Design and method for cross-sequencing platform compatibility of flow cells

Abstract

This disclosure describes methods, non-transitory-computer readable media, and systems that can analyze multiple sets of fiducials within a flow cell to enable imaging by multiple imaging platforms. For example, this disclosure describes a cross-platform compatible flow cell hat comprises a first set of fiducials utilized by a first imaging system and a second set of fiducials utilized by a second imaging system. The disclosed systems can capture a plurality of tiles of the flow cell utilizing the first imaging system. The disclosed systems bypass the second set of fiducials within the flow cell based on utilizing the first, and not the second, imaging system. The disclosed systems can further register the plurality of tiles based on the first set of fiducials.

Description

DESIGN AND METHOD FOR CROSS-SEQUENCING PLATFORM COMPATIBILITY OFFLOW CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional PatentApplication No. 63 / 552,781, entitled, "DESIGN AND METHOD FOR CROSS-SEQUENCINGPLATFORM COMPATIBILITY OF FLOW CELLS," filed on February 13, 2024 (IP-2714-PRV),the entirety of which is hereby incorporated by reference.BACKGROUND

[0002] In recent years, biotechnology firms and research institutions have improved hardwareand software for sequencing nucleotides and determining nucleobase calls for genomic samples.For instance, some existing sequencing machines and sequencing-data-analysis software (together"existing sequencing systems") predict individual nucleobases within sequences by usingconventional Sanger sequencing or sequencing-by-synthesis (SBS) methods. When using SBS,existing sequencing systems can monitor many thousands to billions of oligonucleotides beingsynthesized in parallel from templates to predict nucleobase calls for growing nucleotide reads.During a sequencing run in many existing sequencing systems, a camera captures images ofirradiated fluorescent tags incorporated into oligonucleotides. Existing sequencing system mayutilize unique sequencing platforms that rely on different methods to capture images of thefluorescent tags. For example, existing systems may utilize Time-Delay Integration (TDI), linescanning, Step-and-Shoot (SaS), Structured Illumination (SIM), or other imaging methods. Aftercapturing such images, some existing sequencing systems determine nucleobase calls fornucleotide reads corresponding to respective clusters of oligonucleotides on a flow cell or othernucleotide-sample substrate for a given sequencing run. For example, some existing sequencingsystems utilize sequencing-data-analysis software to analyze image data captured duringsequencing cycles to determine nucleobase calls for given clusters of oligonucleotides andsequence such calls across sequencing cycles to determine nucleotide reads for the given clusters.

[0003] Despite recent advances in imaging flow cells, existing sequencing systems exhibitseveral shortcomings. To illustrate, existing flow cells are often expensive to manufacture andpurchase. More specifically, existing systems require manufacturers to create different types offlow cells corresponding to different capture methods (e.g., SaS, TDI, etc.). To illustrate, eachimaging method (e.g., SaS, TDI, SIM, etc.) requires different flow cells having unique properties.For instance, the flow cells for different sequencing platforms may have different XY dimensions,swath length and width, tile length and width, array pitch, and other properties. Specifically, forpattern flow cells, and in some cases non-pattern flow cells, different imaging methods require fiducial markers to locate each nucleotide sample cluster during sequencing. Fiducial markers canvary in size, shape, number, distance from each other, and location across imaging methods. Insome examples, costly master templates in nanoimprint lithography are used to form fiducialswithin a flow cell. Manufacturing expenses require the utilization of multiple master templates tocreate different types of flow cells. High manufacturing costs to create unique flow cells for eachtype of sequencing platform often result in high operating costs for sequencing platforms.

[0004] Furthermore, existing sequencing systems are often inefficient. Existing sequencingsystems are often subject to operator error in cases where operators have access to multiplesequencing platforms. Operators often spend time identifying and utilizing flow cells for a givensequencing platform. For instance, an operator may erroneously insert a flow cell for a firstsequencing platform into a second sequencing platform. Such operator error often requiresoperators to perform an additional sequencing run on the correct sequencing platform, whichrequires additional consumable materials and time. Existing systems require the expenditure ofadditional time and resources to compensate for operator error.

[0005] Attempts by existing systems to universalize consumables across sequencing platformsoften lead to inaccurate sequencing results. For example, existing systems may attempt to create aflow cell that is compatible across sequencing platforms. As mentioned previously, differentsequencing platforms have different requirements for fiducial marker size, shape, number, distancefrom each other, and location. Existing systems may attempt to create a flow cell thataccommodates fiducial marker requirements across different sequencing platforms by combiningfiducial markers for each sequencing platform. However, this method drastically decreases theamount of sequenceable area within a flow cell, leading to inaccurate base calling.

[0006] There is a need to reduce the cost of sequencing, reduce operator error, and / or reducethe time of sequencing to increase sequencing adoption in the areas of healthcare diagnosis andtreatment, drug discovery and validation, and communicable disease identification and prevention.SUMMARY

[0007] This disclosure describes one or more embodiments of systems, methods, and nontransitory computer readable storage media that solve one or more of the problems described aboveor provide other advantages over the art. The disclosed systems and designs enable flow cellconsumables to be compatible across sequencing platforms. The cross-platform compatible flowcell may have several sets of fiducial markers-each corresponding to a particular sequencingplatform and imaging method. The disclosed systems utilize one of the sequencing platforms tocapture images of the flow cell. For example, in some implementations, the disclosed systems mayutilize sequencing platforms that employ Step-and-Shoot (SaS), Time-Delay Integration (TDI), orother imaging methods to capture images of the flow cell. The disclosed systems can identify,within the flow cell, fiducial markers that correspond to the utilized imaging method whilebypassing fiducial markers corresponding to other imaging methods. The disclosed systems mayfurther register captured images based on the appropriate fiducial markers. In some examples, thedisclosed systems utilize a different imaging method to image the same flow cell.

[0008] To illustrate, in some embodiments, the disclosed system utilizes an imaging system tocapture a plurality of tiles of a flow cell. The flow cell comprises a first set of fiducials utilized bya first imaging system and a second set of fiducials utilized by a second imaging system. Based onimaging the flow cell by utilizing the first imaging system, the disclosed systems may identify,within the flow cell, the first set of fiducials utilized by the first imaging system. In someimplementations, the disclosed system bypasses the second set of fiducials utilized by the secondimaging system. The disclosed system can register the plurality of tiles based on the first set offiducials.

[0009] Additional features and advantages of one or more embodiments of the presentdisclosure will be set forth in the description which follows, and in part will be obvious from thedescription, or may be learned by the practice of such example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The detailed description refers to the drawings briefly described below.

[0011] FIG. 1 illustrates a schematic view of an imaging system that may be used to providebiological or chemical analysis in accordance with one or more embodiments of the presentdisclosure.

[0012] FIGS. 2A-2D illustrates limitations of example tile and fiducial configurations withinflow cells for different types of existing imaging systems.

[0013] FIG. 3 illustrates an overview of the cross-platform compatibility system registering aplurality of tiles based on a first set of fiducials and / or a second set of fiducials in accordance withone or more embodiments of the present disclosure.

[0014] FIG. 4 illustrates an overview of the cross-platform compatibility system determiningselected fiducial and tile configurations for a flow cell in accordance with one or more embodimentsof the present disclosure.

[0015] FIG. 5 illustrates example fiducials within a cross-platform compatible flow cell inaccordance with one or more implementations of the present disclosure.

[0016] FIGS. 6A-6B illustrate example candidate fiducial configurations that incorporatesshared fiducials in flow cells utilized by multiple or a single imaging system in accordance withone or more embodiments of the present disclosure.

[0017] FIGS. 7A-7D illustrate example candidate tile configurations utilizing fiducials for afirst imaging system and shared fiducials for a first and second imaging systems in accordance withone or more embodiments of the present disclosure.

[0018] FIG. 8 illustrates the cross-platform compatibility system determining tile height forcandidate tiles in accordance with one or more implementations of the present disclosure.

[0019] FIGS. 9A-9C illustrate the cross-platform compatibility system determining tilespacing for candidate tiles in accordance with one or more implementations of the presentdisclosure.

[0020] FIGS. 10A-10B illustrate the cross-platform compatibility system determining doubleexposure distance between candidate tiles in accordance with one or more implementations of thepresent disclosure.

[0021] FIG. 11 illustrates example selection parameters for evaluating candidate flow cellconfigurations in accordance with one or more implementations of the present disclosure.

[0022] FIG. 12 illustrates features of a selected flow cell configuration in accordance with oneor more implementations of the present disclosure.

[0023] FIG. 13 illustrates additional example selected flow cell configurations that satisfyrelaxed selection parameters in accordance with one or more implementations of the presentdisclosure.

[0024] FIG. 14 illustrates a schematic view of an example of a system that may be used toprovide biological or chemical analysis in accordance with one or more embodiments of the presentdisclosure.

[0025] FIG. 15 illustrates a schematic view of an example of a set of components that maycooperate to provide a fluid path in the system of FIG. 14 in accordance with one or moreembodiments of the present disclosure.

[0026] FIGS. 16-17 illustrate flowcharts of series of acts for registering a plurality of tilesbased on a first set of fiducials in accordance with one or more embodiments of the presentdisclosure.

[0027] FIG. 18 illustrates a block diagram of an example computing device in accordance withone or more embodiments of the present disclosure.DETAILED DESCRIPTION

[0028] This disclosure describes one or more embodiments of a cross-platform compatibilitysystem that can capture images of and analyze a cross-platform compatible flow cell utilizingdifferent imaging systems. For instance, the cross-platform compatibility system utilizes animaging system (e.g., SaS, TDI, SIM, etc.) to capture tiles of a flow cell. The flow cell can comprisetwo or more sets of fiducials, each utilized by different imaging systems. In one or more embodiments, the cross-platform compatibility system identifies a first set of fiducials on the flowcell that correspond with the utilized imaging system. The cross-platform compatibility system canbypass additional sets of fiducials not corresponding with the utilized imaging system. The crossplatform compatibility system can further register the plurality of tiles based on the first set offiducials.

[0029] As just noted, the cross-platform compatibility system utilizes a cross-platformcompatible flow cell. As described, in some embodiments, the flow cell may comprise multiplesets of fiducials. The flow cell may contain a first set of fiducials utilized by a first imaging systemand a second set of fiducials utilized by a second imaging system. In some implementations, thefirst set of fiducials and the second set of fiducials comprise at least one shared fiducial that can beutilized by both imaging systems.

[0030] In some implementations, the cross-platform compatibility system creates flow cellconfigurations comprising fiducial and tile configurations. Generally, in some embodiments, thecross-platform compatibility system generates candidate fiducial configurations. For instance, thecross-platform compatibility system can determine spacing and size for fiducials within a flow cell.Additionally, the cross-platform compatibility system can determine tile configurationscorresponding to the various imaging systems. More specifically, each imaging system capturescomponent tiles of the flow cell. The sizes and configurations of tiles of the flow cell are differentacross imaging systems. The cross-platform compatibility system can compensate for the variancein fiducial and tiles across imaging systems by evaluating candidate flow cell configurations andselecting fiducial configurations and tile configurations for a flow cell.

[0031] In certain implementations, the cross-platform compatibility system evaluatescandidate fiducial and tile configurations based on determined selection parameters. Morespecifically, the cross-platform compatibility system can generate candidate flow cellconfigurations comprising candidate fiducial configurations and candidate tile configurations. Thecross-platform compatibility system can determine selection parameters, for example, to reducedouble exposure, reduce loss for a particular imaging system, reducing over-scanning, and otherparameters. By specifying selection parameters, the cross-platform compatibility system canoptimize sequencing performance for a selected flow cell.

[0032] Furthermore, in some implementations, the cross-platform compatibility systemcaptures a plurality of tiles of the flow cell. More specifically, the cross-platform compatibilitysystem utilizes an imaging system (e.g., SaS, TDI, etc.) to capture tiles of the flow cell. Typically,each of the imaging systems have different flow cell requirements. To illustrate, by utilizing SaS,the cross-platform compatibility system acquires images one at a time at discrete intervals. In TDIimaging, the cross-platform compatibility system accumulates signals over multiple exposure cycles to enable the capture of continuous images of the flow cell. The cross-platform compatibilitysystem can utilize these or several other imaging systems to capture images of the flow cell.

[0033] The cross-platform compatibility system may further identify a first set of fiducialswithin a flow cell while bypassing a second set of fiducials. The cross-platform compatibilitysystem determines to identify a set of fiducials based on the imaging system used to capture theplurality of tiles of the flow cell. The cross-platform compatibility system may register the pluralityof tiles based on the first set of fiducials.

[0034] As indicated above, the cross-platform compatibility system provides several technicaladvantages relative to existing systems by, for example reducing manufacturing costs, andsubsequently purchasing costs, relative to existing sequencing systems. In some implementations,the cross-platform compatibility system enables a single flow cell to be analyzed using variousimaging systems to generate comprehensive data for a nucleotide sample. In contrast to existingsequencing systems that require different flow cells for different sequencing platforms, the crossplatform compatibility system can design and utilize a single flow cell that is compatible acrossmultiple sequencing platforms. In some implementations, a cross-platform compatible flow cellcan be made using a single master template in nanoimprint lithography. The manufacture of crossplatform compatible flow cells is less costly than the manufacture of existing flow cells becauseproduction of cross-platform compatible flow cells does not require additional lithography mastertemplates. In other examples, the cross-platform compatible flow cells are made using othermethods such as etching, 3D printing, fabrication, molding, and other approaches. The lower costof manufacture of cross-platform compatible flow cells further reduces the cost of purchasing flowcells for and operating sequencing platforms.

[0035] Furthermore, the cross-platform compatible flow cell may also improve efficiencyrelative to existing flow cells. More specifically, use of the cross-platform compatible flow cell caneliminate additional time for an operator to identify a correct flow cell for a given sequencingplatform. Furthermore, utilization of a cross-platform compatible flow cell reduces error insequencing by reducing instances in which erroneous sequencing is performed on the wrong flowcell. Additionally, in cases where an operator utilizes multiple types of sequencing systems, a crossplatform compatible flow cell obviates the need to purchase and stock multiple types of flow cells.Instead, the operator can obtain and utilize a single cross-platform compatible flow cell across allsequencing systems. The use of the cross-platform compatible flow cell can reduce both operatorerror and time required to sequence a genetic sample.

[0036] The cross-platform compatibility system can produce accurate results across multipleimaging systems relative to existing systems that are compatible with a single imaging system. Toillustrate, the cross-platform compatibility system evaluates candidate flow cell configurations and selects flow cell configurations that meet specific selection parameters. Accordingly, the crossplatform compatibility system can create and utilize a flow cell that is compatible with multiplesequencing platforms while also maximizing the sequenceable area on the flow cell. Morespecifically, the cross-platform compatible flow cell comprises fiducials that can be utilized bymultiple sequencing platforms while maintaining flow cell area for genetic samples. In someexamples, the cross-platform compatible flow cell does not compromise flow cell yield andachieves competitive performance to platform-specific flow cells. As described in additional detailbelow, cross-platform compatible flow cells achieve competitive performance relative to platformspecific flow cells.

[0037] As illustrated by the foregoing discussion, the present disclosure utilizes a variety ofterms to describe features and advantages of the cross-platform compatibility system. As usedherein, the term "flow cell" refers to a plate or substrate comprising oligonucleotides for sequencingnucleotide sequences from genomic samples or other sample nucleic-acid polymers. In particular,a flow cell can refer to a substrate containing fluidic channels through which reagents and bufferscan travel as part of sequencing. For example, in one or more embodiments, the flow cell (e.g., apatterned flow cell or non-patterned flow cell) may comprise small fluidic channels andoligonucleotide samples that can be bound to adapter sequences on the substrate. In otherimplementations, a flow cell can be an open substrate with one or more regions for oligonucleotidesamples to be analyzed and the oligonucleotide samples may be positioned using charged pads orother means. In yet another implementation, the nucleotide-sample substrate can be a membranehaving a nanopore through which one or more oligonucleotide samples may pass. As indicatedabove, a flow cell can include tiles and wells (e.g., nanowells) comprising clusters ofoligonucleotides.

[0038] In certain embodiments, a flow cell includes a patterned substrate, which includesdepressions separated by interstitial regions, and surface chemistry positioned in the depressions.Depressions may be in the form of microwells or nanowells. Depressions may be configured tocontain nucleic acid strands or other oligonucleotides and thereby provide a reaction site for SBSand / or for other kinds of processes. In some versions, each depression has a cylindraceousconfiguration, with a generally circular cross-sectional profile. In some other versions, eachdepression has a polygonal (e.g., hexagonal, octagonal, square, rectangular, elliptical, etc.) crosssectional profile. Alternatively, depressions may have any other suitable configuration. It shouldalso be understood that depressions may be arranged in any suitable pattern, including but notlimited to a grid pattern.

