Parallel sample and index sequencing

Abstract

A system and method for identifying nucleobases in a template polynucleotide is disclosed. In an embodiment, such a method may include providing a substrate comprising a plurality of template polynucleotides in a cluster. The method may further include generating light to stimulate fluorescence emission from the cluster. The method may further include receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a first site. The method may further include receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a second site. The method may further include identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.

Description

[Technical field]

[0001] The technology of this disclosure relates to the field of nucleic acid sequencing. More specifically, the technology of this disclosure relates to determining the nucleotide sequence of an index sequence and a corresponding target nucleic acid in the same sequencing run using next generation sequencing. [Background technology]

[0002] In some types of next-generation sequencing techniques, DNA clusters can be created on a flow cell after amplification of a target polynucleotide. Next-generation sequencing cycles can be performed when the complementary strand of the target polynucleotide is being synthesized. In each sequencing cycle, a deoxyribonucleic acid analogue conjugated with a fluorescent label can be hybridized to the target polynucleotide at a certain site, and an excitation light source can be used to excite the fluorescent label on the deoxyribonucleic acid analogue. A detector can capture the fluorescent emission from the fluorescent label to identify the deoxyribonucleic acid analogue. As a result, the sequence of the target polynucleotide can be determined by repeatedly performing sequencing cycles. The speed of sequencing the target polynucleotide can be limited due to the need to perform sequencing cycles and identify the nucleic acid bases of the target polynucleotide in consecutive order. Summary of the Invention

[0003] The disclosed technology relates to systems and methods for efficiently determining the nucleobase sequence of a polynucleotide, more particularly, the disclosed technology relates to systems and methods for determining the nucleobase sequence of different portions of a polynucleotide, such as an index portion and a template portion, in parallel.

[0004] In one aspect, the disclosed technology provides a system and method for identifying nucleobases in a template polynucleotide. In one embodiment, the disclosed method may include providing a substrate comprising a plurality of template polynucleotides in a cluster. The method may further include generating light to stimulate fluorescence emission from the cluster. The method may further include receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a first site. The method may further include receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a second site. The method may further include identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.

[0005] In another aspect, the disclosed technology provides a system and method for determining the sequence of a template polynucleotide including a barcode index. In one embodiment, the disclosed method may include hybridizing an index primer and a lead primer to the template polynucleotide. The method may further include extending the index primer with a first labeled nucleotide including a first label. The method may further include extending the lead primer with a second labeled nucleotide including a second label. The method may further include generating light to stimulate fluorescent emission from the first and second labeled nucleotides. The method may further include determining the sequence of the barcode index and nucleotides in the template polynucleotide by capturing the fluorescent emission.

[0006] The systems, devices, kits, and methods disclosed herein each have several aspects, no one of which is solely responsible for their desirable attributes. Numerous other embodiments are contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled "Detailed Description of the Invention," one will appreciate how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0007] It is understood that any of the features of the systems disclosed herein can be combined in any desired manner and / or configuration. It is further understood that any of the features of the methods disclosed herein can be combined in any desired manner. It is further understood that any combination of the features of the methods and / or systems can be used together and / or combined with any of the embodiments disclosed herein. It is understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below are considered to be part of the inventive subject matter disclosed herein and can be used to achieve the benefits and advantages described herein. [Brief description of the drawings]

