached storage (“NAS”), server message block (“SMB”), or common internet file system (“CIFS”).
  • NAS local network attached storage
  • SMB server message block
  • CIFS common internet file system
  • the sequencing status system 106 is part of the run management and orchestration service 506.
  • the system architecture 500 includes a variant analysis model management service 508.
  • the variant analysis model management service 508 includes one or more containers or pods for managing a variant analysis model for performing genomic analysis.
  • the variant analysis model management service 508 implements a particular diagnostic workflow using a variant analysis model to detect a genetic marker for a certain condition within a sample genomic sequence.
  • the variant analysis model management service 508 manages model peripherals, such as licensing, self-testing, and version authentication for a variant analysis model.
  • the system architecture 500 includes an instrument management service 510.
  • the instrument management service 510 includes one or more containers or pods for pairing and monitoring instruments used as part of a sequencing workflow and/or a genomic analysis workflow after sequencing.
  • the instrument management service 510 manages instruments of a sequencing device and/or a variant analysis model to pair compatible instruments with indicated versions of a variant analysis model (or vice-versa).
  • the system architecture 500 further includes an audit logging service 516 for monitoring and logging performance of instruments, components of a variant analysis model, and/or containers within an application workflow.
  • the audit logging service 516 detects and logs errors or other auditing information associated with the system architecture 500.
  • the sequencing status system 106 can be deployed locally on an edge server (e.g., the local server device 102) or in the cloud such as on cloud-based servers hosting Illumina Connected Analytics (“ICA”) and/or cloud-based servers from Amazon Web Services (“AWS”).
  • edge server e.g., the local server device 102
  • ICA Illumina Connected Analytics
  • AWS Amazon Web Services
  • the sequencing status system 106 can be executed locally on the local server device 102 — or remotely on the server device(s) 110 — as part of planning software that plans resources based on user input for sequencing runs or other assays, and a variant analysis model from the sequencing system 104 can likewise be executed locally on the local server device 102 — or remotely on the server device(s) 110 — to analyze BCL data and determine variant calls or other metrics.
  • FIG. 6A this figure illustrates a flowchart of a series of acts 600a of querying the status of various tasks of a nucleotide sequencing taskset and generating a graphical status summary for the nucleotide sequencing taskset in accordance with one or more embodiments of the present disclosure. While FIG. 6A illustrates acts according to one embodiment, alternative embodiments may omit, add to, reorder, and/or modify any of the acts shown in FIG. 6A. The acts of FIG. 6A can be performed as part of a method.
  • a non -transitory computer readable storage medium can comprise instructions that, when executed by one or more processors, cause a computing device or a system to perform the acts depicted in FIG. 6A.
  • a system comprising at least one processor and a non-transitory computer readable medium comprising instructions that, when executed by one or more processors, cause the system to perform the acts of FIG. 6A.
  • the acts 600a include an act 610a of receiving a status query for a nucleotide sequencing taskset.
  • the act 610a includes receiving, from a computing device, a status query for a nucleotide sequencing taskset depicted by an active sequencing interface.
  • the acts 600a include an act 620a of determining statuses of nucleotide sequencing tasks within the nucleotide sequencing taskset.
  • the act 620a includes determining, for the nucleotide sequencing taskset, statuses of a sequencing run, a data-analysis transfer of base-call data generated during the sequencing run, and a variant analysis of the base-call data.
  • the act 620a includes determining, for the nucleotide sequencing taskset and from a sequencing device, a status of a sequencing run; and determining, for the nucleotide sequencing taskset and from a local server corresponding to the sequencing device, statuses of a data-analysis transfer of base-call data generated during the sequencing run and a variant analysis of the base-call data.
  • determining a status of the data-analysis transfer comprises determining a status of a transfer of base-call data generated during the sequencing run from the sequencing device to the local server.
  • the act 620a includes determining a status of an extemal-call-data transfer of the base-call data generated during the sequencing run to an external storage; and providing the graphical status summary further comprising an extemal- transfer-status icon indicating a status of the extemal-call-data transfer.
  • the act 620a includes determining a status of an extemal-analysis-data transfer of data generated during the variant analysis to an external storage; and providing the graphical status summary further comprising an extemal-transfer-status icon indicating a status of the extemal-analysis-data transfer.
  • the acts 600a include an act 630a of providing for display a graphical status summary for the nucleotide sequencing taskset.
  • the act 630a includes providing, for display within the active sequencing interface, a graphical status summary for the nucleotide sequencing taskset comprising a run status icon indicating a status of the sequencing run, a data-transfer-status icon indicating a status of the data-analysis transfer, and a variant-analysis-status icon indicating a status of the variant analysis.
  • the act 630a includes providing the graphical status summary for display on the sequencing device, local computing device connected to a local server, or an external computing device connected to the server via a network.
  • the acts 600a further include determining a collective base-call-quality metric indicating an accuracy of base calls generated during the sequencing run; and providing, for display within the active sequencing interface, the collective base-call-quality metric. In some cases, the collective base-call-quality metric is displayed proximate to the graphical status summary. Similarly, in some embodiments, the acts 600a further include determining a collective pass filter metric indicating a subset of base calls generated during the sequencing run that satisfy a quality filter; and providing, for display within the active sequencing interface, the collective pass filter metric. In some cases, the collective pass filter metric is displayed proximate to the graphical status summary.
  • the acts 600a further include determining an updated status of one or more of the sequencing run, the data-analysis transfer, or the variant analysis; and providing an updated graphical status summary comprising one or more of an updated run status icon indicating an updated status of the sequencing run, an updated data-transfer-status icon indicating an updated status of the data-analysis transfer, and an updated variant-analysis- status icon indicating an updated status of the variant analysis.
  • the acts 600a include receiving an indication of a user selection of an expand option corresponding to the graphical status summary; and based on the indication of the user selection of the expand option, providing, for display within the active sequencing interface, an expanded graphical status summary comprising a textual status summary for the run status icon, a textual status summary for the data-transfer-status icon, and a textual status summary for the variant-analysis-status icon.
  • the acts 600a include receiving an indication of a user selection of a cancel option corresponding to one or more of the sequencing run, the data-analysis transfer, or the variant analysis; and based on the indication of the user selection of the cancel option, canceling one or more of the sequencing run, the data-analysis transfer, or the variant analysis.
  • the acts 600a include receiving an indication of a user selection of a re-initiate option corresponding to one or more of the sequencing run, the data-analysis transfer, or the variant analysis; and based on the indication of the user selection of the re-initiate option, re-initiating one or more of the sequencing run, the data-analysis transfer, or the variant analysis.
  • FIG. 6B this figure illustrates a flowchart of a series of acts 600b of querying the status of various tasks of nucleotide sequencing tasksets and generating graphical status summaries for the nucleotide sequencing tasksets in accordance with one or more embodiments of the present disclosure.
  • FIG. 6B illustrates acts according to one embodiment
  • alternative embodiments may omit, add to, reorder, and/or modify any of the acts shown in FIG. 6B.
  • the acts of FIG. 6B can be performed as part of a method.
  • a non-transitory computer readable storage medium can comprise instructions that, when executed by one or more processors, cause a computing device or a system to perform the acts depicted in FIG. 6B.
  • a system comprising at least one processor and a non- transitory computer readable medium comprising instructions that, when executed by one or more processors, cause the system to perform the acts of FIG. 6B.
  • the acts 600b include an act 610b of receiving one or more status queries for nucleotide sequencing tasksets.
  • the act 610b includes receiving, from a computing device, one or more status queries for nucleotide sequencing tasksets depicted by an active sequencing interface.
  • the acts 600b include an act 620b of determining statuses of respective nucleotide sequencing tasks for the nucleotide sequencing tasksets.