[0039] As suggested above, a flow cell or other nucleotide-sample substrate can (i) include adevice 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 adetection device that is configured to detect designated reactions that occur at or proximate to thereaction sites. A flow cell or other nucleotide-sample substrate may include a solid-state lightdetection or imaging device, such as a Charge-Coupled Device (CCD) or Complementary MetalOxide Semiconductor (CMOS) (light) detection device. As one specific example, a flow cell maybe 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 cartridgeand / or bioassay system may deliver a reaction solution to reaction sites of a flow cell according toa predetermined protocol (e.g., sequencing-by-synthesis), and perform a plurality of imagingevents. For example, a cartridge and / or bioassay system may direct one or more reaction solutionsthrough the flow channel of the flow cell, and thereby along the reaction sites. At least one of thereaction solutions may include four types of nucleotides having the same or different fluorescentlabels. The nucleotides may bind to the reaction sites of the flow cell, such as to correspondingoligonucleotides at the reaction sites. The cartridge and / or bioassay system may then illuminate thereaction sites using an excitation light source (e.g., solid-state light sources, such as light-emittingdiodes (LEDS)). The excitation light may provide emission signals (e.g., light of a wavelength orwavelengths that differ from the excitation light and, potentially, each other) that may be detectedby the light sensors of the flow cell.

[0040] As used herein, the term "fiducial" refers to a reference point or marker within or on anobject. In particular, a fiducial can be present within a flow cell. A fiducial can be, for example, amark, second object, shape, edge, area, irregularity, channel, pit, post, or the like. Additionally, afiducial may be present within an image of an object, such as a flow cell, or in another data setderived from detecting the object. The fiducial can be specified by an xx and / or y coordinate in aplane of the object. Alternatively or additionally, the fiducial can be specified by a z coordinatethat is orthogonal to the xy plane, for example, being defined by the relative locations of the objectand a detector. In some examples, fiducials are specific to one or more imaging systems.

[0041] As used herein, the term "genomic sample" refers to a target genome or portion of agenome undergoing an assay or sequencing. For example, a genomic sample includes one or moresequences of nucleotides isolated or extracted from a sample organism (or a copy of such anisolated or extracted sequence). In particular, a genomic sample includes a full genome that isisolated or extracted (in whole or in part) from a sample organism and composed of nitrogenousheterocyclic bases. A genomic sample can include a segment of deoxyribonucleic acid (DNA),ribonucleic acid (RNA), or other polymeric forms of nucleic acids or chimeric or hybrid forms ofnucleic acids noted below. In some cases, the genomic sample is found in a sample prepared orisolated by a kit and received by a sequencing device.

[0042] As further used herein, the term "sequencing run" refers to an iterative process on asequencing device to determine a primary structure of nucleotide sequences from a sample (e.g.,genomic sample). In particular, a sequencing run includes cycles of sequencing chemistry andimaging performed by a sequencing device that incorporate nucleobases into growingoligonucleotides to determine nucleotide-fragment reads from nucleotide sequences extracted froma sample (or other sequences within a library fragment) and seeded throughout a nucleotide-sampleslide. In some cases, a sequencing run includes replicating nucleotide sequences from one or moregenome samples seeded in clusters throughout a nucleotide-sample slide (e.g., a flow cell). Uponcompleting a sequencing run, a sequencing device can generate base-call data in a file.

[0043] Relatedly, as used herein, for example, the term "sequencing cycle" refers to aniteration of adding or incorporating one or more nucleobases to one or more oligonucleotidesrepresenting or corresponding to sample's sequence (e.g., a genomic or transcriptomic sequencefrom a sample) or a corresponding adapter sequence. In some cases, a sequencing cycle includesan iteration of both incorporating nucleobases into clusters of oligonucleotides using sequencingchemistry and capturing images of such clusters attached to a flow cell.

[0044] As used herein, the term "image" refers to a visual representation of an object. Inparticular, an image refers to a visual representation of an entire flow cell captured as part of asequencing cycle. For example, an image can comprise fluorescent signals emitted during theincorporation of nucleotides. In some implementations, the image comprises representations offiducials within a flow cell. An image may comprise a compilation of tiles.

[0045] As used herein, the term "tile" refers to an image of a segment of an object. In particular,a tile comprises an image of a segment of a flow cell. More particularly, optical sensors used duringsequencing cycles often have limited fields of view that are smaller than an entire flow cell.Accordingly, the optical sensors capture a plurality of tiles. In some examples, tiles comprise tilesof a flow cell.

[0046] As used herein, the term "imaging system" refers to a system designed to capture,record, and produce visual representations of an object. An imaging system may comprise a systemthat utilizes a particular imaging method to capture tiles of a flow cell during a sequencing run. Forexample, an imaging system may utilize imaging methods such as Step-and-Shoot (SaS), TimeDelay Integration (TDI), Structured Illumination Microscopy (SIM), and other imaging methods.An imaging system may be part of a sequencing device.

[0047] As used herein, the term "register" refers to a process of aligning a plurality of tiles. Inparticular, a plurality of tiles may be registered or aligned to form a composite image of a flow cell.As described previously, tiles may portray different portions of a flow cell. By the process ofregistration, the tiles are aligned and combined into an image of the flow cell. In some implementations, fiducials are utilized in the process of registering the plurality of tiles. Fiducialsare reference points with known coordinates and can be portrayed in the plurality of tiles. Theprocess of registering the images comprises determining the transformation or alignmentparameters needed to bring the observed positions of fiducials into correspondence with theirknown or expected positions. The process of registering fiducials is crucial for accurate calibrationand image analysis.

[0048] As used herein, the term "sequencing device" refers to an instrument or platform usedto perform a sequencing process. In particular, a sequencing device refers to an instrument orplatform used to perform a sequencing process based on sequencing by synthesis (SBS) technology,single-molecule real-time sequencing (SMRT) technology using magnetic beads or nanopores orother suitable medium. For example, a sequencing device may comprise components including,but not limited to, flow cell receptacle, fluidics systems, lasers, imaging systems, and computationalcapabilities for acquiring, processing, and analyzing image data during a sequencing run.

[0049] As mentioned, the cross-platform compatibility system utilizes an imaging system. Thefollowing paragraphs describe an imaging system utilized by the cross-platform compatibilitysystem with respect to illustrative figures that portray example embodiments and implementations.FIG. 1 illustrates a schematic diagram of another example of a system 100 that may be used toperform an analysis on one or more samples of interest. Except as otherwise described below,system 100 of this example may be configured and operable like systems portrayed in FIGS. 14-15 described below. System 100 is configured to perform a large number of parallel reactionswithin a flow cell 110. Flow cell 110 may include one or more flow channels that receive a solutionfrom system 100 and direct the solution toward reaction sites of flow cell 110.

[0050] System 100 includes a system controller 120 that may communicate with the variouscomponents, assemblies, and sub-systems of the system 100. An imaging assembly 122 of system100 includes a light emitting assembly 150 that emits light that reaches reaction sites on flow cell110. Light emitting assembly 150 may include an incoherent light emitter (e.g., emit light beamsoutput by one or more excitation diodes), or a coherent light emitter such as emitter of light outputby one or more lasers or laser diodes. In some implementations, light emitting assembly 150 mayinclude a plurality of different light sources (not shown), each light source emitting light of adifferent wavelength range. Some versions of light emitting assembly 150 may also include one ormore collimating lenses (not shown), a light structuring optical assembly (not shown), a projectionlens (not shown) that is operable to adjust a structured beam shape and path, epifluorescencemicroscopy components, and / or other components. Although system 100 is illustrated as having asingle light emitting assembly 150, multiple light emitting assemblies may be included in someother implementations.

[0051] In the present example, the light from light emitting assembly 150 is directed bydichroic mirror assembly 146 through an objective lens assembly 142 onto a sample of a flow cell110, which is positioned on a sample stage 170. In the case of fluorescent microscopy of a sample,a fluorescent element associated with the sample of interest fluoresces in response to the excitationlight, and the resultant light is collected by objective lens assembly 142 and is directed to an imagesensor of camera system 140 to detect the emitted fluorescence. In some implementations, a tubelens assembly may be positioned between the objective lens assembly 142 and the dichroic mirrorassembly 146 or between the dichroic mirror assembly 146 and the image sensor of the camerasystem 140. A moveable lens element may be translatable along a longitudinal axis of the tube lensassembly to account for focusing on an upper interior surface or lower interior surface of the flowcell 110 and / or spherical aberration introduced by movement of the objective lens assembly 142.

[0052] In the present example, a filter switching assembly 144 is interposed between dichroicmirror assembly 146 and camera system 140. Filter switching assembly 144 includes one or moreemission filters that may be used to pass through particular ranges of emission wavelengths andblock (or reflect) other ranges of emission wavelengths. For example, emission filters may be usedto direct different wavelength ranges of emitted light to different image sensors of the camerasystem 140 of imaging assembly 122. For instance, the emission filters may be implemented asdichroic mirrors that direct emission light of different wavelengths from flow cell 110 to differentimage sensors of camera system 140. In some variations, a projection lens is interposed betweenfilter switching assembly 144 and camera system 140. Filter switching assembly 144 may beomitted in some versions.

[0053] System 100 further includes a fluid delivery assembly 190 that may direct the flow ofreagents (e.g., fluorescently labeled nucleotides, buffers, enzymes, cleavage reagents, etc.) to (andthrough) flow cell 110 and waste valve 180. System 100 of the present example also includes atemperature station actuator 130 and heater / cooler 132 that may optionally regulate the temperatureof conditions of the fluids within the flow cell 110. In some implementations, the heater / cooler 132may be fixed to sample stage 170, upon which the flow cell 110 is placed, and / or may be integratedinto sample stage 170.

[0054] Flow cell 110 may be removably mounted on sample stage 170, which may providemovement and alignment of flow cell 110 relative to objective lens assembly 142. Sample stage170 may have one or more actuators to allow sample stage 170 to move in any of three dimensions.For example, actuators may be provided to allow sample stage 170 to move in the x, y, and zdirections relative to objective lens assembly 142, tilt relative to objective lens assembly 142,and / or otherwise move relative to objective lens assembly 142. Movement of sample stage 170may allow one or more sample locations on flow cell 110 to be positioned in optical alignment with objective lens assembly 142. Movement of sample stage 170 relative to objective lens assembly142 may be achieved by moving sample stage 170 itself, by moving objective lens assembly 142,by moving some other component of imaging assembly 122, by moving some other component ofsystem 100, or any combination of the foregoing. For instance, in some implementations, thesample stage 170 may be actuatable in the x and y directions relative to the objective lens assembly142 while a focus component 162 or z-stage may move the objective lens assembly 142 along thez direction relative to the sample stage 170.

[0055] In some implementations, a focus component 162 may be included to controlpositioning of one or more elements of objective lens assembly 142 relative to the flow cell 110 inthe focus direction (e.g., along the z-axis or z-dimension). Focus component 162 may include oneor more actuators physically coupled to the objective lens assembly 142, the optical stage, thesample stage 170, or a combination thereof, to move flow cell 110 on sample stage 170 relative tothe objective lens assembly 142 to provide proper focusing for the imaging operation. In the presentexample, the focus component 162 utilizes a focus tracking module 160 that is configured to detecta displacement of the objective lens assembly 142 relative to a portion of the flow cell 110 andoutput data indicative of an in-focus position to the focus component 162 or a component thereofor operable to control the focus component 162, such as a system controller 120, to move theobjective lens assembly 142 to position the corresponding portion of the flow cell 110 in focus ofthe objective lens assembly 142.

[0056] In some implementations, an actuator of focus component 162 or for sample stage 170may be physically coupled to objective lens assembly 142, the optical stage, sample stage 170, ora combination thereof, such as, for example, by mechanical, magnetic, fluidic, or other attachmentor contact directly or indirectly to or with the stage or a component thereof. The actuator of focuscomponent 162 may be configured to move objective lens assembly 142 in the z-direction whilemaintaining sample stage 170 in the same plane (e.g., maintaining a level or horizontal attitude,perpendicular to the optical axis). In some implementations, sample stage 170 includes andirection actuator and a y direction actuator to form an x-y stage. Sample stage 170 may also beconfigured to include one or more tip or tilt actuators to tip or tilt sample stage 170 and / or a portionthereof, to account for any slope in its surfaces.X

[0057] Camera system 140 may include one or more image sensors to monitor and track theimaging (e.g., sequencing) of flow cell 110. Camera system 140 may be implemented, for example,as a CCD or CMOS image sensor camera, but other image sensor technologies (e.g., active pixelsensor) may be used. By way of further example only, camera system 140 may include a dualsensor time-delay integration (TDI) camera, a step-and-shoot camera, a single-sensor camera, acamera with one or more two-dimensional image sensors, and / or other kinds of camera technologies. While camera system 140 and associated optical components are shown as beingpositioned above flow cell 110 in FIG. 1, one or more image sensors or other camera componentsmay be incorporated into system 100 in numerous other ways as will be apparent to those skilledin the art in view of the teachings herein. For instance, one or more image sensors may be positionedunder flow cell 110, such as within the sample stage 170 or below the sample stage 170; or mayeven be integrated into flow cell 110. Although the apparatus has been exemplified above withregard to a CCD line scan camera, it will be understood that any of a variety of other detectors canbe used including, but not limited to a detector array configured for TDI operation, a CMOSdetector, APD detector, Geiger-mode photon counter or other detector set forth elsewhere herein.

[0058] In some implementations, the system 100 is operated by the cross-platformcompatibility system. For example, in some embodiments, the system 100 comprises a sequencingdevice for sequencing a genomic sample or other nucleic-acid polymer. In some embodiments, thesequencing device analyzes nucleic-acid segments or oligonucleotides extracted from genomicsamples to generate nucleotide reads or other data utilizing computer implemented methods andsystems (described herein) either directly or indirectly on the sequencing device. More particularly,the sequencing device receives nucleotide-sample substrates (e.g., flow cells) comprisingnucleotide fragments extracted from samples. The system 100 can perform a series of sequencingcycles to determine the nucleotide-base sequence of such extracted nucleotide fragments. In one ormore embodiments, the sequencing device utilizes SBS to sequence nucleic-acid polymers intonucleotide reads.

[0059] As part of performing the series of sequencing cycles, the sequencing device capturesa plurality of tiles of the flow cell. The sequencing device may employ various imaging systems tocapture the plurality of tiles. For example, the sequencing device may utilize an SaS imagingsystem and / or a TDI imaging system to capture a plurality of tiles of the flow cell. The plurality oftiles can depict fiducials within the flow cell. The sequencing device may register the plurality oftiles to form an image of the flow cell. Additionally, the sequencing device can determine basecalls for indexing sequences based on the image of the flow cell.

[0060] In some implementations, the sequencing device can communicate with the systemcontroller 120 or a client device. More specifically, the system controller 120 may register fiducialsto generate an image of a flow cell and generate base calls based on flow cell image data. Morespecifically, the cross-platform compatibility system may utilize the system controller 120 toregister a set of fiducials specific to a utilized imaging system to align tiles.

[0061] In some embodiments, the system 100 may send base call data or flow cell image datato a client device. For example, the system 100 may send data including VCFs or other sequencingrelated information to the client device. The client device can generate, store, receive, and send digital data. In particular, the client device can receive tile data and / or flow cell image data fromthe system 100. Furthermore, the client device may communicate with the system controller 120 toreceive a VCF comprising variant or genotype calls and / or other metrics. The client device canaccordingly present or display information pertaining to variant calls or other genotype calls withina graphical user interface to a user associated with the client device. Furthermore, the client devicecan present imaging system data to the user associated with the client device. For example, theclient device can present data obtained using different imaging systems.

[0062] As mentioned previously, some existing sequencing systems attempt to generatecomprehensive genetic sequencing data by utilizing different types of imaging systems. However,existing sequencing systems are often incapable of utilizing the same flow cells across imagingsystems. Imaging systems often have different fields of view and accordingly capture tiles ofdifferent dimensions. Existing systems are often incapable of creating and utilizing flow cells withcross-platform compatibility. FIGS. 2A-2D illustrate limitations of example tile and fiducialconfigurations within flow cells for different types of imaging systems. FIG. 2A illustrates amismatch of tile size and number between different types of imaging systems. FIG. 2B illustratesexample fiducial configurations within tiles. FIG. 2C illustrates limitations of aligning fiducials fordifferent tiles, and FIG. 2D illustrates limitations of aligning tiles from different types of imagingsystems.