[0008] Features of examples of the present disclosure will become apparent upon reference to the following detailed description and the drawings in which like reference numbers correspond to similar, but possibly not identical, components. For purposes of brevity, reference numbers or features having previously described functions may or may not be described in conjunction with the other drawings in which they appear. [Figure 1] FIG. 1 shows a block diagram that generally illustrates an exemplary sequencing system that can be used to practice the disclosed methods. [Diagram 2]FIG. 2 shows a block diagram that generally illustrates an exemplary imaging system that may be used in conjunction with the exemplary sequencing system of FIG. 1. [Diagram 3] FIG. 2 shows a functional block diagram of an exemplary computer system that may be used in the exemplary sequencing system of FIG. 1. [Figure 4] FIG. 1 shows a schematic diagram of the nucleic acid clusters of an indexed sample and the primers used to sequence the indexed sample, according to one embodiment of the disclosed technology. [Diagram 5] 5 shows an exemplary dye labeling scheme that may be used in conjunction with the embodiment of FIG. 4. [Figure 6] 1 is a plot showing a graphical representation of 16 distributions of signals from a nucleic acid cluster according to one embodiment of the disclosed technology. [Figure 7] 1 is a flow diagram illustrating a method for sequencing a polynucleotide according to one embodiment of the disclosed technology. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] All patents, patent applications, and other publications, including all sequences disclosed therein and referred to herein, are expressly incorporated herein by reference to the same extent as if each publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. All cited documents are, in relevant part, incorporated herein by reference in their entirety for the purposes indicated by the context of the citation herein. However, the citation of any document should not be construed as an admission that it is prior art to the present disclosure.

[0010] Introduction In some next generation sequencing techniques, the addition of a unique identifier or index sequence to DNA fragments generated during library preparation can form indexed template sequences that allow multiple DNA libraries to be pooled and sequenced together. In some systems, each template sequence has a unique index portion. Templates can be bound to a flow cell, and clusters of identical indexed template sequences can be generated at a single location on the flow cell. Thus, DNA clusters on the flow cell can contain identical copies of template polynucleotides with index sequence portions and insert portions derived from the original DNA fragments taken from biological or other samples. Further details regarding indexed sequencing of nucleic acids can be found in U.S. Patent No. 8,822,150, which is incorporated herein by reference.

[0011] In one aspect, the disclosed technology can reduce the time to sequence a template polynucleotide by enabling simultaneous sequencing of the index sequence portion and the insert portion of the template polynucleotide. In one aspect, a primer for sequencing the index sequence portion (i.e., index primer) and a primer for sequencing the insert portion (i.e., lead primer) are annealed to the template polynucleotide in the same reaction step to increase the efficiency of the sequencing-by-synthesis (SBS) workflow. In addition, annealing the index primer and the lead primer to the template strand simultaneously can reduce the fluidic complexity of the entire system. For example, the index primer and the lead primer can be hybridized to the template polynucleotide simultaneously, and then the index sequence portion and the insert portion can be read out through an SBS chemistry cycle. In one embodiment, to separate the signals received from the labeled nucleic acid bases on the index sequence portion and the insert portion, the signal from the index sequence portion is reduced, for example, by 50%, compared to the signal generated by the lead portion. For example, the index primer can be prepared such that a portion (e.g., 50%) of the index primer molecule is blocked and cannot be extended. By blocking the addition of labeled nucleobases to index primers (or lead primers), the signals generated by the labeled nucleobases added to each primer can be differentiated. For example, if half of the index primers have blocked sites, only half of the index primers will be labeled in any SBS sequencing reaction. This reduction in the proportion of index primers that are labeled leads to a proportional reduction in the intensity of the wavelength of light emitted by the index primers in each cluster. By considering not only the wavelength of light but also the intensity of that light from each cluster, the labeled nucleobases attached to the index primers can be differentiated from the labeled nucleobases added to the lead primers.This is explained more fully in the following section.

[0012] Although the following examples may show that some of the index primers are blocked to reduce the intensity of the fluorescent signal received from the nucleobases added to the index primers, it should be understood that any mechanism for distinguishing the intensity of the fluorescent label added to either the index primers or the lead primers is contemplated to be within the scope of the present invention. For example, in one aspect, the index primers are not blocked, but a proportion or percentage of the lead primers contain a blocking group such that the labeled nucleobase cannot be added to the lead primer. In some embodiments, the blocked index primer or the blocked lead primer may have an irreversibly blocked 3' end. For example, the blocked primer may be a nucleic acid that terminates with a dideoxynucleotide or ddNTP (e.g., ddGTP, ddATP, ddTTP, or ddCTP) that lacks the 3'OH group required for further growth of the nucleic acid chain.