  • the act 620b includes determining, for the nucleotide sequencing tasksets, statuses of respective sequencing runs, respective data-analysis transfers of base-call data generated during the respective sequencing runs, and respective variant analyses of the base-call data.
  • the act 620b includes determining, for a first nucleotide sequencing taskset among the nucleotide sequencing tasksets, statuses of a first sequencing run, a first data-analysis transfer of base-call data generated during the first sequencing run, and a first variant analysis of the base-call data; and determining, for a second nucleotide sequencing taskset among the nucleotide sequencing tasksets, statuses of a second sequencing run, a second data- analysis transfer of base-call data generated during the second sequencing run, and a second variant analysis of the base-call data.
  • the acts 600b include an act 630b of providing for display graphical status summaries for the nucleotide sequencing tasksets.
  • the act 630b includes providing, for display within the active sequencing interface, graphical status summaries for the nucleotide sequencing tasksets comprising run status icon indicating statuses of the respective sequencing runs, data-transfer-status icons indicating statuses of the respective data-analysis transfers, and variant-analysis-status icons indicating statuses of the respective variant analyses.
  • providing the graphical status summaries for the nucleotide sequencing tasksets comprises providing, for display within the active sequencing interface: a first graphical status summary for the first nucleotide sequencing taskset comprising a first run status icon indicating a status of the first sequencing run, a first data-transfer-status icon indicating a status of the first data-analysis transfer, and a first variant-analysis-status icon indicating a status of the first variant analysis; and a second graphical status summary for the second nucleotide sequencing taskset comprising a second run status icon indicating a status of the second sequencing run, a second data-transfer-status icon indicating a status of the second data-analysis transfer, and a second variant-analysis-status icon indicating a status of the second variant analysis.
  • the acts 600b further include determining statuses of respective extemal-call-data transfers of the base-call data generated during the respective sequencing runs to an external storage; and providing the graphical status summaries further comprising extemal-transfer-status icons indicating statuses of the respective extemal-call-data transfers.
  • the acts 600b further include determining statuses of respective extemal-analysis-data transfers of data generated during the respective variant analyses to an external storage; and providing the graphical status summaries further comprising extemal-transfer-status icons indicating statuses of the respective extemal- analysis-data transfers.
  • the acts 600b further include receiving an indication of a user selection of an expand option corresponding to a graphical status summary among the graphical status summaries; and based on the indication of the user selection of the expand option, provide, for display within the active sequencing interface, an expanded graphical status summary comprising a textual status summary for a run status icon within the graphical status summary, a textual status summary for a data-transfer-status icon within the graphical status summary, and a textual status summary for a variant-analysis-status icon within the graphical status summary.
  • nucleic acid sequencing techniques can be used in conjunction with a variety of nucleic acid sequencing techniques. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Embodiments in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable.
  • the process to determine the nucleotide sequence of a target nucleic acid i.e., a nucleic-acid polymer
  • Preferred embodiments include sequencing-by-synthesis (SBS) techniques.
  • SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand.
  • a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery.
  • more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.
  • SBS can utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moieties.
  • Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth in further detail below.
  • the number of nucleotides added in each cycle is generally variable and dependent upon the template sequence and the mode of nucleotide delivery.
  • the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.).
  • SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like.
  • a characteristic of the label such as fluorescence of the label
  • a characteristic of the nucleotide monomer such as molecular weight or charge
  • a byproduct of incorporation of the nucleotide such as release of pyrophosphate; or the like.
  • the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be the indistinguishable under the detection techniques being used.
  • the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by
  • Preferred embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P.
  • PPi inorganic pyrophosphate
  • the nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminescent signals that are produced due to incorporation of a nucleotides at the features of the array.
  • An image can be obtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images.
  • the images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.
  • cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference.
  • This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in WO 91/06678 and WO 07/123,744, each of which is incorporated herein by reference.
  • the availability of fluorescently- labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing.
  • Polymerases can also be coengineered to efficiently incorporate and extend from these modified nucleotides.
  • the labels do not substantially inhibit extension under SBS reaction conditions.
  • the detection labels can be removable, for example, by cleavage or degradation. Images can be captured following incorporation of labels into arrayed nucleic acid features.
  • each cycle involves simultaneous delivery of four different nucleotide types to the array and each nucleotide type has a spectrally distinct label. Four images can then be obtained, each using a detection channel that is selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially and an image of the array can be obtained between each addition step.
  • each image will show nucleic acid features that have incorporated nucleotides of a particular type. Different features are present or absent in the different images due the different sequence content of each feature. However, the relative position of the features will remain unchanged in the images. Images obtained from such reversible terminator-SBS methods can be stored, processed and analyzed as set forth herein. Following the image capture step, labels can be removed and reversible terminator moieties can be removed for subsequent cycles of nucleotide addition and detection. Removal of the labels after they have been detected in a particular cycle and prior to a subsequent cycle can provide the advantage of reducing background signal and crosstalk between cycles. Examples of useful labels and removal methods are set forth below.
  • nucleotide monomers can include reversible terminators.
  • reversible terminators/cleavable fluors can include fluor linked to the ribose moiety via a 3' ester linkage (Metzker, Genome Res. 15:1767-1776 (2005), which is incorporated herein by reference).
  • Other approaches have separated the terminator chemistry from the cleavage of the fluorescence label (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), which is incorporated herein by reference in its entirety).
  • Ruparel et al described the development of reversible terminators that used a small 3' allyl group to block extension, but could easily be deblocked by a short treatment with a palladium catalyst.
  • the fluorophore was attached to the base via a photocleavable linker that could easily be cleaved by a 30 second exposure to long wavelength UV light.
  • disulfide reduction or photocleavage can be used as a cleavable linker.
  • Another approach to reversible termination is the use of natural termination that ensues after placement of a bulky dye on a dNTP.
  • the presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and/or electrostatic hindrance.
  • Some embodiments can utilize detection of four different nucleotides using fewer than four different labels.
  • SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Patent Application Publication No. 2013/0079232.
  • a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g. via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair.
  • nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal.
  • one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels.
  • An exemplary embodiment that combines all three examples is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g. dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g.
  • sequencing data can be obtained using a single channel.
  • the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated.
  • the third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.
  • Some embodiments can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.
  • the oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize.
  • images can be obtained following treatment of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features are present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images.
  • Some embodiments can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing.” Trends Biotechnol. 18, 147- 151 (2000); Deamer, D. and D. Branton, “Characterization of nucleic acids by nanopore analysis”. Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entireties).
  • the target nucleic acid passes through a nanopore.
  • the nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin.
  • each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore.
  • Some embodiments can utilize methods involving the real-time monitoring of DNA polymerase activity.
  • Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate- labeled nucleotides as described, for example, in U.S. Pat. No. 7,329,492 and U.S. Pat. No. 7,211,414 (each of which is incorporated herein by reference) or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No.
  • FRET fluorescence resonance energy transfer
  • the illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations.” Science 299, 682-686 (2003); Lundquist, P. M. et al.
  • Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product.
  • sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in US 2009/0026082 Al; US 2009/0127589 Al; US 2010/0137143 Al; or US 2010/0282617 Al, each of which is incorporated herein by reference.
  • Methods set forth herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.
  • the above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously.
  • different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner.
  • the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner.
  • the target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface.
  • the array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PCR as described in further detail below.
  • the methods set forth herein can use arrays having features at any of a variety of densities including, for example, at least about 10 features/cm2, 100 features/cm2, 500 features/cm2, 1,000 features/cm2, 5,000 features/cm2, 10,000 features/cm2, 50,000 features/cm2, 100,000 features/cm2, 1,000,000 features/cm2, 5,000,000 features/cm2, or higher.