[0063] FIG. 2A illustrates differences between two example imaging systems that result invariations in tile dimensions. More particularly, FIG. 2A illustrates differences in a line scanimaging system 202 (e.g., TDI) and a step-and-shoot imaging system 204 (e.g., SaS). As shown,the line scan imaging system 202 captures tiles by sequentially scanning one or more a row ofpixels) at a time during multiple exposure cycles. For example, the number of pixel (i.e., detectionelements in a dimension perpendicular to the scan direction for a rectangular detector array can begreater than 2, 10, 20, 50, 100, 1000, 4000, or higher. The number of rows of pixels in the scandirection can be one or more rows, such as greater than 3, 5, 7, or more. U.S. Patent 8,884,211assigned to Illumina, Inc. discloses embodiments of line scanning which is incorporated in itsentirety by reference. For example, the line scan imaging system 202 utilizes an optical lens thatmoves in a relative scanning motion along a TDI flow cell 222. The line scan imaging system 202operates the optical lens in a continuous fashion-capturing successive lines as it moves across theTDI flow cell 222. As illustrated in FIG. 2A, the line scan imaging system 202 begins its scan atthe bottom left corner of the TDI flow cell 222 and finishes its scan at the top right of the TDI flowcell 222. The line scan imaging system 202 utilizes the optical lens to capture tiles 206 as the opticallens moves up and down the TDI flow cell 222 in a continuous motion. The line scan imaging system 202 captures a series of tiles in a sequential manner. One example of the line scan imagingsystem 202 is Time-Delay Integration (TDI).

[0064] FIG. 2A further illustrates a step-and-shoot imaging system 204 capturing tiles on anSaS flow cell 224.The step-and-shoot imaging system 204 captures tiles by taking discretesnapshots of the entire flow cell at specific intervals or steps. For example, the step-and-shootimaging system 204 positions an optical lens at the top right of the SaS flow cell 224 to capture atile 208. The step-and-shoot imaging system 204 causes relative stepping motion between theoptical lens and the stage to capture a second tile below the tile 208.

[0065] As shown in FIG. 2A, the differences in imaging methods between the line scanimaging system 202 and the step-and-shoot imaging system 204 contributes to varied sequencableareas within flow cells. FIG. 2A illustrates a TDI swath 210 and an SaS swath 212. Swaths indicatea total length of a flow cell along which an optical lens captures tiles. As illustrated, the TDI totallength, or the sequencable length along the TDI swath 210, is shorter than the SAS total length, orthe sequencable length along the SaS swath 212. The TDI total length is shorter because line scanimaging systems, such as TDI, often accelerate the optical lens at the beginning of the TDI swath210 and reserve a portion at the end of the TDI swath 210 to focus the optical lens. TDI swathstypically need a buffer (e.g., 1.25 mm) at the ends while the step-and-shoot imaging system 204can effectively utilize the whole length of the SaS swath.

[0066] The line scan imaging system 202 and the step-and-shoot imaging system 204 haveunique characteristics that contribute to different tile properties. For example, optical lenses for theline scan imaging system 202 and the step-and-shoot imaging system 204 are different sizes. Thefield of view and the number of pixels generated by their respective optical lenses are different. Forexample, the step-and-shoot imaging system 204 captures larger tiles with more pixels than the linescan imaging system 202. Furthermore, the optical field of view (FOV) of the step-and-shootimaging system 204 is larger in order meet scan time requirements.

[0067] The differences between the line scan imaging system 202 and the step-and-shootimaging system 204 results in variances in fiducial configuration requirements between thesystems. FIG. 2B illustrates example fiducial configurations for the different tiles. In particular,FIG. 2B illustrates a line scan imaging system tile 214 and a step-and-shoot imaging system tile216. Typically, fiducials are in the same relative position in every tile. Different imaging systemsrequire different fiducial locations within tiles. As shown in FIG. 2B, TDI fiducials 218 in the linescan imaging system tile 214 are located in different positions than the SaS fiducials 220 step-andshoot imaging system tile 216. Furthermore, different imaging systems require different types offiducials. As shown in FIG. 2B, the TDI fiducials 218 are a different type than the SaS fiducials220. The tiles also have a mismatch in XY dimensions. Furthermore, the usable sequencing area for each tile can be different between SaS and TDI fiducials. More specifically, because the fieldsof view for different imaging systems are of different sizes, the effective sequencing area withineach tile is also different. For example the line scan imaging system tile 214 is significantly shorterand thinner than the step-and-shoot imaging system tile 216.

[0068] One way to create flow cells that are cross-platform compatible is to combine existingfiducials and / or existing tiles for multiple imaging systems. FIG. 2C illustrates shortcomings ofsimply aligning fiducials from different imaging systems. FIG. 2D illustrates shortcomings ofsimply aligning tiles from different imaging systems. However, these proposed methods typicallycompletely fail to function or significantly reduce sequencing performance.

[0069] FIG. 2C illustrates the line scan imaging system tile 214 having the TDI fiducials 218aligning with the SaS fiducials 220 of the step-and-shoot imaging system tile 216. FIG. 2Cillustrates some of the TDI fiducials 218 and the SaS fiducials 220 falling on top of each other.Overlapping fiducials often cannot be registered or uniquely identified. Furthermore, due to themismatch in XY dimension, the tiles are not an exact match for the available flow cell sequencingspace. Thus, sequencing using aligned fiducials is inefficient.

[0070] In another example, the tiles can be aligned. For example, and as shown in FIG. 2D,the XY dimensions of the smaller line scan imaging system tile 214 can be expanded to fit withinthe same sequencing space as the step-and-shoot imaging system tile 216. However, this adjustmenthas its own limitations. In particular, the combined fiducials occupy a prohibitive amount ofsequencing space within a flow cell. Additionally, the adjustment of the size of either of the tilesmay compromise yield. More specifically, because dimensions were not aligned in optimalconditions, one imaging system may have significantly reduced output due to inefficient tile usage.

[0071] FIGS. 2A-2D illustrate shortcomings of existing systems that attempt to create crossplatform compatible flow cells. As mentioned, the cross-platform compatibility system may utilizedifferent imaging systems to capture images of the same flow cell. FIG. 3 illustrates an overviewof the cross-platform compatibility system registering a plurality of tiles based on a first set offiducials and / or a second set of fiducials in accordance with one or more embodiments of thepresent disclosure. By way of overview, FIG. 3 illustrates a series of acts comprising an act 302 ofcapturing a plurality of tiles of a flow cell, an act 306 of identifying a first set of fiducials, an act308 of bypassing the second set of fiducials, and an act 310 of registering the plurality of tiles basedon the first set of fiducials. FIG. 3 also illustrates an act 312 of identifying a second set of fiducials,an act 314 of bypassing the first set of fiducials, and an act 316 of registering the plurality of tilesbased on the second set of fiducials.

[0072] As shown in FIG. 3, the cross-platform compatibility system performs the act 302 ofcapturing a plurality of tiles of a flow cell. The cross-platform compatibility system may utilize several different imaging systems to capture the plurality of tiles. For example, and as illustrated,the cross-platform compatibility system utilizes a TDI imaging system to capture TDI tiles 318 ofa flow cell. Additionally, or alternatively, the cross-platform compatibility system utilizes an SaSimaging system to capture tiles 320 of the flow cell. In some implementations, the cross-platformcompatibility system utilizes various other imaging systems to capture the plurality of tiles. Forinstance, the cross-platform compatibility system may utilize Structured Illumination Microscopу(SIM) or other types of imaging systems to capture the plurality of tiles of the flow cell.

[0073] FIG. 3 illustrates tiles 304 captured by the cross-platform compatibility system. Asshown, the tiles 304 comprise fiducials. Generally, fiducials comprise markers within a flow cellthat are captured by an imaging system. The tiles 304 depict three types of fiducials-first imagingsystem fiducials utilized by a first imaging system, second imaging system fiducials utilized by asecond imaging system, and at least one shared fiducial that can be utilized by both the first andsecond imaging systems. As shown in FIG. 3, the first imaging system fiducials are depicted bylight-colored rings, second imaging system fiducials are depicted by dark-colored rings, and sharedfiducials 330 are depicted by a combination of light- and dark-colored rings. As shown in FIG. 3,a tile can comprise a TDI tile 322 or an SaS tile 324. FIGS. 5-6B further illustrate how fiducialscan be utilized by one or multiple imaging systems in accordance with one or more embodimentsof the present disclosure.

[0074] The cross-platform compatibility system identifies a set of fiducials based on theimaging system utilized to capture the plurality of tiles. Based on determining that a first imagingsystem is used to capture the plurality of tiles, the cross-platform compatibility system identifies afirst set of fiducials used as part of imaging by the first imaging system. In particular, the crossplatform compatibility system canan determine that the first set of fiducials comprises first imagingsystem fiducials. For example, and as shown in FIG. 3, the cross-platform compatibility systemdetermines that a TDI imaging system is utilized to capture the TDI tile 322. Based on thisdetermination, the cross-platform compatibility system identifies first imaging system fiducials 326being the TDI fiducials. First imaging system fiducials may comprise fiducials that are utilizedexclusively by the first imaging system. Additionally, in some implementations, the cross-platformcompatibility system determines that the first set of fiducials comprises (i) first imaging systemfiducials and (ii) one or more shared fiducials. To illustrate, the cross-platform compatibility systemcan determine that the first set of fiducials comprises the first imaging system fiducials 326 and theshared fiducials 330. The shared fiducials 330 can be utilized by both the first imaging system andthe second imaging system.

[0075] Based on identifying the first set of fiducials, the cross-platform compatibility systemperforms the act 308 of bypassing the second set of fiducials. For example, and as shown in FIG. 3, the cross-platform compatibility system analyzes the first set of fiducials (e.g., the first imagingsystem fiducials 326 and, optionally, the shared fiducials 330) and ignores the second set offiducials utilized by the second imaging system.

[0076] As further illustrated in FIG. 3, the cross-platform compatibility system performs theact 310 of registering the plurality of tiles based on the first set of fiducials. Fiducials comprisereference points with known coordinates. The cross-platform compatibility system registers theplurality of tiles by determining the transformation or alignment parameters needed to bring theobserved positions of fiducials into correspondence with their known or expected positions. Thecross-platform compatibility system registers the plurality of tiles based on the first set of fiducialsto generate an image of the flow cell.

[0077] FIG. 3 additionally illustrates the cross-platform compatibility system utilizing asecond set of fiducials. As shown, the cross-platform compatibility system may utilize the sameflow cell and capture a plurality of tiles utilizing a second imaging system. As shown, the secondimaging system comprises an SaS imaging system. The cross-platform compatibility systemanalyzes the tiles 304 and identifies the SaS tile 324. The cross-platform compatibility systemperforms the act 312 of identifying a second set of fiducials. In some embodiments, the second setof fiducials comprises second imaging system fiducials, which are utilized exclusively by thesecond imaging system. For example, the cross-platform compatibility system determines that thesecond set of fiducials comprises second imaging system fiducials 328 that are utilized by the SaSimaging system. In some implementations, the second set of fiducials comprises (i) the secondimaging system fiducials 328 and (ii) the shared fiducials 330.

[0078] The cross-platform compatibility system further performs the act 314 of bypassing thefirst set of fiducials. More specifically because the cross-platform compatibility system utilizes thesecond imaging system to capture the SaS tile 324, the cross-platform compatibility systemdisregards the first imaging system fiducials 326 utilized by the first imaging system. The crossplatform compatibility system performs its analysis utilizing the second set of fiducials comprisingthe second imaging system fiducials 328 and, optionally, the shared fiducials 330. The crossplatform compatibility system performs the act 316 of registering the plurality of tiles based on thesecond set of fiducials.

[0079] FIG. 3 illustrates the cross-platform compatibility system registering tiles based on afirst set of fiducials and a second set of fiducials. In some implementations, the cross-platformcompatibility system analyzes flow cells comprising more sets of fiducials. For instance, a flowcell can be utilized by any number (e.g., 3, 4, 5, etc.) of imaging systems. The cross-platformcompatibility system may utilize flow cells with the corresponding number of imaging systemfiducials.

[0080] As described previously, the cross-platform compatibility system may generate andutilize a cross-platform compatible flow cell that can be imaged using different imaging systems.FIG. 4 illustrates an overview of the cross-platform compatibility system determining selectedfiducial and tile configurations for a flow cell in accordance with one or more embodiments of thepresent disclosure. By way of overview, FIG. 4 illustrates a series of acts comprising an act 402 ofgenerating candidate fiducial and tile configurations, an act 406 of determining selectionparameters, and an act 408 of determining selected fiducial and tile configurations for a flow cell.

[0081] FIG. 4 illustrates the cross-platform compatibility system performing the act 402 ofgenerating candidate fiducial and tile configurations. In particular, the cross-platform compatibilitysystem generates candidate flow cell configurations having different fiducial properties anddifferent tile properties. To limit the number of candidate flow cell configurations, the crossplatform compatibility system generates candidate flow cell configurations complying with varioussets of rules. For example, the cross-platform compatibility system can generate candidate flowcell configurations that comply with rules for properties and configurations of candidate fiducialsand candidate tiles.

[0082] As mentioned, as part of performing the act 402, the cross-platform compatibilitysystem generates candidate flow cell configurations having various fiducial properties such as size,number, absolute position, relative position, and other properties. The cross-platform compatibilitysystem determines fiducial properties for all sets of fiducials. For example, the cross-platformcompatibility system can determine properties of fiducials for a first imaging system, fiducials fora second imaging systems, and / or shared fiducials utilized by both the first and second imagingsystems. FIG. 5 further describes fiducial properties for first imaging system fiducials, secondimaging system fiducials, and shared fiducials in one or more embodiments. FIGS. 6-7D furtherdetail how the cross-platform compatibility system can configure fiducials within a flow cell and,subsequently tiles, in accordance with one or more implementations of the present disclosure.

[0083] As part of performing the act 402, the cross-platform compatibility system alsogenerates candidate tile configurations. For instance, the cross-platform compatibility systemiterates through different sizes, numbers, relative positions, and other properties of tiles for allimaging systems. In some implementations, the cross-platform compatibility system generates thecandidate fiducial and tile configurations based on configuration rules. FIGS. 8-10C detail how thecross-platform compatibility system configures candidate tiles in accordance with one or moreimplementations of the present disclosure. As shown in FIG. 4, the cross-platform compatibilitysystem generates candidate configurations 410a, 410b, and 410c. The candidate configurations410a-410c can vary properties of fiducials 412 and the tiles.

[0084] The cross-platform compatibility system further performs the act 406 of determiningselection parameters. Candidate fiducial and tile configurations can correspond with variousstrengths and shortcomings. The cross-platform compatibility system determines selectionparameters to identify candidate flow cell configurations that optimize certain parameters. FIG. 11illustrates example selection parameters for evaluating candidate flow cell configurations inaccordance with one or more implementations of the present disclosure. The cross-platformcompatibility system may also consider image system loss.

[0085] More specifically, certain flow cell configurations may increase the performance of oneimaging system while negatively impacting the performance of another imaging system. The crossplatform compatibility system can select criteria to minimize the loss because of using one or bothimaging systems. As further illustrated in FIG. 4, the cross-platform compatibility system candetermine a selection parameter comprising the numbers of tiles for each imaging system. Inparticular, the cross-platform compatibility system may determinea number of tiles within a swathor within a flow cell for eachof the imaging systems. Furthermore, the cross-platform compatibilitysystem can evaluate over scanning that results from various candidate fiducial and tileconfigurations. The cross-platform compatibility system may determine these or other selectionparameters utilized to evaluate the candidate fiducial and tile configurations.

[0086] FIG. 4 further illustrates the act 408 of determining selected fiducial and tileconfigurations for a flow cell. The cross-platform compatibility system utilizes the determinedselection parameters to evaluate the candidate fiducial and tile configurations. Based on thisevaluation, the cross-platform compatibility system selects a candidate fiducial and tileconfiguration for the flow cell. In some implementations, the cross-platform compatibility systemcreates the selected fiducial and tile configurations for the flow cell. For example, the crossplatform compatibility system can utilize imaging systems to capture images of tiles havingconfigurations consistent with the selected fiducial and tile configurations. For instance, and asillustrated in FIG. 4, the cross-platform compatibility system selects the candidate fiducial and tileconfiguration 410a as the selected fiducial and tile configuration for a flow cell. FIGS. 12-13illustrate example selected fiducial and tile configurations in certain embodiments based ondifferent selection parameters.

[0087] As mentioned previously, the cross-platform compatibility system can utilize variousmethods to create and identify the first set of fiducials and the second set of fiducials. FIG. 5illustrates example fiducials within a cross-platform compatible flow cell in accordance with oneor more implementations of the present disclosure. Generally, FIG. 5 illustrates examples of firstimaging system fiducials 502, second imaging system fiducials 504, and fiducials on a candidatecross-platform compatible flow cell 506.

[0088] In one example illustrate in FIG. 5, the cross-platform compatibility system utilizes thesame fiducial types for each imaging system. For example, the cross-platform compatibility systemmay utilize a fiducial 508a for a first imaging system and a fiducial 508b for a second imagingsystem. The fiducial 508a and the fiducial 508b are the same fiducial type being identical in size,form, and shape. The cross-platform compatibility system can train the different imaging systemsto recognize the fiducials 508a-508b and register tiles based on the fiducials 508a-508b. The crossplatform compatibility system can create a candidate cross-platform compatible flow cell 506having a fiducial 508c that is recognized by both imaging systems.