  • an advantage of the methods set forth herein is that they provide for rapid and efficient detection of a plurality of target nucleic acid in parallel. Accordingly the present disclosure provides integrated systems capable of preparing and detecting nucleic acids using techniques known in the art such as those exemplified above.
  • an integrated system of the present disclosure can include fluidic components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, the system comprising components such as pumps, valves, reservoirs, fluidic lines and the like.
  • a flow cell can be configured and/or used in an integrated system for detection of target nucleic acids. Exemplary flow cells are described, for example, in US 2010/0111768 Al and US Ser. No.
  • one or more of the fluidic components of an integrated system can be used for an amplification method and for a detection method.
  • one or more of the fluidic components of an integrated system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in a sequencing method such as those exemplified above.
  • an integrated system can include separate fluidic systems to carry out amplification methods and to carry out detection methods.
  • Examples of integrated sequencing systems that are capable of creating amplified nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeqTM platform (Illumina, Inc., San Diego, CA) and devices described in US Ser. No. 13/273,666, which is incorporated herein by reference.
  • sample and its derivatives, is used in its broadest sense and includes any specimen, culture and the like that is suspected of including a target.
  • the sample comprises DNA, RNA, PNA, LNA, chimeric or hybrid forms of nucleic acids.
  • the sample can include any biological, clinical, surgical, agricultural, atmospheric or aquatic-based specimen containing one or more nucleic acids.
  • the term also includes any isolated nucleic acid sample such a genomic DNA, fresh-frozen or formalin-fixed paraffin-embedded nucleic acid specimen.
  • the sample can be from a single individual, a collection of nucleic acid samples from genetically related members, nucleic acid samples from genetically unrelated members, nucleic acid samples (matched) from a single individual such as a tumor sample and normal tissue sample, or sample from a single source that contains two distinct forms of genetic material such as maternal and fetal DNA obtained from a maternal subject, or the presence of contaminating bacterial DNA in a sample that contains plant or animal DNA.
  • the source of nucleic acid material can include nucleic acids obtained from a newborn, for example as typically used for newborn screening.
  • the nucleic acid sample can include high molecular weight material such as genomic DNA (gDNA).
  • the sample can include low molecular weight material such as nucleic acid molecules obtained from FFPE or archived DNA samples.
  • low molecular weight material includes enzymatically or mechanically fragmented DNA.
  • the sample can include cell-free circulating DNA.
  • the sample can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture micro-dissections, surgical resections, and other clinical or laboratory obtained samples.
  • the sample can be an epidemiological, agricultural, forensic or pathogenic sample.
  • the sample can include nucleic acid molecules obtained from an animal such as a human or mammalian source.
  • the sample can include nucleic acid molecules obtained from a non-mammalian source such as a plant, bacteria, virus or fungus.
  • the source of the nucleic acid molecules may be an archived or extinct sample or species.
  • forensic samples can include nucleic acids obtained from a crime scene, nucleic acids obtained from a missing persons DNA database, nucleic acids obtained from a laboratory associated with a forensic investigation or include forensic samples obtained by law enforcement agencies, one or more military services or any such personnel.
  • the nucleic acid sample may be a purified sample or a crude DNA containing lysate, for example derived from a buccal swab, paper, fabric or other substrate that may be impregnated with saliva, blood, or other bodily fluids.
  • the nucleic acid sample may comprise low amounts of, or fragmented portions of DNA, such as genomic DNA.
  • target sequences can be present in one or more bodily fluids including but not limited to, blood, sputum, plasma, semen, urine and serum.
  • target sequences can be obtained from hair, skin, tissue samples, autopsy or remains of a victim.
  • nucleic acids including one or more target sequences can be obtained from a deceased animal or human.
  • target sequences can include nucleic acids obtained from non-human DNA such a microbial, plant or entomological DNA.
  • target sequences or amplified target sequences are directed to purposes of human identification.
  • the disclosure relates generally to methods for identifying characteristics of a forensic sample.
  • the disclosure relates generally to human identification methods using one or more target specific primers disclosed herein or one or more target specific primers designed using the primer design criteria outlined herein.
  • a forensic or human identification sample containing at least one target sequence can be amplified using any one or more of the target-specific primers disclosed herein or using the primer criteria outlined herein.
  • the components of the sequencing status system 106 can include software, hardware, or both.
  • the components of the sequencing status system 106 can include one or more instructions stored on a computer-readable storage medium and executable by processors of one or more computing devices (e.g., the client device 114). When executed by the one or more processors, the computer-executable instructions of the sequencing status system 106 can cause the computing devices to perform the bubble detection methods described herein.
  • the components of the sequencing status system 106 can comprise hardware, such as special purpose processing devices to perform a certain function or group of functions. Additionally, or alternatively, the components of the sequencing status system 106 can include a combination of computer-executable instructions and hardware.
  • the components of the sequencing status system 106 performing the functions described herein with respect to the sequencing status system 106 may, for example, be implemented as part of a stand-alone application, as a module of an application, as a plug-in for applications, as a library function or functions that may be called by other applications, and/or as a cloud-computing model.
  • components of the sequencing status system 106 may be implemented as part of a stand-alone application on a personal computing device or a mobile device.
  • the components of the sequencing status system 106 may be implemented in any application that provides sequencing services including, but not limited to Illumina BaseSpace, Illumina DRAGEN, or Illumina TruSight software.
  • Embodiments of the present disclosure may comprise or utilize a special purpose or general-purpose computer including computer hardware, such as, for example, one or more processors and system memory, as discussed in greater detail below.
  • Embodiments within the scope of the present disclosure also include physical and other computer-readable media for carrying or storing computer-executable instructions and/or data structures.
  • one or more of the processes described herein may be implemented at least in part as instructions embodied in anon-transitory computer-readable medium and executable by one or more computing devices (e.g., any of the media content access devices described herein).
  • a processor e.g., a microprocessor
  • Computer-readable media can be any available media that can be accessed by a general purpose or special purpose computer system.
  • Computer-readable media that store computerexecutable instructions are non-transitory computer-readable storage media (devices).
  • Computer- readable media that carry computer-executable instructions are transmission media.
  • embodiments of the disclosure can comprise at least two distinctly different kinds of computer-readable media: non-transitory computer-readable storage media (devices) and transmission media.
  • Non-transitory computer-readable storage media includes RAM, ROM, EEPROM, CD-ROM, solid state drives (SSDs) (e.g., based on RAM), Flash memory, phasechange memory (PCM), other types of memory, other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computer.
  • SSDs solid state drives
  • PCM phasechange memory
  • a “network” is defined as one or more data links that enable the transport of electronic data between computer systems and/or modules and/or other electronic devices.
  • a network or another communications connection can include a network and/or data links which can be used to carry desired program code means in the form of computer-executable instructions or data structures and which can be accessed by a general purpose or special purpose computer. Combinations of the above should also be included within the scope of computer- readable media.
  • program code means in the form of computer-executable instructions or data structures can be transferred automatically from transmission media to non-transitory computer-readable storage media (devices) (or vice versa).
  • computer-executable instructions or data structures received over a network or data link can be buffered in RAM within a network interface module (e.g., a NIC), and then eventually transferred to computer system RAM and/or to less volatile computer storage media (devices) at a computer system.
  • a network interface module e.g., a NIC
  • non-transitory computer- readable storage media (devices) can be included in computer system components that also (or even primarily) utilize transmission media.
  • Computer-executable instructions comprise, for example, instructions and data which, when executed at a processor, cause a general-purpose computer, special purpose computer, or special purpose processing device to perform a certain function or group of functions.
  • computer-executable instructions are executed on a general-purpose computer to turn the general-purpose computer into a special purpose computer implementing elements of the disclosure.
  • the computer executable instructions may be, for example, binaries, intermediate format instructions such as assembly language, or even source code.
  • the disclosure may be practiced in network computing environments with many types of computer system configurations, including, personal computers, desktop computers, laptop computers, message processors, hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, mobile telephones, PDAs, tablets, pagers, routers, switches, and the like.
  • the disclosure may also be practiced in distributed system environments where local and remote computer systems, which are linked (either by hardwired data links, wireless data links, or by a combination of hardwired and wireless data links) through a network, both perform tasks.
  • program modules may be located in both local and remote memory storage devices.
  • Embodiments of the present disclosure can also be implemented in cloud computing environments.
  • “cloud computing” is defined as a model for enabling on-demand network access to a shared pool of configurable computing resources.
  • cloud computing can be employed in the marketplace to offer ubiquitous and convenient on-demand access to the shared pool of configurable computing resources.
  • the shared pool of configurable computing resources can be rapidly provisioned via virtualization and released with low management effort or service provider interaction, and then scaled accordingly.
  • a cloud-computing model can be composed of various characteristics such as, for example, on-demand self-service, broad network access, resource pooling, rapid elasticity, measured service, and so forth.
  • a cloud-computing model can also expose various service models, such as, for example, Software as a Service (SaaS), Platform as a Service (PaaS), and Infrastructure as a Service (laaS).
  • SaaS Software as a Service
  • PaaS Platform as a Service
  • laaS Infrastructure as a Service
  • a cloud-computing model can also be deployed using different deployment models such as private cloud, community cloud, public cloud, hybrid cloud, and so forth.
  • a “cloud-computing environment” is an environment in which cloud computing is employed.
  • FIG. 7 illustrates a block diagram of a computing device 700 that may be configured to perform one or more of the processes described above.
  • the computing device 700 may implement the sequencing status system 106 and the sequencing system 104.
  • the computing device 700 can comprise a processor 702, a memory 704, a storage device 706, an I/O interface 708, and a communication interface 710, which may be communicatively coupled by way of a communication infrastructure 712.
  • the computing device 700 can include fewer or more components than those shown in FIG. 7. The following paragraphs describe components of the computing device 700 shown in FIG. 7 in additional detail.
  • the processor 702 includes hardware for executing instructions, such as those making up a computer program.
  • the processor 702 may retrieve (or fetch) the instructions from an internal register, an internal cache, the memory 704, or the storage device 706 and decode and execute them.
  • the memory 704 may be a volatile or nonvolatile memory used for storing data, metadata, and programs for execution by the processor(s).
  • the storage device 706 includes storage, such as a hard disk, flash disk drive, or other digital storage device, for storing data or instructions for performing the methods described herein.
  • the I/O interface 708 allows a user to provide input to, receive output from, and otherwise transfer data to and receive data from computing device 700.
  • the I/O interface 708 may include a mouse, a keypad or a keyboard, a touch screen, a camera, an optical scanner, network interface, modem, other known I/O devices or a combination of such I/O interfaces.
  • the I/O interface 708 may include one or more devices for presenting output to a user, including, but not limited to, a graphics engine, a display (e.g., a display screen), one or more output drivers (e.g., display drivers), one or more audio speakers, and one or more audio drivers.
  • the VO interface 708 is configured to provide graphical data to a display for presentation to a user.
  • the graphical data may be representative of one or more graphical user interfaces and/or any other graphical content as may serve a particular implementation.
  • the communication interface 710 can include hardware, software, or both. In any event, the communication interface 710 can provide one or more interfaces for communication (such as, for example, packet-based communication) between the computing device 700 and one or more other computing devices or networks.
  • the communication interface 710 may include a network interface controller (NIC) or network adapter for communicating with an Ethernet or other wire-based network or a wireless NIC (WNIC) or wireless adapter for communicating with a wireless network, such as a WI-FI.
  • NIC network interface controller
  • WNIC wireless NIC
  • the communication interface 710 may facilitate communications with various types of wired or wireless networks.
  • the communication interface 710 may also facilitate communications using various communication protocols.
  • the communication infrastructure 712 may also include hardware, software, or both that couples components of the computing device 700 to each other.
  • the communication interface 710 may use one or more networks and/or protocols to enable a plurality of computing devices connected by a particular infrastructure to communicate with each other to perform one or more aspects of the processes described herein.
  • the sequencing process can allow a plurality of devices (e.g., a client device, sequencing device, and server device(s)) to exchange information such as sequencing data and error notifications.
  • objects includes all things that are suitable for imaging, viewing, analyzing, inspecting, or profiling with the optical systems described herein.
  • objects may include semiconductor wafers or chips, recordable media, samples, flow cells, microparticles, slides, or microarrays.
  • Objects generally include one or more surfaces and/or one or more interfaces that a user may desire to image, view, analyze, inspect, and/or determine a profile thereof.
  • the objects may have surfaces or interfaces with relief features such as wells, pits, ridges, bumps, beads or the like.
  • a sample may be imaged or scanned for subsequent analysis.
  • a sample may include biological or chemical substances of interests and, optionally, an optical substrate that supports the biological or chemical substances.
  • a sample may or may not include an optical substrate.
  • biological or chemical substances is not intended to be limiting, but may include a variety of biological or chemical substances that are suitable for being imaged or examined with the optical systems described herein.
  • biological or chemical substances include biomolecules, such as nucleosides, nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes, polypeptides, antibodies, antigens, ligands, receptors, polysaccharide, carbohydrate, polyphosphates, nanopores, organelles, lipid layers, cells, tissues, organisms, and biologically active chemical compound(s) such as analogs or mimetics of the aforementioned species.
  • biomolecules such as nucleosides, nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes, polypeptides, antibodies, antigens, ligands, receptors, polysaccharide, carbohydrate, polyphosphates, nanopores, organelles, lipid layers, cells, tissues, organisms, and biologically active chemical compound(s) such as analogs or mimetics of the aforementioned species.
  • the biological or chemical substances may be supported by an optical substrate.
  • the term “optical substrate” is not intended to be limiting, but may include various materials that support the biological or chemical substances and permit the biological or chemical substances to be at least one of viewed, imaged, and examined.
  • the optical substrate may comprise a transparent material that reflects a portion of incident light and refracts a portion of the incident light.
  • the optical substrate may be, for example, a mirror that reflects the incident light entirely such that no light is transmitted through the optical substrate.
  • the optical substrate has a flat surface.
  • the optical substrate can have a surface with relief features such as wells, pits, ridges, bumps, beads or the like.
  • the optical substrate is a flow cell having flow channels where nucleic acids are sequenced.
  • the optical substrate may include one or more slides, planar chips (such as those used in microarrays), or microparticles.
  • the microparticles may be held by another optical substrate, such as a slide or grooved plate.
  • the optical substrate includes diffraction grating based encoded optical identification elements similar to or the same as those described in pending U.S. patent application Ser. No. 10/661,234, entitled Diffraction Grating Based Optical Identification Element, filed Sep.
  • a bead cell or plate for holding the optical identification elements may be similar to or the same as that described in pending U.S. patent application Ser. No. 10/661,836, entitled “Method and Apparatus for Aligning Microbeads in Order to Interrogate the Same”, filed Sep. 12, 2003, and U.S. Pat. No. 7,164,533, entitled “Hybrid Random Bead/Chip Based Microarray”, issued Jan. 16, 2007, as well as U.S. patent application Ser. No. 60/609,583, entitled “Improved Method and Apparatus for Aligning Microbeads in Order to Interrogate the Same”, filed Sep. 13, 2004, Ser. No. 60/1010,910, entitled “Method and Apparatus for Aligning Microbeads in Order to Interrogate the Same”, filed Sep. 17, 2004, each of which is incorporated herein by reference in its entirety.