[0089] FIG. 5 further illustrates an example where the cross-platform compatibility systemuses different fiducial types for each imaging system. As shown, the first imaging system utilizesa fiducial 510 and the second imaging system utilizes a fiducial 512. In this example, the crossplatform compatible flow cell contains both the fiducial 510 and the fiducial 512. The crossplatform compatibility system may utilize each of the first imaging system and the second imagingsystem to analyze the cross-platform compatible flow cell.

[0090] In a preferred embodiment of the present disclosure, the cross-platform compatibilitysystem creates a shared fiducial that can be utilized by both imaging systems. For example, thecross-platform compatibility system superimposes fiducials for different imaging systems to createa shared fiducial. As shown in FIG. 5, the cross-platform compatibility system can use a fiducial514 utilized by the first imaging system and a fiducial 516 utilized by the second imaging system.The cross-platform compatibility system further superimposes the fiducial 514 and the fiducial 516to create a shared fiducial 518. The shared fiducial 518 can be utilized by both the first imagingsystem and the second imaging system. Utilization of the shared fiducial 518 can improveefficiency relative to the previously mentioned examples. For example, each fiducial within a flowcell reduces the sequenceable area (e.g., number of clusters and / or number of nanowells) of theflow cell. By utilizing the shared fiducial 518, the cross-platform compatibility system canminimize the number of fiducials present within a flow cell.

[0091] In some implementations, the fiducial 514, the fiducial 516, and the shared fiducial 518are all different fiducial types. For example, they can all have different properties andcharacteristics. However, in some examples, the cross-platform compatibility system utilizes onlytwo, instead of three, fiducial types for two imaging systems. For example, a first imaging systemcan utilize both the fiducial 514 and the shared fiducial 518. The second imaging system can utilizethe shared fiducial 518.

[0092] As mentioned, the cross-platform compatibility system may superimpose fiducialsutilized by different imaging systems to create shared fiducials. FIG. 6A illustrates an example candidate fiducial configuration that incorporates shared fiducials in accordance with one or moreembodiments of the present disclosure.

[0093] In some embodiments, one, several, or all of fiducials 508, 510, 512, 514, 516, 518 arepositioned on a nanowell grid of a patterned flow cell.

[0094] In some embodiments, one, several, or all of the fiducials 508, 510, 512, 514, 516, 518comprises one or more bright rings or other shapes that appear bright during imaging. For ease ofdescription, the term "ring" refers to any shape. For example, a bright ring comprises a regionhaving fluorescent material and / or reflective material. In some embodiments, a bright ringcomprises a trench of a patterned flow cell. In some embodiments, a bright ring comprisesnanowells of the flow cell (i.e., a portion of the nanowells formed in a ring). In some embodiments,a bright ring comprises nanowells of the flow cell having a sample therein. In certain aspects, abright ring comprising nanowells having a sample therein which is sequencable. For example, thesample comprises fluorescent material during sequencing.

[0095] In some embodiments, one, several, or all of the fiducials 508, 510, 512, 514, 516, 518comprises one or more dark rings or other shapes that appear dark during imaging. For example,the dark rings may comprise a region devoid of fluorescent material and / or reflective material. Insome embodiments, a dark ring comprises an interstitial region of a patterned flow cell.

[0096] In some embodiments, one, several, or all of the fiducials 508, 510, 512, 514, 516, 518comprises one or more bright rings and one or more dark rings.

[0097] In some embodiments, one, several, or all of the fiducials 508, 510, 512, 514, 516, 518comprises one or more bright rings comprising a reflective material, such a metal and alloysthereof, that reflective light at the wavelengths utilized during imaging.

[0098] In some embodiments, all of fiducials 508, 510, 512, 514, 516, 518 are positioned on ananowell grid of a patterned flow cell. For example, light rings are formed from the nanowells ofa patterned flow cell. For example, dark rings are formed from the interstitial regions of a patternedflow cell. In certain aspects, having all of the fiducials positioned on a nanowell grid of a patternedflow cell reduces the cost of manufacturing of the flow cell to have two sets of fiducials on thesame flow cell in comparison to having two sets of fiducials on two separate flow cells. Forexample, lithography is used to form the nanowells and corresponding interstitial regions of theflow cells. In the case of nanoimprint lithography, the same master template can be used to formtwo sets of fiducials on the same flow cell. A master template in nanoimprint lithography isexpensive and used to create a working stamp in which the working stamp imprints the features ofthe flow cell into a nanoimprint lithography resin. In the case of photolithography, the same UVmask or EUV mirror may be used to form two sets of fiducials on the same flow cell. A UV maskor EUV is expensive and used to form the features of the flow cell into a photoresist material.WO 2025 / 174708

[0099] PCT / US2025 / 015300The cross-platform compatibility system may determine selection parameters as part ofdetermining a fiducial configuration within a flow cell. In some implementations, the crossplatform compatibility system considers the numbers of fiducials required by each imaging system.For instance, TDI imaging systems may require more fiducials per tile than SaS imaging systems.Considering these restrictions, the cross-platform compatibility system may determine a selectionparameter of minimizing the total number of fiducials within the flow cell while achieving a targetimage system loss. In the example of creating a flow cell that is compatible with multiple imagingsystems, the cross-platform compatibility system can make the following determinations: (i)fiducials for the first imaging system consume significantly more sequenceable area than fiducialsfor the second imaging system; (ii) the first imaging system requires several more fiducials thanthe second imaging system; and (iii) using the fiducials for the second imaging system throughoutthe flow cell would decrease the sequenceable area for both imaging systems. Based on thesedeterminations, the cross-platform compatibility system can determine to place fiducials using thefollowing rules: (1) place a fiducial for the first imaging system at locations where the first imagingsystem requires a fiducial and the second imaging system does not require a fiducial; (2) place afiducial for the second imaging system at locations where the second imaging system requires afiducial and the first imaging system does not require a fiducial; and (3) place a shared fiducialwhere both the first imaging system and the second imaging system require a fiducial. Furthermore,in some implementations, the cross-platform compatibility system ensures that fiducials fordifferent imaging systems do not overlap.

[0100] In some implementations, the cross-platform compatibility system determinesadditional rules for the placement of fiducials within candidate flow cells. In particular, the crossplatform compatibility system may determine that fiducial patterns for each imaging system haveconsistent positions within each tile for all tiles. In other words, each tile comprises a consistentpattern of each set of fiducials. This ensures that the cross-platform compatibility system canaccurately register the tiles.

[0101] In some embodiments, the cross-platform compatibility system determines anadditional rule precluding the placement of fiducials in the center of the field of view of anyimaging system. More specifically, the cross-platform compatibility system can determine thatmoving objectives for certain imaging systems (such as SaS) means that focus tracking spots willbe in the center of the field of view. Accordingly, the cross-platform compatibility system maydetermine to keep the center of tiles and / or FOV clear of fiducials for all imaging systems.

[0102] Utilizing these rules, the cross-platform compatibility system can create variouscandidate flow cell configurations comprising first imaging system fiducials, second imagingfiducials, and shared fiducials. FIGS. 6A-6B illustrate example candidate flow cell configurations for flow cells imaged by multiple imaging systems or a single imaging system in accordance withone or more implementations of the present disclosure. More particularly, FIG. 6A illustrates anexample fiducial layout 628 for a flow cell imaged by multiple imaging systems, and FIG. 6Billustrates an example fiducial layout 626 for a flow cell imaged by a single imaging system inaccordance with one or more embodiments of the present disclosure.

[0103] FIG. 6A illustrates a portion of a swath. The portion includes first imaging systemfiducials 616a-616j that are utilized by the first imaging system, second imaging system fiducials614a-614c that are utilized by the second imaging system, and shared fiducials 612a-612f. FIG. 6Aalso includes depictions of first imaging system tiles 602a-602b and second imaging system tile604.

[0104] As shown in FIG. 6A, the cross-platform compatibility system may utilize the firstimaging system (e.g., TDI) to capture first imaging system tiles 602a-602b. The first imagingsystem tiles 602a-602b contain consistent configurations of the first imaging system fiducials 616а616j and the shared fiducials 612a-612f. The cross-platform compatibility system may utilize thefirst imaging system to capture the first imaging system tile 602a and register the first imagingsystem tile 602a based on the first imaging system fiducials 616a-616j and the shared fiducials612a-612f.

[0105] The cross-platform compatibility system may also utilize the second imaging system(e.g., SaS) to capture the second imaging system tile 604 portrayed by FIG. 6A. The second imagingsystem tile 604 comprises second imaging system fiducials 614a-614b and the shared fiducials612a-612d. The cross-platform compatibility system may utilize the second imaging system toidentify the second imaging system fiducials 614a-614b and the shared fiducials 612a-612d andbypass the second imaging system fiducials.

[0106] FIG. 6A illustrates the example fiducial layout 628 of a flow cell that can be imaged bymultiple imaging systems. More specifically, the flow cell illustrated in FIG. 6A comprises threetypes of fiducials: fiducials for a first imaging system, fiducials for a second imaging system, andshared fiducials. In some embodiments, a single imaging system can access and image a flow cellhaving multiple types of fiducials. Furthermore, in some implementations, instead of bypassing theone or more sets of fiducials, the imaging system can utilize all types of fiducials. FIG. 6Billustrates an example fiducial layout 626 of a flow cell having multiple types of fiducials foranalysis by a single imaging system.

[0107] FIG. 6B illustrates an example fiducial layout 626 of a flow cell. The flow cell maycomprise different types of fiducials. The flow cell illustrated in FIG. 6B contains anchoringfiducials 620a-620d portrayed by a combination of light- and dark-colored rings. The flow cell alsocontains fine-alignment fiducials 624 portrayed by dark-colored rings. FIG. 6B further illustrates imaging system tiles 622a-622b. The imaging system tiles 622a-622b comprise images capturedby a given imaging system.

[0108] As mentioned, the flow cell portrayed in FIG. 6B contains the anchoring fiducials 620a620d. Generally, the anchoring fiducials 620a-620d are used in a preliminary or coarse-alignmentprocess during tile registration. For instance, the anchoring fiducials 620a-620d can be used toroughly align images of tiles before performing more complex image registration procedures. Insome existing systems, coarse-alignment fiducials are placed along a central axis of a flow cell(e.g., the second imaging system fiducials 614a-614c illustrated in FIG. 6A). Such placement offiducials can the total sequenceable area.

[0109] In some implementations, the system can design a flow cell having different types offiducials to maximize the total sequenceable area within a flow cell. The system can utilize a singlefiducial type for different purposes. Namely, the system can utilize an anchoring fiducial duringboth coarse and fine alignment processes. As shown in FIG. 6B, instead of placing the anchoringfiducials 620a-620d along a central axis, the anchoring fiducials 620a-620d are incorporated as partof the regular imaging system fiducials or the fine-alignment fiducials. The imaging system canuse the anchoring fiducials 620a-620d during its coarse alignment process. Additionally, duringthe fine alignment process, the imaging system can use one or both of the anchoring fiducials 620a620d and the fine-alignment fiducials 624.

[0110] In some implementations, the cross-platform compatibility system utilizes fiducials fora first imaging system and shared fiducials while excluding fiducials used exclusively by a secondimaging system. FIGS. 7A-7D illustrate example candidate tile configurations utilizing fiducialsfor a first imaging system and shared fiducials for a first and second imaging systems in accordancewith one or more embodiments of the present disclosure.

[0111] FIG. 7A illustrates tile configuration 702 displaying a configuration of fiducials for afirst imaging system and shared fiducials within a flow cell. For example, FIG. 7A illustratesfiducials for a first imaging system 716a. More specifically the first imaging system (e.g., a TDIimaging system that requires more fiducials) utilizes all fiducials portrayed within the tileconfiguration 702. Additionally, the tile configuration 702 comprises shared fiducials 720a-7200.Both the first imaging system and the second imaging system (e.g., an SaS imaging system) usethe shared fiducials 720a-7200. FIG. 7A further illustrates a field of view 710a of an optical sensor,an SAS scan start location 714a and a TDI scan start location 712a. As discussed previously, theTDI scan start location 712a is not aligned with the SaS scan start location 714a because of a bufferrequired for Integration Through Focus (ITF) or an acceleration region for TDI. FIG. 7A furtherincludes indications of a first imaging system tile 724 and a second imaging system tile 726. FIG. 13 illustrates example dimensions for the tile configuration 702 illustrated in FIG. 7A in accordancewith one or more implementations.

[0112] FIGS. 7B-7D illustrate additional example tile configurations in accordance with oneor more implementations of the present disclosure. FIG. 7B illustrates a tile configuration 704, FIG.7C illustrates a tile configuration 706, and FIG. 7D illustrates a tile configuration 708. Each of thetile configurations portrayed in FIGS. 7B-7D include fiducials for the first imaging system 716b716d. The fiducials for the first imaging system 716b-716d comprises all fiducials depicted in thetile configurations. In some implementations, some of the fiducials depicted in the tileconfiguration comprises shared fiducials utilized by both imaging systems in a configuration likethe shared fiducials 720a-7200 illustrated in FIG. 7A. FIGS. 7B-7D also include indications of thefield of view 710b-710d, TDI scan start locations 712b-712d, and SaS scan start locations 714b714d.

[0113] As mentioned, the cross-platform compatibility system can generate candidate tileconfigurations having different properties. FIGS. 8-10B illustrate various rules for tileconfigurations that the cross-platform compatibility system can utilize as part of generatingcandidate image segment configurations. More specifically, FIG. 8 illustrates the cross-platformcompatibility system determining tile height for candidate tiles in accordance with one or moreimplementations of the present disclosure. FIGS. 9A-9C illustrate the cross-platform compatibilitysystem determining tile spacing for candidate tiles in accordance with one or more implementationsof the present disclosure. FIGS. 10A-10B illustrate the cross-platform compatibility systemdetermining double exposure distance between candidate tiles in accordance with one or moreimplementations of the present disclosure.

[0114] As mentioned, FIG. 8 illustrates the cross-platform compatibility system determiningtile height for candidate tiles in accordance with one or more implementations of the presentdisclosure. In some embodiments, the cross-platform compatibility system determines that thenumber of smaller tiles grouped for one larger tile should divide evenly into the total number of thelarger tiles. More specifically, any left-over fractional tiles within a swath result in a direct loss. Inone example, line scan tiles are smaller than and can be grouped into a larger SaS tile. The crossplatform compatibility system can divide the number of line scan tiles (e.g., 78) by the number ofSaS tiles (e.g., 4) in which the line scan tiles are contained. The cross-platform compatibility systemdetermines that each SaS tile contains 19.5 line scan tiles where the cross-platform compatibilitysystem would lose 0.5 line scan tiles of data when utilizing a line scan imaging system forapproximately 2.5% loss. The principles for determining tile heigh of both tiles can be expressedusing the following equation: taller tile height + gutter height = x*shorter tile height, where xcomprises an integer (e.g., 2, 3, etc.).

[0115] FIG. 8 illustrates an example side-by-side comparison of SaS tiles 806 and TDI tiles810. The SaS tiles 806 comprise the taller tiles and the TDI tiles 810 comprise the shorter tiles.FIG. 8 illustrates a taller tile height 802 and a gutter height 804 for the SaS tiles 806. FIGS. 9A-9Cfurther describe the use of gutters between tiles. The TDI tiles 810 further have a shorter tile height808. As shown in FIG. 8, a full number (i.e., 2) of the shorter tile height 808 fit into the combinedheight of the taller tile height 802 and the gutter height 804.

[0116] As mentioned, the cross-platform compatibility system may also determine tile spacing.FIGS. 9A-9C illustrate the cross-platform compatibility system determining tile spacing inaccordance with one or more implementations of the present disclosure. FIGS. 9A-9C illustraterules for candidate tile spacing in the y-direction for all tiles regardless of imaging system.

[0117] FIG. 9A illustrates tiles 920a-920b with fiducials 910. As shown in FIG. 9A, the crossplatform compatibility system determines tile spacing based on a distance between fiducials 902,a distance between a fiducial and a tile boundary 904, and a gutter distance 906. Generally, thegutter distance equals the distance between tiles. In certain embodiments, the gutter represents aspace or region on a flow cell that is unimaged in certain systems, such as in a step-and-shootsystem. In certain embodiments, the gutter represents a space or region on a flow cell that isunimaged in certain systems, such as step-and-shoot systems, but is imaged and sequenced in othercertain systems, such as in a line scanning system. For example, the gutter regions may comprisenanowells that are not imaged and sequenced in a step-and-shoot system but are imaged andsequenced in a line scanning system. The gutter can serve as a buffer zone between tiles to preventinterference, over sequencing, or double exposure. In some examples, the cross-platformcompatibility system may determine that each of the fiducials 910 is positioned on a nanowell gridof a patterned flow cell. Thus, the distance between fiducials 902 can be an integer number of afixed value. Furthermore, the cross-platform compatibility system determines that the tiles can bespaced to include a gutter. These requirements can be expressed using the following equations:Distance between fiducials (r) = 2 1+gSegment Image Height (H) = (n-1) r+2.1H= n*r -gWhere / represents a distance between a fiducial and a tile boundary (e.g., 904), g represents thegutter distance (e.g., 906), r represents a distance between fiducials (e.g., 902), and n represents anumber of fiducials per tile.

[0118] FIGS. 9B-9C illustrate additional example tile spacing in accordance with one or moreimplementations of the present disclosure. More specifically, the tiles in FIGS. 9B-9C arecompliant with the principles described above with respect to FIG. 9B. More particularly, FIGS.9B-9C illustrate greater gutter distances between tiles than the gutter distance depicted in FIG. 9A.

[0119] FIG. 9B illustrates tiles 918a-918b. FIG. 9B illustrates a gutter distance 914 thatencompasses fiducial. FIG. 9B also portrays a distance between fiducials 916 and a distancebetween a fiducial and a tile boundary 912, and a gutter distance 906. As shown in FIG. 9B, thegutter distance 914 can be expanded to encompass the height of a fiducial so long as the principlesdescribed above in relation to FIG. 9A are satisfied.

[0120] FIG. 9C illustrates tiles 922a-922b. A gutter distance 926 between the tiles 918a-918bencompasses the heights of two fiducials. FIG. 9C further portrays a distance between fiducials924 and a distance between a fiducial and a tile boundary 928. In some implementations, the crossplatform compatibility system can determine that spacing portrayed in FIGS. 9A-9C is eligible forcandidate flow cell configurations.

[0121] As described previously, the cross-platform compatibility system may also formatcandidate flow cell configurations to decrease or eliminate double exposure. FIGS. 10A-10Billustrate the cross-platform compatibility system determining double exposure distance betweencandidate tiles in accordance with one or more implementations of the present disclosure. Doubleexposure can introduce signal decay due to degradation of the fluorescent label due to the increasedexposure to the excitation light source. For example, degraded fluorescent labels may appeardimmer or may appear photobleached during imaging. The cross-platform compatibility systemmay determine to space tiles to reduce or eliminate double exposure.

[0122] Generally, double occurs if a portion of a tile is exposed in successive fluorescent andimage capture cycles. FIG. 10A illustrates double exposure and the impact of gutter usage inaccordance with one or more implementations of the present disclosure. Typically, during imaging,the cross-platform compatibility system illuminates the flow cell using a circular field of view. Theillumination excites fluorophores within the field of view, which causes them to emit fluorescence.The optical lens captures the emitted fluorescence and records the emitted fluorescence as part ofan image. Double exposure causes an area of two consecutive tiles to emit fluorescence more thanonce, which may cause signal decay during in imaging. Accordingly, areas of double exposure areoften unusable.

[0123] Fields of view may create unused areas within tiles because of double exposure. Forexample, FIG. 10A illustrates tiles 1002a-1002b during imaging. The tile 1002a is fluoresced andcaptured first in a field of view 1008a. The tile 1002b is fluoresced and captured second in a fieldof view 1008b. FIG 10A illustrates an area of double exposure 1006 within the tile 1002b. Morespecifically the area of double exposure 1006 comprises an area that is captured by both the fieldsof view 1008a-1008b.

[0124] The circular nature of fields of view can also create unused areas within segment areasbecause of areas that do not fall within the FOV. For example, FIG. 10A further illustrates an unused area 1004 of the tile 1002a that is not captured by the view 1008a. The cross-platformcompatibility system can space and configure tiles to avoid unused areas resulting from both doubleexposure and falling outside of the FOV.

[0125] The cross-platform compatibility system could completely eliminate the unused area1004 by increasing the size of the field of view 1008a or shrinking the tile 1002a. However, thesemodifications have the downside of introducing over scanning. Over scanning occurs when a FOVilluminates and captures an area larger than the tile. Over scanning can contribute to unused areawithin a flow cell by creating or expanding areas of double exposure. In some implementations,the cross-platform compatibility system determines tile sizes and configurations that reduce oreliminate over scanning.

[0126] FIG. 10B illustrates how the cross-platform compatibility system can space andconfigure tiles to reduce or eliminate unused areas within tiles in accordance with one or moreimplementations of the present disclosure. For example, FIG. 10B illustrates tiles 1012a-1012bduring imaging. A field of view 1014 covers the tile 1012a. As shown, the field of view 1014 alsocaptures a portion of the tile 1012b. The captured portion is unused as it is an area of doubleexposure 1016.

[0127] The inclusion of Y gutters reduces or eliminates unused areas of tiles. As shown in FIG.10B, tiles 1020a-1020b are separated by Y gutters 1018a-1018b. The Y gutters 1018a-1018bincrease the distance of illumination of the field of views 1010a-101b to eliminate areas of doubleexposure. Furthermore, the addition of the Y gutters 1018a-1018b ensures that the tiles 1020a1020b are entirely captured within the fields of view 1010a-1010Ь.

[0128] In some implementations, the cross-platform compatibility system can blockillumination within a field of view in both the x- and y- directions to further reduce the risk ofdouble exposure. For example, during imaging, the cross-platform compatibility system can blockillumination in the x-direction in area 1022a and area 1022b. Similarly, the cross-platformcompatibility system can block illumination in the y-direction in area 1022c.

[0129] As described previously, the cross-platform compatibility system evaluates candidateflow cell configurations (having varied fiducial and tile configurations) to determine a selectedflow cell configuration. FIG. 11 illustrates example selection parameters for evaluating candidateflow cell configurations in accordance with one or more implementations of the present disclosure.Generally, the cross-platform compatibility system determines one or more selection parametersbased on requirements for individual imaging applications. For instance, the cross-platformcompatibility system can dictate selection parameters that increase the performance of one imagingsystem over another, maximize sequenceable area within a flow cell, or meet other imaging goals. The cross-platform compatibility system may dictate specific values for any combination of theselection parameters.

[0130] As shown in FIG. 11, the cross-platform compatibility system can dictate tile height.The cross-platform compatibility system can vary tile height parameters to accommodate FOVs ofdifferent sizes. For example, some imaging platforms or systems may have larger or smaller FOVs.The cross-platform compatibility system can dictate tile height parameters to optimize performanceof individual imaging systems.

[0131] The cross-platform compatibility system may also dictate tile number. Morespecifically, the cross-platform compatibility system can determine a number of tiles within a flowcell or swath. The cross-platform compatibility system can determine the tile number selectionparameter based on FOV size relative to the flow cell size.

[0132] As further illustrated in FIG. 11, the cross-platform compatibility system can determinescan length as a selection parameter. As described previously, SaS imaging systems have longerscan lengths than TDI imaging systems. More specifically, TDI scan lengths are typically shorterbecause of integration through focus (ITF) and acceleration regions. The cross-platformcompatibility system can determine scan length selection parameters based on individualsequencing systems.

[0133] FIG. 11 further illustrates a selection parameter of fiducial number. The cross-platformcompatibility system can determine fiducial number selection parameters based on competinginterests of maximizing sequenceable area and including the requisite number of fiducials per tile.For example, the cross-platform compatibility system can determine a target number of fiducialswithin tiles for both imaging systems.

[0134] FIG. 11 further illustrates the cross-platform compatibility system determining gutterlength selection parameters. The cross-platform compatibility system can specify a gutter lengthbetween neighboring image segments based on the shape or size of the FOV of different imagingsystems.

[0135] FIG. 11 also illustrates the cross-platform compatibility system determining targetimaging system outputs. Generally, different imaging systems may have different outputsdepending on the flow cell configurations. The cross-platform compatibility system can determinetarget imaging system outputs for each utilized imaging system. Utilization of this selectionparameter eliminates candidate flow cells falling below target imaging system outputs. The crossplatform compatibility system can also utilize this selection parameter to prioritize one imagingsystem over another imaging system by deliberately prescribing a lower output for a particularimaging system.

[0136] As further illustrated in FIG. 11, the cross-platform compatibility system can determineselection parameters comprising tile usage. Generally, the cross-platform compatibility system candetermine a percentage of tiles that are successfully utilized. For example, a first imaging systemmay achieve 100% tile usage while the second imaging system achieves a lower percent tile usage.The cross-platform compatibility system can utilize the tile usage selection parameter to prioritizeone imaging system over another imaging system.

[0137] As described, the cross-platform compatibility system utilizes selection parameters toevaluate candidate flow cell configurations. The cross-platform compatibility system determinesselected fiducial and tile configurations that meet the one or more selection parameters. FIGS. 12-13 illustrate example selected fiducial and tile configurations in accordance with one or moreimplementations of the present disclosure. FIGS. 12-13 illustrate approximations for variousfiducial layout parameters. For example, the variable a illustrated in FIGS. 12-13 can representdifferent values across different examples.

[0138] FIG. 12 illustrates features of a selected flow cell configuration in accordance with oneor more implementations of the present disclosure. The features illustrated in FIG. 12 correspondwith the fiducial configuration illustrated in FIG. 7A. As shown, the cross-platform compatibilitysystem determined selection criteria to accomplish the following objectives: 1) minimize TDI loss(< 1%), 2) optimize SAS output, 3) eliminate double exposure (Y gutter > 10um), 4) ensure aneven number of TDI and SAS tiles, and 4) no over scanning of SaS and TDI tiles. The table in FIG.12 includes example measurements and experiment outcomes for tile height, number of tiles perswath (i.e., tile number), total scan length, loss to max usage, number of fiducials per tile, gutterlength in the X- and Y- directions, output, tile usage (i.e., tile usage), and ratio of SAS to TDI tiles.

[0139] Cross-platform compatible flow cells can achieve competitive performance in multipleimaging systems relative to platform-specific flow cells. As shown in FIG. 12, the loss for maxusage in both TDI and SaS imaging systems is less than 1%. Furthermore, the output for both theTDI and SaS imaging systems is high with 100% output for TDI and 97% output for SaS. Similarly,tile usage is high with TDI tile usage at 100% and SaS tile usage at 95%.

[0140] Even with relaxed requirements for selection parameters, the cross-platformcompatibility system can generate selected flow cell configurations with competitive performance.FIG. 13 illustrates additional example selected flow cell configurations in accordance with one ormore implementations of the present disclosure. More specifically, for the selected flow cellconfigurations illustrated in FIG. 13, the cross-platform compatibility system determined selectionparameters that accomplish the following objectives: 1) eliminate double exposure (Y gutter >10um), 2) ensure that TDI loss is < 3%, 3) ensure even numbers of TDI tiles and SaS tiles, and 4)allow for slight over-scanning of SaS and TDI.

[0141] FIG. 13 illustrates a table 1302 and a table 1304 including features of selected flow cellconfigurations. In particular, the table 1302 demonstrates features for a flow cell resulting in SaSoverscan (144 um) with lower TDI loss (1.6%), and the table 1304 demonstrates features for noSaS overscan with higher TDI loss (2.7%). Even given these limitations, the cross-platformcompatibility system has created flow cells demonstrating high output and tile usage. Morespecifically, the ouput in all cases is more than 99.5%, and the tile usage, for all cases, is greaterthan 93%.

[0142] Aspects of the present disclosure relate generally to devices, systems, and methodsproviding biological or chemical analysis. Various protocols in biological or chemical researchinvolve performing a large number of controlled reactions on local support surfaces or withinpredefined reaction chambers. The designated reactions may then be observed or detected, andsubsequent analysis may help identify or reveal properties of chemicals involved in the reaction.For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g.,fluorescent label) may be exposed to thousands of known probes under controlled conditions. Eachknown probe may be deposited into a corresponding well of a flow cell channel. Observing anychemical reactions that occur between the known probes and the unknown analyte within the wellsmay help identify or reveal properties of the analyte. Other examples of such protocols includeknown DNA sequencing processes, such as sequencing-by-synthesis (SBS) or cyclic-arraysequencing.

[0143] While a variety of devices, systems, and methods have been made and used to performbiological or chemical analysis, it is believed that no one prior to the inventor(s) has made or usedthe devices and techniques described herein.

[0144] FIG. 14 illustrates a schematic diagram of an example of a system (1400) that may beused to perform an analysis on one or more samples of interest. In some implementations, thesample may include one or more clusters of nucleotides (e.g., DNA) that have been linearized toform a single stranded DNA (sstDNA). In the implementation shown, system (1400) is configuredto receive a flow cell cartridge assembly (1402) including a flow cell assembly (1403) and a samplecartridge (1404). System (1400) includes a flow cell receptacle (1422) that receives flow cellcartridge assembly (1402), a vacuum chuck (1424) that supports flow cell assembly (1403), and aflow cell interface (1426) that is used to establish a fluidic coupling between system (1400) andflow cell assembly (1403). Flow cell interface (1426) may include one or more manifolds. System(1400) further includes a sipper manifold assembly (1406), a sample loading manifold assembly(1408), and a pump manifold assembly (1410). System (1400) also includes a drive assembly(1412), a controller (1414), an imaging system (1416), and a waste reservoir (1418). Controller(1414) is electrically and / or communicatively coupled to drive assembly (1412) and to imaging system (1416); and is configured to cause drive assembly (1412) and / or the imaging system (1416)to perform various functions as disclosed herein.

[0145] In the present example, flow cell assembly (1403) includes a flow cell (1428) havingachannel (1430) and defining a plurality of first openings (1432), which are fluidically coupled tothe channel (1430) and arranged on a first side (1434) of the channel (1430). Flow cell (1428)further includes a plurality of second openings (1436) fluidically coupled to the channel (1430) andarranged on a second side (1438) of the channel (1430). Fluid may flow through flow cell (1428)via channel. While the flow cell (1428) is shown including one channel (1430), flow cell (1428)may include two or more channels (1430). Flow cell assembly (1403) also includes a flow cellmanifold assembly (1440) coupled to flow cell (1428) and having a first manifold fluidic line(1442) and a second manifold fluidic line (1444). Flow cell manifold assembly (1440) may be inthe form of a laminate including a plurality of layers as discussed in more detail below.

[0146] In the implementation shown, first manifold fluidic line (1442) has a first fluidic lineopening (1446) and is fluidically coupled to each of the plurality of first openings (1432) of flowcell (1428); and second manifold fluidic line (1444) has a second fluidic line opening (1448) andis fluidically coupled to each of the plurality of second openings (1436). As shown, flow cellassembly (1403) includes gaskets (1450) coupled to flow cell manifold assembly (1440) andfluidically coupled to fluidic line openings (1446, 1448). In some implementations where floww cell(1428) includes a plurality of channels (1430), flow cell manifold assembly (1440) may includeadditional fluidic lines (1452) that couple the first fluidic line opening (1446) to a single manifoldport (1454). In such implementations, a single gasket of the gaskets (1450) may be coupled to flowcell manifold assembly (1440) that surrounds the single manifold port (1454) and is in fluidiccommunication with a plurality of channels (1430). In operation, flow cell interface (1426) engageswith corresponding gaskets (1450) to establish a fluidic coupling between system (1400) and flowcell (1428). The engagement between flow cell interface (1426) and gaskets (1450) reduces oreliminates fluid leakage between flow cell interface (1426) and flow cell (1428).

[0147] In the implementation shown, first manifold fluidic line (1442) has a portion (1456)that is substantially parallel to a longitudinal axis (1458) of channel (1430); and second manifoldfluidic line (1444) has a portion (1460) that is substantially parallel to longitudinal axis (1458) ofchannel (1430). Additionally, first manifold fluidic line (1442) is shown being at least partiallyadjacent a first end (1462) of flow cell (1428) and spaced from a second end (1464) of flow cell(1428); and second manifold fluidic line (1444) is shown being at least partially adjacent secondend (1464) of flow cell (1428) and spaced from first end (1462). Other arrangements of manifoldfluidic lines (1442, 1444) may prove suitable, however.

[0148] In the implementation shown, system (1400) includes a sample cartridge receptacle(1466) that receives sample cartridge (1404) that carries one or more samples of interest (e.g., ananalyte). System (1400) also includes a sample cartridge interface (1468) that establishes a fluidicconnection with sample cartridge (1404). Sample loading manifold assembly (1408) includes oneor more sample valves (1470). Pump manifold assembly (1410) includes one or more pumps(1472), one or more pump valves (1474), and a cache (1476). Valves (1470, 1474) and pumps(1472) may take any suitable form. Cache (1476) may include a serpentine cache and maytemporarily store one or more reaction components during, for example, bypass manipulations ofthe system (1400). While cache (1476) is shown being included in pump manifold assembly (1410),cache (1476) may alternatively be located elsewhere (e.g., in sipper manifold assembly (1406) orin another manifold downstream of a bypass fluidic line (1478), etc.).