  • optical components or “focus components” includes various elements that affect the transmission of light.
  • Optical components may be, for example, reflectors, dichroics, beam splitters, collimators, lenses, filters, wedges, prisms, mirrors, and the like.
  • optical systems described herein may be constructed to include various components and assemblies as described in PCT application PCT/US07/07991, entitled “System and Devices for Sequence by Synthesis Analysis”, filed Mar. 30, 2007 and/or to include various components and assemblies as described in PCT application PCT/US2008/077850, entitled “Fluorescence Excitation and Detection System and Method”, filed Sep. 26, 2008, both of which the complete subject matter are incorporated herein by reference in their entirety.
  • optical systems can include various components and assemblies as described in U.S. Pat. No. 7,329,860, of which the complete subject matter is incorporated herein by reference in its entirety.
  • Optical systems can also include various components and assemblies as described in U.S. patent application Ser. No. 12/638,770, filed on Dec. 15, 2009, of which the complete subject matter is incorporated herein by reference in its entirety.
  • methods, and optical systems described herein may be used for sequencing nucleic acids.
  • sequencing-by-synthesis (SBS) protocols are particularly applicable.
  • SBS sequencing-by-synthesis
  • a plurality of fluorescently labeled modified nucleotides are used to sequence dense clusters of amplified DNA (possibly millions of clusters) present on the surface of an optical substrate (e.g., a surface that at least partially defines a channel in a flow cell).
  • the flow cells may contain nucleic acid samples for sequencing where the flow cells are placed within the appropriate flow cell holders.
  • the samples for sequencing can take the form of single nucleic acid molecules that are separated from each other so as to be individually resolvable, amplified populations of a nucleic acid molecules in the form of clusters or other features, or beads that are attached to one or more molecules of nucleic acid.
  • the nucleic acids can be prepared such that they comprise an oligonucleotide primer adjacent to an unknown target sequence.
  • a fluid flow subsystem (not shown).
  • Either a single type of nucleotide can be added at a time, or the nucleotides used in the sequencing procedure can be specially designed to possess a reversible termination property, thus allowing each cycle of the sequencing reaction to occur simultaneously in the presence of several types of labeled nucleotides (e.g., A, C, T, G).
  • the nucleotides can include detectable label moieties such as fluorophores. Where the four nucleotides are mixed together, the polymerase is able to select the correct base to incorporate and each sequence is extended by a single base. One or more lasers may excite the nucleic acids and induce fluorescence.
  • the fluorescence emitted from the nucleic acids is based upon the fluorophores of the incorporated base, and different fluorophores may emit different wavelengths of emission light.
  • Exemplary sequencing methods are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123,744; U.S. Pat. No. 7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat. No. 7,315,019; U.S. Pat. No. 7,405,281, and US 2008/0108082, each of which is incorporated herein by reference.
  • sequencing techniques that are applicable for use of the methods and systems set forth herein are pyrosequencing, nanopore sequencing, and sequencing by ligation.
  • Exemplary pyrosequencing techniques and samples that are particularly useful are described in U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320 and Ronaghi, Genome Research 11:3- 11 (2001), each of which is incorporated herein by reference.
  • Exemplary nanopore techniques and samples that are also useful are described in Deamer et al., Acc. Chem. Res. 35:817-825 (2002); Li et al., Nat. Mater.
  • optical systems described herein may be utilized for detection of samples that include microarrays.
  • a microarray may include a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location.
  • An array can include different probe molecules, or populations of the probe molecules, that are each located at a different addressable location on a substrate.
  • a microarray can include separate optical substrates, such as beads, each bearing a different probe molecule, or population of the probe molecules, that can be identified according to the locations of the optical substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid.
  • Exemplary arrays in which separate substrates are located on a surface include, without limitation, a Sentrix® Array or Sentrix® BeadChip Array available from Illumina®, Inc. (San Diego, Calif) or others including beads in wells such as those described in U.S. Pat. Nos. 6,266,459, 6,355,431, 6,770,441, and 6,859,570; and PCT Publication No. WO 00/63437, each of which is hereby incorporated by reference.
  • Other arrays having particles on a surface include those set forth in US 2005/0227252; WO 05/033681; and WO 04/024328, each of which is hereby incorporated by reference.
  • a typical microarray contains sites, sometimes referred to as features, each having a population of probes.
  • the population of probes at each site is typically homogenous having a single species of probe, but in some embodiments the populations can each be heterogeneous.
  • Sites or features of an array are typically discrete, being separated with spaces between each other.
  • the size of the probe sites and/or spacing between the sites can vary such that arrays can be high density, medium density or lower density. High density arrays are characterized as having sites separated by less than about 15 pm.
  • Medium density arrays have sites separated by about 15 to 30 pm, while low density arrays have sites separated by greater than 30 pm.
  • An array useful in the invention can have sites that are separated by less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, or 0.5 pm.
  • An apparatus or method of an embodiment of the invention can be used to image an array at a resolution sufficient to distinguish sites at the above densities or density ranges.
  • microarrays that can be used include, for example, an Affymetrix® GeneChip® microarray or other microarray synthesized in accordance with techniques sometimes referred to as VLSIPSTM (Very Large Scale Immobilized Polymer Synthesis) technologies as described, for example, in U.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659;
  • VLSIPSTM Very Large Scale Immobilized Polymer Synthesis
  • a spotted microarray can also be used in a method according to an embodiment of the invention.
  • An exemplary spotted microarray is a CodeLinkTM Array available from Amersham Biosciences.
  • Another microarray that is useful is one that is manufactured using inkjet printing methods such as SurePrintTM Technology available from Agilent Technologies.
  • the systems and methods set forth herein can be used to detect the presence of a particular target molecule in a sample contacted with the microarray. This can be determined, for example, based on binding of a labeled target analyte to a particular probe of the microarray or due to a target-dependent modification of a particular probe to incorporate, remove, or alter a label at the probe location. Any one of several assays can be used to identify or characterize targets using a microarray as described, for example, in U.S. Patent Application Publication Nos. 2003/0108867; 2003/0108900; 2003/0170684; 2003/0207295; or 2005/0181394, each of which is hereby incorporated by reference.
  • Exemplary labels that can be detected in accordance with embodiments of the invention, for example, when present on a microarray include, but are not limited to, a chromophore; luminophore; fluorophore; optically encoded nanoparticles; particles encoded with a diffraction-grating; electrochemiluminescent label such as Ru(bpy) 32+ ; or moiety that can be detected based on an optical characteristic.
  • Fluorophores that may be useful include, for example, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, alexa dyes, phycoerythin, bodipy, and others known in the art such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or WO 98/59066, each of which is hereby incorporated by reference.
  • fluorescent lanthanide complexes including those of Europium and Terbium, fluorescein,
  • the optical system can be configured for Time Delay Integration (TDI) for example in line scanning embodiments as described, for example, in U.S. Pat. No. 7,329,860, of which the complete subject matter is incorporated herein by reference in its entirety.
  • TDI Time Delay Integration
  • the optical assembly may have a 0.75 NA lens and a focus accuracy of +/-125 to 500 nm.
  • the resolution can be 50 to 100 nm.
  • the system may be able to obtain 1,000-10,000 measurements/second unfiltered.
  • exemplary samples include, but are not limited to, biological specimens such as cells or tissues, electronic chips such as those used in computer processors, or the like.
  • biological specimens such as cells or tissues
  • electronic chips such as those used in computer processors, or the like.
  • examples of some of the applications include microscopy, satellite scanners, high-resolution reprographics, fluorescent image acquisition, analyzing and sequencing of nucleic acids, DNA sequencing, sequencing-by-synthesis, imaging of microarrays, imaging of holographically encoded microparticles and the like.