[0149] Sample loading manifold assembly (1408) and pump manifold assembly (1410) flowone or more samples of interest from sample cartridge (1404) through a fluidic line (1480) towardflow cell cartridge assembly (1402). In some implementations, sample loading manifold assembly(1408) may individually load or address each channel (1430) of flow cell (1428) with a respectivesample of interest. The process of loading channel (1430) with a sample of interest may occurautomatically using system (1400). As shown in FIG. 14, sample cartridge (1404) and sampleloading manifold assembly (1408) are positioned downstream of flow cell cartridge assembly(1402). In the implementation shown, sample loading manifold assembly (1408) is coupledbetween flow cell cartridge assembly (1402) and pump manifold assembly (1410). To draw asample of interest from sample cartridge (1404) and toward pump manifold assembly (1410),sample valves (1470), pump valves (1474), and / or pumps (1472) may be selectively actuated tourge the sample of interest toward pump manifold assembly (1410). Sample cartridge (1404) mayinclude a plurality of sample reservoirs that are selectively fluidically accessible via thecorresponding sample valves (1470). To individually flow the sample of interest toward channel(1430) of flow cell (1428) and away from pump manifold assembly (1410), sample valves (1470),pump valves (1474), and / or pumps (1472) may be selectively actuated to urge the sample of interesttoward flow cell cartridge assembly (1402) and into respective channels (1430) of flow cell (1428).

[0150] Drive assembly (1412) interfaces with sipper manifold assembly (1406) and pumpmanifold assembly (1410) to flow one or more reagents that interact with the sample within flowcell (1428). In some scenarios, a reversible terminator is attached to the reagent to allow a singlenucleotide to be incorporated onto a growing DNA strand. In some such implementations, one ormore of the nucleotides has a unique fluorescent label that emits a color when excited. The color(or absence thereof) is used to detect the corresponding nucleotide. In the implementation shown,imaging system (1416) excites one or more of the identifiable labels (e.g., a fluorescent label) and thereafter obtains image data for the identifiable labels. The labels may be excited by incident lightand / or a laser and the image data may include one or more colors emitted by the respective labelsin response to the excitation. The image data (e.g., detection data) may be analyzed by system(1400). Examples of features and functionalities that may be incorporated into imaging system(1416) will be described in greater detail below.

[0151] After the image data is obtained, drive assembly (1412) interfaces with sipper manifoldassembly (1406) and pump manifold assembly (1410) to flow another reaction component (e.g., areagent) through flow cell (1428) that is thereafter received by waste reservoir (1418) via a primarywaste fluidic line (1482) and / or otherwise exhausted by system (1400). Some reaction componentsmay perform a flushing operation that chemically cleaves the fluorescent label and the reversibleterminator from the sstDNA. The sstDNA may then be ready for another cycle.

[0152] The primary waste fluidic line (1482) is coupled between pump manifold assembly(1410) and waste reservoir (1418). In some implementations, pumps (1472) and / or pump valves(1474) of pump manifold assembly (1410) selectively flow the reaction components from flow cellcartridge assembly (1402), through fluidic line (1480) and sample loading manifold assembly(1408) to primary waste fluidic line (1482). Flow cell cartridge assembly (1402) is coupled to acentral valve (1484) via flow cell interface (1426). Central valve (1484) is coupled with flow cellinterface (1426) via a fluidic line (1485). An auxiliary waste fluidic line (1486) is coupled to centralvalve (1484) and to waste reservoir (1418). In some implementations, auxiliary waste fluidic line(1486) receives excess fluid of a sample of interest from flow cell cartridge assembly (1402), viacentral valve (1484), and flows the excess fluid of the sample of interest to waste reservoir (1418)when back loading the sample of interest into flow cell (1428), as described herein.

[0153] Sipper manifold assembly (1406) includes a shared line valve (1488) and a bypass valve(1490). Shared line valve (1488) may be referred to as a reagent selector valve. Central valve (1484)and the valves (1488, 1490) of sipper manifold assembly (1406) may be selectively actuated tocontrol the flow of fluid through fluidic lines (1492, 1494, 1496). Sipper manifold assembly (1406)may be coupled to a corresponding number of reagent reservoirs (1498) via reagent sippers (1500).Reagent reservoirs (1498) may contain fluid (e.g., reagent and / or another reaction component). Insome implementations, sipper manifold assembly (1406) includes a plurality of ports. Each port ofsipper manifold assembly (1406) may receive one of the reagent sippers (1500). Reagent sippers(1500) may be referred to as fluidic lines. Some forms of reagent sippers (1500) may include anarray of sipper tubes extending downwardly along the z-dimension from ports in the body of sippermanifold assembly (1406). Reagent reservoirs (1498) may be provided in a cartridge, and the tubesof reagent sippers (1500) may be configured to be inserted into corresponding reagent reservoirs (1498) in the reagent cartridge so that liquid reagent may be drawn from each reagent reservoir(1498) into the sipper manifold assembly (1406).

[0154] Shared line valve (1488) of sipper manifold assembly (1406) is coupled to central valve(1484) via shared reagent fluidic line (1496). Different reagents may flow through shared reagentfluidic line (1496) at different times. In some versions, when performing a flushing operation beforechanging between one reagent and another, pump manifold assembly (1410) may draw wash bufferthrough shared reagent fluidic line (1496), central valve (1484), and flow cell cartridge assembly(1402).

[0155] Bypass valve (1490) of sipper manifold assembly (1406) is coupled to central valve(1484) via dedicated reagent fluidic lines (1494, 1496). Each of the dedicated reagent fluidic lines(1494, 1496) may be associated with a single reagent. The fluids that may flow through dedicatedreagent fluidic lines (1494, 1496) may be used during sequencing operations and may include acleave reagent, an incorporation reagent, a scan reagent, a cleave wash, and / or a wash buffer.

[0156] Bypass valve (1490) is also coupled to cache (1476) of pump manifold assembly (1410)via bypass fluidic line (1478). One or more reagent priming operations, hydration operations,mixing operations, and / or transfer operations may be performed using bypass fluidic line (1478).The priming operations, the hydration operations, the mixing operations, and / or the transferoperations may be performed independent of flow cell cartridge assembly (1402). The operationsusing bypass fluidic line (1478) may occur during, for example, incubation of one or more samplesof interest within flow cell cartridge assembly (1402). That is, shared line valve (1488) may beutilized independently of bypass valve (1490) such that bypass valve (1490) may utilize bypassfluidic line (1478) and / or cache (1476) to perform one or more operations while shared line valve(1488) and / or central valve (1484) simultaneously, substantially simultaneously, or offsetsynchronously perform other operations.

[0157] Drive assembly (1412) includes a pump drive assembly (1502) and a valve driveassembly (1504). Pump drive assembly (1502) may be adapted to interface with one or more pumps(1472) to pump fluid through flow cell (1428) and / or to load one or more samples of interest intoflow cell (1428). Valve drive assembly (1504) may be adapted to interface with one or more of thevalves (1470, 1474, 1484, 1488, 1490) to control the position of the corresponding valves (1470,1474, 1484, 1488, 1490).

[0158] FIG. 15 shows an example of a fluidic arrangement (1520) that may be incorporatedinto a variation of system (1400). Fluidic arrangement (1520) of this example includes a pumpmanifold assembly (1522), which may operate similar to pump manifold assembly (1410)described above; a sample loading manifold assembly (1528), which may operate similar to sampleloading manifold assembly (1408) described above; a flow cell interface (1540), which may operate similar to flow cell interface (1426) described above; a sipper manifold assembly (1550), whichmay operate similar to sipper manifold assembly (1406) described above; and a waste reservoir(1570), which may operate similar to waste reservoir (1418) described above. Pump manifoldassembly (1522) is coupled with a port assembly (1558) of sipper manifold assembly (1550) via afluidic line (1524), which may be similar to fluidic line (1478); and with sample loading manifoldassembly (1528) via a fluidic line (1526). Sample loading manifold assembly (1528) is coupledwith flow cell interface (1540) via fluidic line (1530), which may be similar to fluidic line (1480);and with port assembly (1558) via fluidic lines (1532, 1534). Flow cell interface (1540) is coupledwith sipper manifold assembly (1550) via fluidic line (1542), which may be similar to fluidic line(1485). Sipper manifold assembly (1550) includes a manifold body (1552) and a common outputport (1556), which provides fluid communication via fluidic line (1485). A valve assembly (1554)controls fluid flow through common output port (1556) and may operate similar to central valve(1484). Port assembly (1558) of sipper manifold assembly (1550) is coupled with waste reservoir(1570) via fluidic line (1572), which may be similar to fluidic line (1486).

[0159] A plurality of reagent sippers (1560) extend from manifold body (1552) and arefluidically coupled with valve assembly (1554) via respective fluid channels (1562) in manifoldbody (1552). The plurality of reagent sippers (1560) may operate similar to reagent sippers (1500).Valve assembly (1554) is operable to selectively couple fluid channels (1562) with flow cellinterface (1540) via common output port (1556) and fluidic line (1530), to thereby selectivelyprovide various reagents to flow cell interface (1540). In other words, when each of the pluralityof reagent sippers (1560) is disposed in a different respective reagent (e.g., in a respective reagentreservoir (1498)), a flow cell (e.g., like flow cell (1428)) that is coupled with flow cell interface(1540) may selectively receive those different reagents based on control of valve assembly (1554).

[0160] A plurality of reagent sippers (1560) extend from manifold body (1552) and arefluidically coupled with valve assembly (1554) via respective fluid channels (1562) in manifoldbody (1552). The plurality of reagent sippers (1560) may operate similar to reagent sippers (1500).Valve assembly (1554) is operable to selectively couple fluid channels (1562) with flow cellinterface (1540) via common output port (1556) and fluidic line (1530), to thereby selectivelyprovide various reagents to flow cell interface (1540). In other words, when each of the pluralityof reagent sippers (1560) is disposed in a different respective reagent (e.g., in a respective reagentreservoir (1498), a flow cell (e.g., like flow cell (1428)) that is coupled with flow cell interface(1540) may selectively receive those different reagents based on control of valve assembly (1554).

[0161] Referring back to FIG. 14, controller (1414) of the present example includes a userinterface (1506), a communication interface (1508), one or more processors (1510), and a memory(1512) storing instructions executable by the one or more processors (1510) to perform various functions including the disclosed implementations. User interface (1506), communication interface(1433), and memory (1512) are electrically and / or communicatively coupled to the one or moreprocessors (1510). User interface (1506) may be adapted to receive input from a user and to provideinformation to the user associated with the operation of system (1400) and / or an analysis takingplace. User interface (1506) may include a touch screen, a display, a keyboard, a speaker(s), amouse, a track ball, and / or a voice recognition system.

[0162] Communication interface (1508) is adapted to enable communication between system(1400) and a remote system(s) (e.g., computers) via a network(s) (e.g., the Internet, an intranet, alocal-area network (LAN), a wide-area network (WAN), a coaxial-cable network, a wirelessnetwork, a wired network, a satellite network, a digital subscriber line (DSL) network, a cellularnetwork, a Bluetooth connection, a near field communication (NFC) connection, etc.). Some of thecommunications provided to the remote system may be associated with analysis results, imagingdata, etc. generated or otherwise obtained by system (1400). Some of the communications providedto system (1400) may be associated with a fluidics analysis operation, patient records, and / or aprotocol(s) to be executed by system (1400).

[0163] The one or more processors (1510) and / or system (1400) may include one or more ofa processor-based system(s) or a microprocessor-based system(s). In some implementations, theone or more processors (1510) and / or system (1400) includes one or more of a programmableprocessor, a programmable controller, a microprocessor, a microcontroller, a graphics processingunit (GPU), a digital signal processor (DSP), a reduced-instruction set computer (RISC), anapplication specific integrated circuit (ASIC), a field programmable gate array (FPGA), a fieldprogrammable logic device (FPLD), a logic circuit, and / or another logic-based device executingvarious functions including the ones described herein.

[0164] Memory (1512) may include one or more of a semiconductor memory, a magneticallyreadable memory, an optical memory, a hard disk drive (HDD), an optical storage drive, a solidstate storage device, a solid-state drive (SSD), a flash memory, a read-only memory (ROM),erasable programmable read-only memory (EPROM), electrically erasable programmable readonly memory (EEPROM), a random-access memory (RAM), a non-volatile RAM (NVRAM)memory, a compact disc (CD), a compact disc read-only memory (CD-ROM), a digital versatiledisk (DVD), a Blu-ray disk, a redundant array of independent disks (RAID) system, a cache and / orany other storage device or storage disk in which information is stored for any duration (e.g.,permanently, temporarily, for extended periods of time, for buffering, for caching).

[0165] FIGS. 1-15, the corresponding text, and the examples provide a number of differentmethods, systems, devices, and non-transitory computer-readable media of the cross-platformcompatibility system. In addition to the foregoing, one or more implementations can also be described in terms of flowcharts comprising acts for accomplishing a particular result, as shown inFIGS. 16-17. FIG. 16 illustrates a flowchart of a series of acts 1600 for registering a plurality oftiles based on a first set of fiducials in accordance with one or more embodiments of the presentdisclosure. FIG. 17 illustrates a flowchart of a series of acts 1700 for registering a plurality of tilesbased on a first set of fiducials in accordance with one or more embodiments of the presentdisclosure. While FIGS. 16-17 illustrate acts according to one embodiment, alternativeembodiments may omit, add to, reorder, and / or modify any of the acts shown in FIGS. 16-17. Theacts of FIGS. 16-17 can be performed as part of a method. Alternatively, a non-transitory computerreadable 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 FIGS. 16-17. In still furtherembodiments, an instrument comprising; a stage to receive a flow cell comprising a first set offiducials and a second set of fiducials, a detector, an optical lens assembly between the stage andthe detector, the optical lens assembly to direct light from the flow cell to the detector, at least oneprocessor coupled to the detector, and a non-transitory computer readable medium comprisinginstructions that, when executed by the at least one processor, cause the system to perform the actsof FIGS. 16-17.

[0166] As shown in FIG. 16, the series of acts 1600 includes an act 1602 of capturing aplurality of tiles, an act 1604 of identifying a first set of fiducials, an act 1606 of bypassing a secondset of fiducials, and an act 1608 of registering the plurality of tiles based on the first set of fiducials.For example, the series of acts 1600 can include acts to perform any of the operations described inthe following clauses:CLAUSE 1. A method comprising:capturing a plurality of tiles of the flow cell;identifying, within the flow cell, the first set of fiducials;bypassing, within the flow cell, the second set of fiducials; andregistering the plurality of tiles based on the first set of fiducials.CLAUSE 2. The method of clause 1, wherein the at least one processor is furthercoupled to the stage or the optical lens assembly to cause a relative stepping motion between thestage and the optical lens assembly to capture the plurality of tiles in a step-and-shoot manner.CLAUSE 3. The method of clause 1, wherein the at least one processor is furthercoupled to the stage or the optical lens assembly to cause a relative scanning motion between thestage and the optical lens assembly to capture the plurality of tiles in a scanning manner.CLAUSE 4. The method of clause 1, wherein the second set of fiducials is utilized by asecond instrument. CLAUSE 5. The method of clause 1, further comprising registering the plurality of tilesby aligning the plurality of tiles based on the first set of fiducials.CLAUSE 6. The method of clause 1, wherein fiducials from the first set of fiducials andfiducials from the second set of fiducials do not overlap on the flow cell.CLAUSE 7. The method of clause 1, wherein a tile of the plurality of tiles comprises aconsistent pattern of the first set of fiducials.

[0167] As shown in FIG. 17, the series of acts 1700 comprises an act 1702 of receiving a flowcell comprising a first set of fiducials and a set of fiducials, an act 1704 of capturing a plurality oftiles of the flow cell utilizing a first imaging system, an act 1706 of identifying the first set offiducials, and an act 1708 of registering the plurality of tiles based on the first set of fiducials. Forexample, the series of acts 1700 can include acts to perform any of the operations described in thefollowing clauses:CLAUSE 8. A method comprising:receiving a flow cell comprising a first set of fiducials utilized by a first imaging systemand a second set of fiducials utilized by a second imaging system;capturing a plurality of tiles of the flow cell by utilizing the first imaging system;identifying, within the flow cell, the first set of fiducials corresponding to the first imagingsystem; andregistering the plurality of tiles based on the first set of fiducials.CLAUSE 9. The method of clause 8, further comprising:capturing a second plurality of tiles of the flow cell by utilizing the second imaging system;identifying, within the flow cell, the second set of fiducials corresponding to the secondimaging system; andregistering the second plurality of tiles based on the second set of fiducials.CLAUSE 10. The method of clause 9, further comprising:comparing expected positions of the first set of fiducials with observed positions of the firstset of fiducials in the plurality of tiles;determining, based on comparing the expected positions of the first set of fiducials with theobserved positions of the first set of fiducials, alignment parameters for the plurality of tiles; andcombining the plurality of tiles utilizing the alignment parameters.CLAUSE 11. The method of clause 9, wherein:andeach tile of the plurality of tiles comprises a consistent pattern of the first set of fiducials;each tile of the second plurality of tiles comprises a consistent pattern of the second set offiducials. CLAUSE 12. The method of clause 9, further comprising registering the second pluralityof tiles by:comparing expected positions of the second set of fiducials with observed positions of thesecond set of fiducials in the second plurality of tiles;determining, based on comparing the expected positions of the second set of fiducials withthe observed positions of the second set of fiducials, alignment parameters for the second pluralityof tiles; andcombining the second plurality of tiles utilizing the alignment parameters.CLAUSE 13. The method of clause 8, wherein a number of segments in the secondplurality of tiles divides evenly into a number of segments in the plurality of tiles.CLAUSE 14. The method of clause 8, wherein the first imaging system comprises a stepand-shoot (SaS) system and the second imaging system comprises a time-delay integration (TDI)system.gutters.cell:CLAUSE 15. The method of clause 8, wherein the plurality of tiles are separated byCLAUSE 16. A method comprising:capturing an initial set of images of the flow cell;identifying, based on the initial set of images, the first set of fiducials within the flowadjusting an alignment between the stage and the detector based on the first set offiducials:capturingaplurality of segment images of the flow cell; andregistering the plurality of segment images based on the first set of fiducials and the secondset of fiducials.CLAUSE 17. The method of clause 16, wherein:the first set of fiducials comprises a first fiducial type; andthe second set of fiducials comprises a second fiducial type.CLAUSE 18. The method of clause 16, further comprising adjusting the alignmentbetween the stage and the detector by:determining locations of the first set of fiducials; andbypassing the second set of fiducials.

[0168] In addition to the foregoing, one or more implementations can also be described interms of a flow cell comprising components for accomplishing a particular result. For example, aflow cell can be described in the following clauses:WO 2025 / 174708CLAUSE 19. A flow cell comprising:a first set of fiducials utilized by a first imaging system; anda second set of fiducials utilized by a second imaging system;PCT / US2025 / 015300wherein the first set of fiducials and the second set of fiducials comprise at least one sharedfiducial that is utilized by both the first imaging system and the second imaging system.CLAUSE 20. The flow cell of clause 19, wherein:the first set of fiducials comprise a first fiducial type;the second set of fiducials comprise a second fiducial type; andthe at least one shared fiducial comprises the second fiducial type.CLAUSE 21. The flow cell of clause 19, wherein the first set of fiducials and the secondset of fiducials fall on a nanowell grid.CLAUSE 22. The flow cell of clause 19, wherein the first imaging system comprises astep-and-shoot (SaS) system and the second imaging system comprises a time-delay integration(TDI) system.CLAUSE 23. The flow cell of clause 19, wherein:areas corresponding to a first set of tiles of the flow cell comprise a consistent pattern ofthe first set of fiducials: andsecond areas corresponding to a second set of tiles of the flow cell comprise a consistent pattern ofthe second set of fiducials, wherein the areas and the second areas are different sizes.

[0169] The methods described herein can be used in conjunction with a variety of nucleic acidsequencing techniques. Particularly applicable techniques are those wherein nucleic acids areattached at fixed locations in an array such that their relative positions do not change and whereinthe array is repeatedly imaged. Embodiments in which images are obtained in different colorchannels, for example, coinciding with different labels used to distinguish one nucleobase typefrom another are particularly applicable. In some embodiments, the process to determine thenucleotide sequence of a target nucleic acid (i.e., a nucleic-acid polymer) can be an automatedprocess. Preferred embodiments include sequencing-by-synthesis (SBS) techniques.

[0170] SBS techniques generally involve the enzymatic extension of a nascent nucleic acidstrand through the iterative addition of nucleotides against a template strand. In traditional methodsof SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of apolymerase in each delivery. However, in the methods described herein, more than one type ofnucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in adelivery.

[0171] SBS can utilize nucleotide monomers that have a terminator moiety or those that lackany terminator moieties. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using y-phosphate-labeled nucleotides, as set forth infurther detail below. In methods using nucleotide monomers lacking terminators, the number ofnucleotides added in each cycle is generally variable and dependent upon the template sequenceand the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers havinga terminator moiety, the terminator can be effectively irreversible under the sequencing conditionsused as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methods developed by Solexa (nowIllumina, Inc.).

[0172] SBS techniques can utilize nucleotide monomers that have a label moiety or those thatlack a label moiety. Accordingly, incorporation events can be detected based on a characteristic ofthe label, such as fluorescence of the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of the nucleotide, such as release ofpyrophosphate; or the like. In embodiments, where two or more different nucleotides are presentin a sequencing reagent, the different nucleotides can be distinguishable from each other, oralternatively, the two or more different labels can be the indistinguishable under the detectiontechniques being used. For example, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriate optics as exemplified by thesequencing methods developed by Solexa (now Illumina, Inc.).

[0173] Preferred embodiments include pyrosequencing techniques. Pyrosequencing detectsthe release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into thenascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)"Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." GenomeRes. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method based onreal-time pyrophosphate." Science 281(5375), 363; U.S. Pat. No. 6,210,891; U.S. Pat. No.6,258,568 and U.S. Pat. No. 6,274,320, the disclosures of which are incorporated herein byreference in their entireties). In pyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated isdetected via luciferase-produced photons. The nucleic acids to be sequenced can be attached tofeatures in an array and the array can be imaged to capture the chemiluminescent signals that areproduced due to incorporation of a nucleotides at the features of the array. An image can beobtained after the array is treated with a particular nucleotide type (e.g., A, T, C or G). Imagesobtained after addition of each nucleotide type will differ with regard to which features in the arrayare detected. These differences in the image reflect the different sequence content of the featureson 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 inthe same way as exemplified herein for images obtained from different detection channels forreversible terminator-based sequencing methods.

[0174] In another exemplary type of SBS, cycle sequencing is accomplished by stepwiseaddition of reversible terminator nucleotides containing, for example, a cleavable or fluorescentdye label as described, for example, in WO 04 / 018497 and U.S. Pat. No. 7,057,026, the disclosuresof 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 isincorporated herein by reference. The availability of fluorescently-labeled terminators in whichboth the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficientlyincorporate and extend from these modified nucleotides.

[0175] Preferably in reversible terminator-based sequencing embodiments, the labels do notsubstantially inhibit extension under SBS reaction conditions. However, the detection labels can beremovable, for example, by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. In particular embodiments, each cycleinvolves simultaneous delivery of four different nucleotide types to the array and each nucleotidetype has a spectrally distinct label. Four images can then be obtained, each using a detection channelthat is selective for one of the four different labels. Alternatively, different nucleotide types can beadded sequentially and an image of the array can be obtained between each addition step. In suchembodiments, each image will show nucleic acid features that have incorporated nucleotides of aparticular type. Different features are present or absent in the different images due the differentsequence content of each feature. However, the relative position of the features will remainunchanged in the images. Images obtained from such reversible terminator-SBS methods can bestored, processed and analyzed as set forth herein. Following the image capture step, labels can beremoved and reversible terminator moieties can be removed for subsequent cycles of nucleotideaddition and detection. Removal of the labels after they have been detected in a particular cycleand prior to a subsequent cycle can provide the advantage of reducing background signal andcrosstalk between cycles. Examples of useful labels and removal methods are set forth below.

[0176] In particular embodiments some or all of the nucleotide monomers can includereversible terminators. In such embodiments, reversible terminators / cleavable fluors can includefluor 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 terminatorchemistry 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 describedthe development of reversible terminators that used a small 3' allyl group to block extension, butcould easily be deblocked by a short treatment with a palladium catalyst. The fluorophore wasattached to the base via a photocleavable linker that could easily be cleaved by a 30 second exposureto long wavelength UV light. Either disulfide reduction or photocleavage can be used as a cleavablelinker. Another approach to reversible termination is the use of natural termination that ensues afterplacement of a bulky dye on a dNTP. The presence of a charged bulky dye on the dNTP can act asan effective terminator through steric and / or electrostatic hindrance. The presence of oneincorporation event prevents further incorporations unless the dye is removed. Cleavage of the dyeremoves the fluor and effectively reverses the termination. Examples of modified nucleotides arealso described in U.S. Pat. No. 7,427,673, and U.S. Pat. No. 7,057,026, the disclosures of whichare incorporated herein by reference in their entireties.

[0177] Additional exemplary SBS systems and methods which can be utilized with themethods and systems described herein are described in U.S. Patent Application Publication No.2007 / 0166705, U.S. Patent Application Publication No. 2006 / 0188901, U.S. Pat. No. 7,057,026,U.S. Patent Application Publication No. 2006 / 0240439, U.S. Patent Application Publication No.2006 / 0281109, РСТ Publication No. WO 05 / 065814, U.S. Patent Application Publication No.2005 / 0100900, PCT Publication No. WO 06 / 064199, РCT Publication No. WO 07 / 010,251, U.S.Patent Application Publication No. 2012 / 0270305 and U.S. Patent Application Publication No.2013 / 0260372, the disclosures of which are incorporated herein by reference in their entireties.

[0178] Some embodiments can utilize detection of four different nucleotides using fewer thanfour different labels. For example, SBS can be performed utilizing methods and systems describedin the incorporated materials of U.S. Patent Application Publication No. 2013 / 0079232. As a firstexample, a pair of nucleotide types can be detected at the same wavelength, but distinguished basedon a difference in intensity for one member of the pair compared to the other, or based on a changeto one member of the pair (e.g. via chemical modification, photochemical modification or physicalmodification) that causes apparent signal to appear or disappear compared to the signal detectedfor the other member of the pair. As a second example, three of four different nucleotide types canbe detected under particular conditions while a fourth nucleotide type lacks a label that is detectableunder those conditions, or is minimally detected under those conditions (e.g., minimal detectiondue to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleicacid can be determined based on presence of their respective signals and incorporation of the fourthnucleotide type into the nucleic acid can be determined based on absence or minimal detection ofany signal. As a third example, one nucleotide type can include label(s) that are detected in twodifferent channels, whereas other nucleotide types are detected in no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and canbe used in various combinations. An exemplary embodiment that combines all three examples, isa 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 excitationwavelength), a second nucleotide type that is detected in a second channel (e.g. dCTP having alabel that is detected in the second channel when excited by a second excitation wavelength), athird nucleotide type that is detected in both the first and the second channel (e.g. dTTP having atleast one label that is detected in both channels when excited by the first and / or second excitationwavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected ineither channel (e.g. dGTP having no label).

[0179] Further, as described in the incorporated materials of U.S. Patent ApplicationPublication No. 2013 / 0079232, sequencing data can be obtained using a single channel. In such socalled one-dye sequencing approaches, the first nucleotide type is labeled but the label is removedafter the first image is generated, and the second nucleotide type is labeled only after a first imageis generated. The third nucleotide type retains its label in both the first and second images, and thefourth nucleotide type remains unlabeled in both images.

[0180] Some embodiments can utilize sequencing by ligation techniques. Such techniquesutilize DNA ligase to incorporate oligonucleotides and identify the incorporation of sucholigonucleotides. The oligonucleotides typically have different labels that are correlated with theidentity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As withother SBS methods, images can be obtained following treatment of an array of nucleic acid featureswith the labeled sequencing reagents. Each image will show nucleic acid features that haveincorporated labels of a particular type. Different features are present or absent in the differentimages due the different sequence content of each feature, but the relative position of the featureswill remain unchanged in the images. Images obtained from ligation-based sequencing methodscan be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methodswhich can be utilized with the methods and systems described herein are described in U.S. Pat. No.6,969,488, U.S. Pat. No. 6,172,218, and U.S. Pat. No. 6,306,597, the disclosures of which areincorporated herein by reference in their entireties.

[0181] 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. А.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). In such embodiments, the target nucleic acid passes through a nanopore. The nanoporecan be a synthetic pore or biological membrane protein, such as a-hemolysin. As the target nucleicacid passes through the nanopore, each base-pair can be identified by measuring fluctuations in theelectrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, G. V. & Meller, "A. Progresstoward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007);Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007);Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. "A single-molecule nanopore device detectsDNA polymerase activity with single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820(2008), the disclosures of which are incorporated herein by reference in their entireties). Dataobtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. Inparticular, the data can be treated as an image in accordance with the exemplary treatment of opticalimages and other images that is set forth herein.

[0182] Some embodiments can utilize methods involving the real-time monitoring of DNApolymerase activity. Nucleotide incorporations can be detected through fluorescence resonanceenergy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphatelabeled 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 bedetected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019 (whichis incorporated herein by reference) and using fluorescent nucleotide analogs and engineeredpolymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Patent ApplicationPublication No. 2008 / 0108082 (each of which is incorporated herein by reference). Theillumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerasesuch 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. "Parallel confocal detection of singlemolecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminumpassivation for targeted immobilization of single DNA polymerase molecules in zero-modewaveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosuresof which are incorporated herein by reference in their entireties). Images obtained from suchmethods can be stored, processed and analyzed as set forth herein.

[0183] Some SBS embodiments include detection of a proton released upon incorporation ofa nucleotide into an extension product. For example, sequencing based on detection of releasedprotons can use an electrical detector and associated techniques that are commercially availablefrom Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods andsystems described in US 2009 / 0026082 A1; US 2009 / 0127589 A1; US 2010 / 0137143 A1; or US 2010 / 0282617 A1, each of which is incorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used fordetecting protons. More specifically, methods set forth herein can be used to produce clonalpopulations of amplicons that are used to detect protons.

[0184] The above SBS methods can be advantageously carried out in multiplex formats suchthat multiple different target nucleic acids are manipulated simultaneously. In particularembodiments, different target nucleic acids can be treated in a common reaction vessel or on asurface of a particular substrate. This allows convenient delivery of sequencing reagents, removalof unreacted reagents and detection of incorporation events in a multiplex manner. In embodimentsusing surface-bound target nucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to a surface in a spatiallydistinguishable 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 attachedto the surface. The array can include a single copy of a target nucleic acid at each site (also referredto 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 oremulsion PCR as described in further detail below.

[0185] The methods set forth herein can use arrays having features at any of a variety ofdensities including, for example, at least about 10 features / cm2, 100 features / cm2, 500features / 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.

[0186] An advantage of the methods set forth herein is that they provide for rapid and efficientdetection of a plurality of target nucleic acid in parallel. Accordingly the present disclosureprovides integrated systems capable of preparing and detecting nucleic acids using techniquesknown in the art such as those exemplified above. An integrated system of the present disclosurecan include fluidic components capable of delivering amplification reagents and / or sequencingreagents to one or more immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell can be configured and / or used inan integrated system for detection of target nucleic acids. Exemplary flow cells are described, forexample, in US 2010 / 0111768 A1 and US Ser. No. 13 / 273,666, each of which is incorporatedherein by reference. As exemplified for flow cells, one or more of the fluidic components of anintegrated system can be used for an amplification method and for a detection method. Taking anucleic acid sequencing embodiment as an example, one or more of the fluidic components of anintegrated system can be used for an amplification method set forth herein and for the delivery ofsequencing reagents in a sequencing method such as those exemplified above. Alternatively, an integrated system can include separate fluidic systems to carry out amplification methods and tocarry out detection methods. Examples of integrated sequencing systems that are capable ofcreating 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 describedin US Ser. No. 13 / 273,666, which is incorporated herein by reference.

[0187] The sequencing system described above sequences nucleic-acid polymers present insamples received by a sequencing device. As defined herein, "sample" and its derivatives, is usedin its broadest sense and includes any specimen, culture and the like that is suspected of includinga target. In some embodiments, the sample comprises DNA, RNA, PNA, LNA, chimeric or hybridforms 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 alsoincludes any isolated nucleic acid sample such a genomic DNA, fresh-frozen or formalin-fixedparaffin-embedded nucleic acid specimen. It is also envisioned that the sample can be from a singleindividual, a collection of nucleic acid samples from genetically related members, nucleic acidsamples from genetically unrelated members, nucleic acid samples (matched) from a singleindividual such as a tumor sample and normal tissue sample, or sample from a single source thatcontains two distinct forms of genetic material such as maternal and fetal DNA obtained from amaternal subject, or the presence of contaminating bacterial DNA in a sample that contains plantor animal DNA. In some embodiments, the source of nucleic acid material can include nucleicacids obtained from a newborn, for example as typically used for newborn screening.