  • the optical systems may be configured to inspect an object to determine certain features or structures of the object.
  • the optical systems may be used to inspect a surface of the object, (e.g., semiconductor chip, silicon wafer) to determine whether there are any deviations or defects on the surface.
  • FIG. 8 illustrates a block diagram of an optical system 800 formed in accordance with one embodiment.
  • the optical system 800 may be a sampler imager that images a sample of interest for analysis.
  • the optical system 800 may be a profilometer that determines a surface profile (e.g., topography) of an object.
  • various other types of optical systems may use the mechanisms and systems described herein.
  • the optical system 800 includes an optical assembly 806, an object holder 802 for supporting an object 810 near a focal plane FP of the optical assembly 806, and a stage controller 815 that is configured to move the object holder 802 in a lateral direction (along an X- axis and/or a Y-axis that extend into the page) or in a vertical/elevational direction along a Z-axis.
  • the optical system 800 may also include a system controller or computing system 820 that is operatively coupled to the optical assembly 806, the stage controller 815, and/or the object holder 802.
  • the optical system 800 is a sample imager configured to image samples.
  • a sample imager may include other sub-systems or devices for performing various assay protocols.
  • the sample may include a flow cell having flow channels.
  • the sample imager may include a fluid control system that includes liquid reservoirs that are fluidically coupled to the flow channels through a fluidic network.
  • the sample imager may also include a temperature control system that may have a heater/cooler configured to regulate a temperature of the sample and/or the fluid that flows through the sample.
  • the temperature control system may include sensors that detect a temperature of the fluids.
  • the optical assembly 806 is configured to direct input light to an object 810 and receive and direct output light to one or more detectors.
  • the output light may be input light that was at least one of reflected and refracted by the object 810 and/or the output light may be light emitted from the object 810.
  • the optical assembly 806 may include at least one reference light source 812 and at least one excitation light source 814 that direct light, such as light beams having predetermined wavelengths, through one or more optical components of the optical assembly 806.
  • the optical assembly 806 may include various optical components, including a conjugate lens 818, for directing the input light toward the object 810 and directing the output light toward the detector(s).
  • the reference light source 812 may be used by a distance measuring system or a focus-control system (or focusing mechanism) of the optical system 800 and the excitation light source 814 may be used to excite the biological or chemical substances of the object 810 when the object 810 includes a biological or chemical sample.
  • the excitation light source 814 may be arranged to illuminate a bottom surface of the object 810, such as in TIRF imaging, or may be arranged to illuminate a top surface of the obj ect 810, such as in epi-fluorescent imaging.
  • the conjugate lens 818 directs the input light to a focal region 822 lying within the focal plane FP.
  • the lens 818 has an optical axis 824 and is positioned a working distance WDi away from the object 810 measured along the optical axis 824.
  • the stage controller 815 may move the object 810 in the Z-direction to adjust the working distance WDi so that, for example, a portion of the object 810 is within the focal region 822.
  • the optical assembly 806 is configured to direct at least one pair of light beams to the focal region 822 where the object 810 is approximately located.
  • the object 810 reflects the light beams. More specifically, an exterior surface of the object 810 or an interface within the object 810 reflects the light beams. The reflected light beams then return to and propagate through the lens 818. As shown, each light beam has an optical path that includes a portion that has not yet been reflected by the obj ect 810 and a portion that has been reflected by the object 810.
  • the portions of the optical paths prior to reflection are designated as incident light beams 830A and 832A and are indicated with arrows pointing toward the object 810.
  • the portions of the optical paths that have been reflected by the object 810 are designated as reflected light beams 830B and 832B and are indicated with arrows pointing away from the object 810.
  • the light beams 830A, 830B, 832A, and 832B are shown as having different optical paths within the lens 818 and near the object 810.
  • the light beams 830A and 832B propagate in opposite directions and are configured to have the same or substantially overlapping optical paths within the lens 818 and near the object 810
  • the light beams 830B and 832A propagate in opposite directions and are configured to have the same or substantially overlapping optical paths within the lens 818 and near the object 810.
  • light beams 830A, 830B, 832A, and 832B pass through the same lens that is used for imaging.
  • the light beams used for distance measurement or focus determination can pass through a different lens that is not used for imaging.
  • the lens 818 is dedicated to passing beams 830A, 830B, 832A, and 832B for distance measurement or focus determination, and a separate lens (not shown) is used for imaging the object 810.
  • the systems and methods set forth herein for focus determination and distance measurement can occur using a common objective lens that is shared with the imaging optics or, alternatively, the objective lenses exemplified herein can be dedicated to focus determination or distance measurement.
  • the reflected light beams 830B and 832B propagate through the lens 818 and may, optionally, be further directed by other optical components of the optical assembly 806. As shown, the reflected light beams 830B and 832B are detected by at least one focus detector 844. In the illustrated embodiment, both reflected light beams 830B and 832B are detected by a single focus detector 844.
  • the reflected light beams may be used to determine relative separation RSi. For example, the relative separation RSi may be determined by the distance separating the beam spots from the impinging reflected light beams 830B and 832B on the focus detector 844 (i.e., a separation distance).
  • the relative separation RSi may be used to determine a degree-of-focus of the optical system 800 with respect to the object 810.
  • each reflected light beam 830B and 832B may be detected by a separate corresponding focus detector 844 and the relative separation RSi may be determined based upon a location of the beam spots on the corresponding focus detectors 844.
  • the computing system 820 may operate the stage controller 815 to move the object holder 802 to a desired position.
  • the optical assembly 806 may be moved in the Z-direction and/or along the XY plane.
  • the object 810 may be relatively moved a distance AZi toward the focal plane FP if the object 810 is located above the focal plane FP (or focal region 822), or the object 810 may be relatively moved a distance Z2 toward the focal plane FP if the object 810 is located below the focal plane FP (or focal region 822).
  • the optical system 800 may substitute the lens 818 with another lens 818 or other optical components to move the focal region 822 of the optical assembly 806.
  • the example set forth above and in FIG. 8 has been presented with respect to a system for controlling focus or for determining degree-of-focus.
  • the system is also useful for determining the working distance WDibetween the object 810 and the lens 818.
  • the focus detector 844 can function as a working distance detector and the distance separating the beam spots on the working distance detector can be used to determine the working distance between the object 810 and the lens 818.
  • various embodiments of the systems and methods are exemplified herein with regard to controlling focus or determining degree-of-focus. It will be understood that the systems and methods can also be used to determine the working distance between an object and a lens. Likewise, the systems and methods may also be used to determine a surface profile of an object.
  • the excitation light source 814 directs input light (not shown) onto the object 810 to excite fluorescently-labeled biological or chemical substances.
  • the labels of the biological or chemical substances provide light signals 840 (also called light emissions) having predetermined wavelength(s).
  • the light signals 840 are received by the lens 818 and then directed by other optical components of the optical assembly 806 to at least one object detector 842.
  • the object detector 842 may comprise multiple detectors.
  • the object detector 842 may include a first detector configured to detect one or more wavelengths of light and a second detector configured to detect one or more different wavelengths of light.
  • the optical assembly 806 may include a lens/filter assembly that directs different light signals along different optical paths toward the corresponding object detectors.
  • a lens/filter assembly that directs different light signals along different optical paths toward the corresponding object detectors.
  • the object detector 842 communicates object data relating to the detected light signals 840 to the computing system 820.
  • the computing system 820 may then record, process, analyze, and/or communicate the data to other users or computing systems, including remote computing systems through a communication line (e.g., Internet).
  • the object data may include imaging data that is processed to generate an image(s) of the object 810.
  • the images may then be analyzed by the computing system and/or a user of the optical system 800.
  • the object data may not only include light emissions from the biological or chemical substances, but may also include light that is at least one of reflected and refracted by the optical substrate or other components.
  • the light signals 840 may include light that has been reflected by encoded microparticles, such as the holographically encoded optical identification elements described above.
  • a single detector may provide both functions as described above with respect to the object and focus detectors 842 and 844.
  • a single detector may detect the reflected light beams 830B and 832B and also the light signals 840.
  • the optical system 800 may include a user interface 825 that interacts with the user through the computing system 820.
  • the user interface 825 may include a display (not shown) that shows and requests information from a user and a user input device (not shown) to receive user inputs.
  • the computing system 820 may include, among other things, an object analysis module 850 and a focus-control module 852.
  • the focus-control module 852 is configured to receive focus data obtained by the focus detector 844.
  • the focus data may include signals representative of the beam spots incident upon the focus detector 844.
  • the data may be processed to determine relative separation (e.g., separation distance between the beam spots).
  • a degree-of-focus of the optical system 800 with respect to the object 810 may then be determined based upon the relative separation.
  • the working distance WDi between the object 810 and lens 818 can be determined.
  • the object analysis module 850 may receive object data obtained by the object detectors 842.
  • the object analysis module may process or analyze the object data to generate images of the object.
  • the computing system 820 may include any processor-based or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate array (FPGAs), logic circuits, and any other circuit or processor capable of executing functions described herein.
  • RISC reduced instruction set computers
  • ASICs application specific integrated circuits
  • FPGAs field programmable gate array
  • the computing system 820 executes a set of instructions that are stored in one or more storage elements, memories, or modules in order to at least one of obtain and analyze object data.
  • Storage elements may be in the form of information sources or physical memory elements within the optical system 800.
  • the set of instructions may include various commands that instruct the optical system 800 to perform specific protocols.
  • the set of instructions may include various commands for performing assays and imaging the object 810 or for determining a surface profile of the object 810.
  • the set of instructions may be in the form of a software program.
  • the terms “software” and “firmware” are interchangeable, and include any computer program stored in memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory.
  • RAM memory random access memory
  • ROM memory read only memory
  • EPROM memory electrically erasable programmable read-only memory
  • EEPROM memory electrically erasable programmable read-only memory
  • NVRAM non-volatile RAM
  • the excitation light source 814 generates an excitation light that is directed onto the obj ect 810.
  • the excitation light source 814 may generate one or more laser beams at one or more predetermined excitation wavelengths.
  • the light may be moved in a raster pattern across portions of the object 810, such as groups in columns and rows of the object 810.
  • the excitation light may illuminate one or more entire regions of the object 810 at one time and serially stop through the regions in a “step and shoot” scanning pattern. Line scanning can also be used as described, for example, in U.S. Pat. No. 7,329,860, of which the complete subject matter is incorporated herein by reference in its entirety.
  • the object 810 produces the light signals 840, which may include light emissions generated in response to illumination of a label in the object 810 and/or light that has been reflected or refracted by an optical substrate of the object 810.
  • the light signals 840 may be generated, without illumination, based entirely on emission properties of a material within the object 810 (e.g., a radioactive or chemiluminescent component in the object).
  • the object and focus detectors 842 and 844 may be, for example photodiodes or cameras.
  • the detectors 842 and 844 may comprise a camera that has a 1 mega pixel CCD-based optical imaging system such as a 1002x1004 CCD camera with 8 gm pixels, which at 20x magnification can optionally image an area of 0.4x0.4 mm per tile using an excitation light that has a laser spot size of 0.5x0.5 mm (e.g., a square spot, or a circle of 0.5 mm diameter, or an elliptical spot, etc.).
  • Cameras can optionally have more or less than 1 million pixels, for example a 4 mega pixel camera can be used.
  • the readout rate of the camera should be as fast as possible, for example the transfer rate can be 10 MHz or higher, for example 20 or 30 MHz. More pixels generally mean that a larger area of surface, and therefore more sequencing reactions or other optically detectable events, can be imaged simultaneously for a single exposure.
  • the CCD camera/TIRF lasers may collect about 6400 images to interrogate 1600 tiles (since images are optionally done in 4 different colors per cycle using combinations of fdters, dichroics and detectors as described herein).
  • certain images optionally can contain between about 5,000 to 50,000 randomly spaced unique nucleic acid clusters (i.e., images upon the flow cell surface).
  • the systems herein can optionally quantify about 45 million features per hour.
  • the imaging rate can be improved.
  • a readout rate of a 20 MHz camera, and a resolved cluster every 20 pixels the readout can be 1 million clusters per second.
  • a detector can be configured for Time Delay Integration (TDI) for example in line scanning embodiments as described, for example, in U.S. Pat. No. 7,329,860, of which the complete subject matter is incorporated herein by reference in its entirety.
  • TDI Time Delay Integration
  • detectors include, but are not limited, to an optical quadrant photodiode detector, such as those having a 2x2 array of individual photodiode active areas fabricated on a single chip, examples of which are available from Pacific Silicon Sensor (Westlake Village, Calif), or a position sensitive detector such as those having a monolithic PIN photodiode with a uniform resistance in one or two dimensions, examples of which are available from Hamamatsu Photonics, K.K., (Hamamatsu City, Japan).
  • an optical quadrant photodiode detector such as those having a 2x2 array of individual photodiode active areas fabricated on a single chip, examples of which are available from Pacific Silicon Sensor (Westlake Village, Calif)
  • a position sensitive detector such as those having a monolithic PIN photodiode with a uniform resistance in one or two dimensions, examples of which are available from Hamamatsu Photonics, K.K., (Hamamatsu City, Japan).
  • FIG. 9 is a perspective view of a sample imager 900 formed in accordance with one embodiment.
  • the sample imager 900 includes an imager base 902 that supports a stage 904 having a sample holder 906 thereon.
  • the sample holder 906 is configured to support one or more optical substrates 908 during an imaging session.
  • the optical substrates 908 are illustrated as flow cells in FIG. 9. However, other samples may be used.
  • the sample imager 900 also includes a housing 910 (illustrated in phantom) and a strut 912 that supports the housing 910.
  • the housing 910 can enclose at least a portion of an optical assembly 914 therein.
  • the optical assembly 914 may include a focus assembly 916 and a sampledetecting assembly 930.
  • the focus assembly 916 may include an auto-focus line scan camera that receives reflected light beams for determining a degree-of-focus of the sampler imager 900.
  • the sample imager 900 may also include a fdter wheel 922 and an alignment mirror 924 that directs light toward a sample detector 932, which is shown as a K4 camera in FIG. 9.
  • FIG. 10 illustrates an implementation of a sequencing system 1010 configured to process molecular samples that may be sequenced to determine their components, the component ordering, and generally the structure of the sample.
  • the system includes an instrument 1012 that receives and processes a biological sample.
  • a sample source 1014 provides the sample 1016 which in many cases will include a tissue sample.
  • the sample source may include, for example, an individual or subject, such as a human, animal, microorganism, plant, or other donor (including environmental samples), or any other subject that includes organic molecules of interest, the sequence of which is to be determined.
  • the system may be used with samples other than those taken from organisms, including synthesized molecules.
  • the molecules will include DNA, RNA, or other molecules having base pairs the sequence of which may define genes and variants having particular functions of ultimate interest.
  • the sample 1016 is introduced into a sample/library preparation system 1018. This system may isolate, break, and otherwise prepare the sample for analysis.
  • the resulting library includes the molecules of interest in lengths that facilitate the sequencing operation.
  • the resulting library is then provided to the instrument 1012 where the sequencing operation is performed.