[0188] The nucleic acid sample can include high molecular weight material such as genomicDNA (gDNA). The sample can include low molecular weight material such as nucleic acidmolecules obtained from FFPE or archived DNA samples. In another embodiment, low molecularweight material includes enzymatically or mechanically fragmented DNA. The sample can includecell-free circulating DNA. In some embodiments, the sample can include nucleic acid moleculesobtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, lasercapture micro-dissections, surgical resections, and other clinical or laboratory obtained samples. Insome embodiments, the sample can be an epidemiological, agricultural, forensic or pathogenicsample. In some embodiments, the sample can include nucleic acid molecules obtained from ananimal such as a human or mammalian source. In another embodiment, the sample can includenucleic acid molecules obtained from a non-mammalian source such as a plant, bacteria, virus orfungus. In some embodiments, the source of the nucleic acid molecules may be an archived orextinct sample or species.

[0189] Further, the methods and compositions disclosed herein may be useful to amplify anucleic acid sample having low-quality nucleic acid molecules, such as degraded and / or fragmented genomic DNA from a forensic sample. In one embodiment, forensic samples caninclude nucleic acids obtained from a crime scene, nucleic acids obtained from a missing personsDNA database, nucleic acids obtained from a laboratory associated with a forensic investigation orinclude forensic samples obtained by law enforcement agencies, one or more military services orany such personnel. The nucleic acid sample may be a purified sample or a crude DNA containinglysate, for example derived from a buccal swab, paper, fabric or other substrate that may beimpregnated with saliva, blood, or other bodily fluids. As such, in some embodiments, the nucleicacid sample may comprise low amounts of, or fragmented portions of DNA, such as genomic DNA.In some embodiments, target sequences can be present in one or more bodily fluids including butnot limited to, blood, sputum, plasma, semen, urine and serum. In some embodiments, targetsequences can be obtained from hair, skin, tissue samples, autopsy or remains of a victim. In someembodiments, nucleic acids including one or more target sequences can be obtained from adeceased animal or human. In some embodiments, target sequences can include nucleic acidsobtained from non-human DNA such a microbial, plant or entomological DNA. In someembodiments, target sequences or amplified target sequences are directed to purposes of humanidentification. In some embodiments, the disclosure relates generally to methods for identifyingcharacteristics of a forensic sample. In some embodiments, the disclosure relates generally tohuman identification methods using one or more target specific primers disclosed herein or one ormore target specific primers designed using the primer design criteria outlined herein. In oneembodiment, a forensic or human identification sample containing at least one target sequence canbe amplified using any one or more of the target-specific primers disclosed herein or using theprimer criteria outlined herein.

[0190] The components of the cross-platform compatibility system can include software,hardware, or both. For example, the components of the cross-platform compatibility system caninclude one or more instructions stored on a computer-readable storage medium and executable byprocessors of one or more computing devices (e.g., system 100, the local connected device, localserver device, cloud server device). When executed by the one or more processors, the computerexecutable instructions of the cross-platform compatibility system can cause the computing devicesto perform the tile registration processes described herein. Alternatively, the components of thecross-platform compatibility system can comprise hardware, such as special purpose processingdevices to perform a certain function or group of functions. Additionally, or alternatively, thecomponents of the cross-platform compatibility system can include a combination of computerexecutable instructions and hardware.

[0191] Furthermore, the components of the cross-platform compatibility system performingthe functions described herein with respect to the cross-platform compatibility system may, for example, be implemented as part of a stand-alone application, as a module of an application, as aplug-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 cross-platform compatibility system maybe implemented as part of a stand-alone application on a personal computing device or a mobiledevice. Additionally, or alternatively, the components of the cross-platform compatibility systemmay be implemented in any application that provides sequencing services including, but not limitedto Illumina BaseSpace, Illumina MiSeq, Illumina NovaSeq, Illumina NextSeq, Illumina TruSeq, orIllumina TruSight software. "Illumina," "BaseSpace," "MiSeq," "NovaSeq," "NextSeq,""TruSeq," and "TruSight," are either registered trademarks or trademarks of Illumina, Inc. in theUnited States and / or other countries.

[0192] Embodiments of the present disclosure may comprise or utilize a special purpose orgeneral-purpose computer including computer hardware, such as, for example, one or moreprocessors and system memory, as discussed in greater detail below. Embodiments within the scopeof the present disclosure also include physical and other computer-readable media for carrying orstoring computer-executable instructions and / or data structures. In particular, one or more of theprocesses described herein may be implemented at least in part as instructions embodied in a nontransitory computer-readable medium and executable by one or more computing devices (e.g., anyof the media content access devices described herein). In general, a processor (e.g.,microprocessor) receives instructions, from a non-transitory computer-readable medium, (e.g., amemory, etc.), and executes those instructions, thereby performing one or more processes,including one or more of the processes described herein.a

[0193] Computer-readable media can be any available media that can be accessed by a generalpurpose or special purpose computer system. Computer-readable media that store computerexecutable instructions are non-transitory computer-readable storage media (devices). Computerreadable media that carry computer-executable instructions are transmission media. By way ofexample, and not limitation, embodiments of the disclosure can comprise at least two distinctlydifferent kinds of computer-readable media: non-transitory computer-readable storage media(devices) and transmission media.

[0194] Non-transitory computer-readable storage media (devices) 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 storageor other magnetic storage devices, or any other medium which can be used to store desired programcode means in the form of computer-executable instructions or data structures and which can beaccessed by a general purpose or special purpose computer.

[0195] A "network" is defined as one or more data links that enable the transport of electronicdata between computer systems and / or modules and / or other electronic devices. When informationis transferred or provided over a network or another communications connection (either hardwired,wireless, or a combination of hardwired or wireless) to a computer, the computer properly viewsthe connection as a transmission medium. Transmissions media can include a network and / or datalinks which can be used to carry desired program code means in the form of computer-executableinstructions or data structures and which can be accessed by a general purpose or special purposecomputer. Combinations of the above should also be included within the scope of computerreadable media.

[0196] Further, upon reaching various computer system components, program code means inthe form of computer-executable instructions or data structures can be transferred automaticallyfrom transmission media to non-transitory computer-readable storage media (devices) (or viceversa). For example, computer-executable instructions or data structures received over a networkor data link can be buffered in RAM within a network interface module (e.g., a NIC), and theneventually transferred to computer system RAM and / or to less volatile computer storage media(devices) at a computer system. It should be understood that non-transitory computer-readablestorage media (devices) can be included in computer system components that also (or evenprimarily) utilize transmission media.

[0197] Computer-executable instructions comprise, for example, instructions and data which,when executed at a processor, cause a general-purpose computer, special purpose computer, orspecial purpose processing device to perform a certain function or group of functions. In someembodiments, computer-executable instructions are executed on a general-purpose computer toturn the general-purpose computer into a special purpose computer implementing elements of thedisclosure. The computer executable instructions may be, for example, binaries, intermediateformat instructions such as assembly language, or even source code. Although the subject matterhas been described in language specific to structural features and / or methodological acts, it is to beunderstood that the subject matter defined in the appended claims is not necessarily limited to thedescribed features or acts described above. Rather, the described features and acts are disclosed asexample forms of implementing the claims.

[0198] Those skilled in the art will appreciate that the disclosure may be practiced in networkcomputing environments with many types of computer system configurations, including, personalcomputers, 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.In a distributed system environment, program modules may be located in both local and remotememory storage devices.

[0199] Embodiments of the present disclosure can also be implemented in cloud computingenvironments. In this description, "cloud computing" is defined as a model for enabling on-demandnetwork access to a shared pool of configurable computing resources. For example, cloudcomputing can be employed in the marketplace to offer ubiquitous and convenient on-demandaccess to the shared pool of configurable computing resources. The shared pool of configurablecomputing resources can be rapidly provisioned via virtualization and released with lowmanagement effort or service provider interaction, and then scaled accordingly.

[0200] A cloud-computing model can be composed of various characteristics such as, forexample, 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 Infrastructureas a Service (laaS). A cloud-computing model can also be deployed using different deploymentmodels such as private cloud, community cloud, public cloud, hybrid cloud, and so forth. In thisdescription and in the claims, a "cloud-computing environment" is an environment in which cloudcomputing is employed.

[0201] FIG. 18 illustrates a block diagram of a computing device 1800 that may be configuredto perform one or more of the processes described above. One will appreciate that one or morecomputing devices such as the computing device 1800 may implement the cross-platformcompatibility system. As shown by FIG. 18, the computing device 1800 can comprise a processor1802, a memory 1804, a storage device 1806, an I / O interface 1808, and a communication interface1810, which may be communicatively coupled by way of a communication infrastructure 1812. Incertain embodiments, the computing device 1800 can include fewer or more components than thoseshown in FIG. 18. The following paragraphs describe components of the computing device 1800shown in FIG. 18 in additional detail.

[0202] In one or more embodiments, the processor 1802 includes hardware for executinginstructions, such as those making up a computer program. As an example, and not by way oflimitation, to execute instructions for dynamically modifying workflows, the processor 1802 mayretrieve (or fetch) the instructions from an internal register, an internal cache, the memory 1804, orthe storage device 1806 and decode and execute them. The memory 1804 may be a volatile or nonvolatile memory used for storing data, metadata, and programs for execution by the processor(s). The storage device 1806 includes storage, such as a hard disk, flash disk drive, or other digitalstorage device, for storing data or instructions for performing the methods described herein.

[0203] The I / O interface 1808 allows a user to provide input to, receive output from, andotherwise transfer data to and receive data from computing device 1800. The I / O interface 1808may include a mouse, a keypad or a keyboard, a touch screen, a camera, an optical scanner, networkinterface, modem, other known I / O devices or a combination of such I / O interfaces. The I / Ointerface 1808 may include one or more devices for presenting output to a user, including, but notlimited 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. In certainembodiments, the I / O interface 1808 is configured to provide graphical data to a display forpresentation to a user. The graphical data may be representative of one or more graphical userinterfaces and / or any other graphical content as may serve a particular implementation.

[0204] The communication interface 1810 can include hardware, software, or both. In anyevent, the communication interface 1810 can provide one or more interfaces for communication(such as, for example, packet-based communication) between the computing device 1800 and oneor more other computing devices or networks. As an example, and not by way of limitation, thecommunication interface 1810 may include a network interface controller (NIC) or network adapterfor communicating with an Ethernet or other wire-based network or a wireless NIC (WNIC) orwireless adapter for communicating with a wireless network, such as a WI-FI.

[0205] Additionally, the communication interface 1810 may facilitate communications withvarious types of wired or wireless networks. The communication interface 1810 may also facilitatecommunications using various communication protocols. The communication infrastructure 1812may also include hardware, software, or both that couples components of the computing device1800 to each other. For example, the communication interface 1810 may use one or more networksand / or protocols to enable a plurality of computing devices connected by a particular infrastructureto communicate with each other to perform one or more aspects of the processes described herein.To illustrate, 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 errornotifications.

[0206] In the foregoing specification, the present disclosure has been described with referenceto specific exemplary embodiments thereof. Various embodiments and aspects of the presentdisclosure(s) are described with reference to details discussed herein, and the accompanyingdrawings illustrate the various embodiments. The description above and drawings are illustrativeof the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the presentdisclosure.

[0207] The present disclosure may be embodied in other specific forms without departing fromits spirit or essential characteristics. The described embodiments are to be considered in all respectsonly as illustrative and not restrictive. For example, the methods described herein may beperformed with less or more steps / acts or the steps / acts may be performed in differing orders.Additionally, the steps / acts described herein may be repeated or performed in parallel with oneanother or in parallel with different instances of the same or similar steps / acts. The scope of thepresent application is, therefore, indicated by the appended claims rather than by the foregoingdescription. All changes that come within the meaning and range of equivalency of the claims areto be embraced within their scopе.

Claims

CLAIMSWe Claim:

1. An instrument comprising:a stage to receive a flow cell comprising a first set of fiducials and a second set of fiducials;a detector;an optical lens assembly between the stage and the detector, the optical lens assembly todirect light from the flow cell to the detector;at least one processor coupled to the detector; anda non-transitory computer readable medium comprising instructions that, when executedby the at least one processor, causes the instrument to:capture a plurality of tiles of the flow cell;identify, within the flow cell, the first set of fiducials;bypass, within the flow cell, the second set of fiducials; andregister the plurality of tiles based on the first set of fiducials.

2. The instrument of claim 1, wherein the at least one processor is further coupled tothe stage or the optical lens assembly to cause a relative stepping motion between the stage and theoptical lens assembly to capture the plurality of tiles in a step-and-shoot manner.

3. The instrument of claim 1, wherein the at least one processor is further coupled tothe stage or the optical lens assembly to cause a relative scanning motion between the stage and theoptical lens assembly to capture the plurality of tiles in a scanning manner.

4. The instrument of claim 1, wherein the second set of fiducials is utilized by asecond instrument.

5. The instrument of claim 1, further comprising instructions that, when executed bythe at least one processor, causes the instrument to register the plurality of tiles by aligning theplurality of tiles based on the first set of fiducials.

6. The instrument of claim 1, wherein fiducials from the first set of fiducials andfiducials from the second set of fiducials do not overlap on the flow cell.

7. The instrument of claim 1, wherein a tile of the plurality of tiles comprises aconsistent pattern of the first set of fiducials.

8. A method comprising:receiving a flow cell comprising a first set of fiducials utilized by a first imaging systemand a second set of fiducials utilized by a second imaging system;capturing a plurality of tiles of the flow cell by utilizing the first imaging system;identifying, within the flow cell, the first set of fiducials corresponding to the first imagingsystem; and registering the plurality of tiles based on the first set of fiducials.

9. The method of claim 8, further comprising:capturing a second plurality of tiles of the flow cell by utilizing the second imaging system;identifying, within the flow cell, the second set of fiducials corresponding to the secondimaging system; andregistering the second plurality of tiles based on the second set of fiducials.

10. The method of claim 9, further comprising registering the plurality of tiles by:comparing expected positions of the first set of fiducials with observed positions of the firstset of fiducials in the plurality of tiles;determining, based on comparing the expected positions ofthe first set of fiducials with theobserved positions of the first set of fiducials, alignment parameters for the plurality of tiles; andcombining the plurality of tiles utilizing the alignment parameters.

11. The method of claim 9, wherein:andeach tile of the plurality of tiles comprises a consistent pattern of the first set of fiducials;each tile of the second plurality of tiles comprises a consistent pattern of the second set offiducials.

12. The method of claim 9 further comprising registering the second plurality of tilesby:comparing expected positions of the second set of fiducials with observed positions of thesecond set of fiducials in the second plurality of tiles;determining, based on comparing the expected positions of the second set of fiducials withthe observed positions of the second set of fiducials, alignment parameters for the second pluralityof tiles; andcombining the second plurality of tiles utilizing the alignment parameters.

13. The method of claim 9, wherein a number of segments in the second plurality oftiles divides evenly into a number of segments in the plurality of tiles.

14. The method of claim 8, wherein the first imaging system comprises a step-andshoot (SaS) system and the second imaging system comprises a time-delay integration (TDI)system.

15. The method of claim 8, wherein tiles ofthe plurality of tiles are separated by gutters.

16. A flow cell comprising:a first set of fiducials utilized by a first imaging system; anda second set of fiducials utilized by a second imaging system; wherein the first set of fiducials and the second set of fiducials comprise at least one sharedfiducial that is utilized by both the first imaging system and the second imaging system.

17. The flow cell of claim 16, wherein:the first set of fiducials comprise a first fiducial type;the second set of fiducials comprise a second fiducial type; andthe at least one shared fiducial comprises a third fiducial type.

18. The flow cell of claim 16, wherein:the first set of fiducials comprise a first fiducial type;the second set of fiducials comprise a second fiducial type; andthe at least one shared fiducial comprises the second fiducial type.

19. The flow cell of claim 16, wherein the first imaging system comprises a step-andshoot (SaS) system and the second imaging system comprises a time-delay integration (TDI)system.

20. The flow cell of claim 16, wherein:areas corresponding to a first set of tiles of the flow cell comprise a consistent pattern ofthe first set of fiducials; andsecond areas corresponding to a second set of tiles of the flow cell comprise a consistentpattern of the second set of fiducials, wherein the areas and the second areas are different sizes.

21. An instrument comprising:a stage to receive a flow cell comprising a first set of fiducials and a second set offiducials:a detector:an optical lens assembly between the stage and the detector, the optical lens assembly todirect light from the flow cell to the detector;at least one processor coupled to the detector, anda non-transitory computer readable medium comprising instructions that, when executedby the at least one processor, causes the instrument to:capture an initial set of images of the flow cell;identify, based on the initial set of images, the first set of fiducials within the flowcell:adjust an alignment between the stage and the detector based on the first set offiducials;capture a plurality of segment images of the flow cell; andregister the plurality of segment images based on the first set of fiducials and thesecond set of fiducials22. The instrument of claim 21, wherein:the first set of fiducials comprises a first fiducial type; andthe second set of fiducials comprises a second fiducial type.

23. The instrument of claim 21, further comprising instructions that, when executedby the at least one processor, cause the instrument to adjust the alignment between the stage andthe detector by:determining locations of the first set of fiducials; andbypassing the second set of fiducials.

Images