  • the library which may sometimes be referred to as a template, is combined with reagents in an automated or semi-automated process, and then introduced to the flow cell prior to sequencing.
  • the instrument includes a flow cell or array 1020 that receives the sample library.
  • the flow cell includes one or more fluidic channels that allow for sequencing chemistry to occur, including attachment of the molecules of the library, and amplification at locations or sites that can be detected during the sequencing operation.
  • the flow cell/array 1020 may include sequencing templates immobilized on one or more surfaces at the locations or sites.
  • a “flow cell” may include a patterned array, such as a microarray, a nanoarray, and so forth. In practice, the locations or sites may be disposed in a regular, repeating pattern, a complex non-repeating pattern, or in a random arrangement on one or more surfaces of a support.
  • the flow cell also allows for introduction of substances, such as including various reagents, buffers, and other reaction media, that are used for reactions, flushing, and so forth.
  • substances such as including various reagents, buffers, and other reaction media, that are used for reactions, flushing, and so forth.
  • the substances flow through the flow cell and may contact the molecules of interest at the individual sites.
  • the flow cell 1020 is mounted on a movable stage 1022 that, in this implementation, may be moved in one or more directions as indicated by reference numeral 1024.
  • the flow cell 1020 may, for example, be provided in the form of a removable and replaceable cartridge that may interface with ports on the movable stage 1022 or other components of the system in order to allow reagents and other fluids to be delivered to or from the flow cell 1020.
  • the stage is associated with an optical detection system 1026 that can direct radiation or light 1028 to the flow cell during sequencing.
  • the optical detection system may employ various methods, such as fluorescence microscopy methods, for detection of the analytes disposed at the sites of the flow cell.
  • the optical detection system 1026 may employ confocal line scanning to produce progressive pixilated image data that can be analyzed to locate individual sites in the flow cell and to determine the type of nucleotide that was most recently attached or bound to each site.
  • Other imaging techniques may also suitably be employed, such as techniques in which one or more points of radiation are scanned along the sample or techniques employing “step and shoot” imaging approaches.
  • the optical detection system 1026 and the stage 1022 may cooperate to maintain the flow cell and detection system in a static relationship while obtaining an area image, or, as noted, the flow cell may be scanned in any suitable mode (e.g., point scanning, line scanning, “step-and-shoof ’ scanning).
  • the optical detection system 1026 is configured to capture such signals, to process pixelated image data at a resolution that allows for analysis of the signal-emitting sites, and to process and store the resulting image data (or data derived from it).
  • a sequencing operation cyclic operations or processes are implemented in an automated or semi-automated fashion in which reactions are promoted, such as with single nucleotides or with oligonucleotides, followed by flushing, imaging and de-blocking in preparation for a subsequent cycle.
  • the sample library prepared for sequencing and immobilized on the flow cell, may undergo a number of such cycles before all useful information is extracted from the library.
  • the optical detection system 1026 may generate image data from scans of the flow cell (and its sites) during each cycle of the sequencing operation by use of electronic detection circuits (e.g., cameras or imaging electronic circuits or chips).
  • the resulting image data may then be analyzed to locate individual sites in the image data, and to analyze and characterize the molecules present at the sites, such as by reference to a specific color or wavelength of light (a characteristic emission spectrum of a particular fluorescent tag) that was detected at a specific location, as indicated by a group or cluster of pixels in the image data at the location.
  • a specific color or wavelength of light a characteristic emission spectrum of a particular fluorescent tag
  • the four common nucleotides may be represented by distinguishable fluorescence emission spectra (wavelengths or wavelength ranges of light). Each emission spectrum, then, may be assigned a value corresponding to that nucleotide. Based upon this analysis, and tracking the cyclical values determined for each site, individual nucleotides and their orders may be determined for each site.
  • sequences may then be further processed to assemble longer segments including genes, chromosomes, and so forth.
  • automated and “semi-automated” mean that the operations are performed by system programming or configuration with little or no human interaction once the operations are initiated, or once processes including the operations are initiated.
  • reagents 1030 are drawn or aspirated into the flow cell through valving 1032.
  • the valving may access the reagents from recipients or vessels in which they are stored, such as through pipettes or sippers (not shown in FIG. 10).
  • the valving 1032 may allow for selection of the reagents based upon a prescribed sequence of operations performed.
  • the valving may further receive commands for directing the reagents through flow paths 1034 into the flow cell 1020. Exit or effluent flow paths 1036 direct the used reagents from the flow cell.
  • a pump 1038 serves to move the reagents through the system.
  • the pump may also serve other useful functions, such as measuring reagents or other fluids through the system, aspirating air or other fluids, and so forth. Additional valving 1040 downstream of pump 1038 allows for appropriately directing the used reagent to disposal vessels or recipients 1042.
  • the instrument further includes a range of circuitry that aids in commanding the operation of the various system components, monitoring their operation by feedback from sensors, collecting image data, and at least partially processing the image data.
  • a control/supervisory system 1044 includes a control system 1046 and a data acquisition and analysis system 1048.
  • Both systems will include one or more processors (e.g., digital processing circuits, such as microprocessors, multi-core processors, FPGA's, or any other suitable processing circuitry) and associated memory circuitry 1050 (e.g., solid state memory devices, dynamic memory devices, on and/or off-board memory devices, and so forth) that may store machine-executable instructions for controlling, for example, one or more computers, processors, or other similar logical devices to provide certain functionality.
  • Application-specific or general purpose computers may at least partially make up the control system and the data acquisition and analysis system.
  • the control system may include, for example, circuitry configured (e.g., programmed) to process commands for fluidics, optics, stage control, and any other useful functions of the instrument.
  • the data acquisition and analysis system 1048 interfaces with the optical detection system to command movement of the optical detection system or the stage, or both, the emission of light for cyclic detection, receiving and processing of returned signals, and so forth.
  • the instrument may also include various interfaces as indicated at reference 1052, such as an operator interface that permits control and monitoring of the instrument, transfer of samples, launching of automated or semi-automated sequencing operations, generation of reports, and so forth.
  • external networks or systems 1054 may be coupled to and cooperate with the instrument, for example, for analysis, control, monitoring, servicing, and other operations.
  • flow cell and fluidics path and a single optical detection system 1026 are illustrated in FIG. 10, in some instruments more than one flow cell and fluidics path may be accommodated. For example, in a presently contemplated implementation, two such arrangements are provided to enhance sequencing and throughput. In practice, any number of flow cells and paths may be provided. These may make use of the same or different reagent receptacles, disposal receptacles, control systems, image analysis systems, and so forth. Where provided, the multiple fluidics systems may be individually controlled or controlled in a coordinated fashion.

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Abstract

La présente divulgation concerne des procédés, des supports non transitoires lisibles par ordinateur et des systèmes qui peuvent interroger l'état de divers stades dans un processus de séquençage de bout en bout et générer un résumé d'état graphique pour le processus de séquençage qui représente des icônes indiquant les états des divers stades. Par exemple, les systèmes divulgués peuvent générer un résumé d'état graphique pour un ensemble de tâches de séquençage de nucléotides qui comprend des icônes représentant des états d'un cycle de séquençage, un transfert de données d'appel de base à un dispositif d'analyse de variants et l'analyse de variants, chacun faisant partie du même ensemble de tâches de séquençage de nucléotides. En échangeant des données avec un dispositif de séquençage de de données lues et un ou plusieurs serveurs d'analyse de variants, le système divulgué peut fournir rapidement un résumé d'état graphique d'un processus de séquençage de bout en bout marqué par diverses tâches au sein d'un ensemble de tâches de séquençage de nucléotides.
PCT/US2022/077422 2021-12-23 2022-09-30 Résumés dynamiques d'état graphique pour séquençage de nucléotides WO2023122363A1 (fr)

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