[0013] In some embodiments, the disclosed techniques include obtaining sequence information using Illumina sequencing by synthesis and reversible terminator-based sequencing chemistry with removable fluorescent dyes (e.g., as described in Bentley et al., Nature 6:53-59

[2009] ). Short sequence reads of about tens to hundreds of base pairs can be aligned to a reference genome and unique mappings of the short sequence reads to the reference genome can be identified. After the first forward sequencing is completed, the template can be regenerated in situ to allow for a second reverse sequencing from the opposite end of the fragment. Thus, either single-end or paired-end sequencing of DNA fragments can be used. Further information regarding paired-end sequencing can be found in U.S. Pat. No. 7,601,499 and U.S. Pat. Pub. No. 2012 / 0053063, which are incorporated by reference. Further details regarding sequencing-by-synthesis and dye labeling methods that may be used with the disclosed technology are described in U.S. Patent Application Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2005 / 0100900, 2013 / 0079232, U.S. Patent No. 7,057,026, PCT Publication Nos. 2005 / 065814, 2006 / 064199, 2007 / 010251, and 2018 / 165099, and U.S. Patent Application No. 17 / 338590, the disclosures of which are incorporated herein by reference in their entireties.

[0014] Example Sequencer Referring to FIG. 1, a schematic diagram of an exemplary sequencing system 10 is shown including a sequencer 12 designed to sequence the genetic material of a sample 14. The sequencer can function in a variety of ways and can be based on a variety of techniques, including sequencing by primer extension using labeled nucleotides, as in the embodiments contemplated in the present invention, as well as other sequencing techniques (e.g., sequencing by ligation or pyrosequencing). In some embodiments, the sequencer 12 moves the sample progressively through reaction and imaging cycles to progressively build oligonucleotides by binding nucleotides to templates at individual sites on the sample. In some embodiments, the sample can be prepared by a sample preparation system 16. This process can include amplification of DNA or RNA fragments on a support to create multiple sites of DNA or RNA fragments that are sequenced by the sequencing process.Exemplary methods for generating sites of amplified nucleic acid suitable for sequencing include, but are not limited to, rolling circle amplification (RCA) (Lizardi et al., Nat Genet. 19:225-232 (1998)), bridge PCR (Adams and Kron, Method for Performing Amplification of Nucleic Acid with Two Primers Bound to a Single Solid Support, Mosaic Technologies, Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research, Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000); Pemov et al., Nucl. Acids Res. 33:ell (2005); or U.S. Pat. No. 5,641,658), polony generation (Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra et al., Anal. Biochem. 320:55-65 (2003)), or clonal amplification on beads using emulsions (Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003)) or ligation to bead-based adapter libraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. USA 97:1665-1670 (2000); Reinartz, et al., Brief Funct. Genomic Proteomic 1:95-104 (2002)), the foregoing publications being incorporated by reference. The sample preparation system 16 may place the sample in a sample container for processing and imaging, where the sample may be in the form of an array of sites.

[0015] In some embodiments, the sequencer 12 includes a fluid control / delivery system 18 and a detection system 20. The fluid control / delivery system 18 may receive a plurality of process fluids, as indicated by reference numeral 22, for circulation through a sample vessel, designated by reference numeral 24, of the sample in process. As will be appreciated by those skilled in the art, the process fluids may vary depending on the particular stage of sequencing. For example, in sequencing by synthesis (SBS) using labeled nucleotides, the process fluids introduced to the sample may include a polymerase and tagged nucleotides of four common DNA types, each nucleotide having a unique fluorescent tag and a blocking agent linked to it. The fluorescent tag allows the detection system 20 to detect which nucleotide was last added to a primer hybridized to the template nucleic acid at each site in the array, and the blocking agent prevents the addition of more than one nucleotide per cycle at each site.

[0016] At other stages of the sequencing cycle, the process fluid 22 may contain other fluids and reagents, such as reagents for removing extension blocks from nucleotides or cleaving nucleotide linkers to release newly extendable primer ends. For example, once the reaction has occurred at individual sites in the array of samples, the initial process fluids containing the tagged nucleotides may be washed from the sample in one or more flushing operations. The sample may then undergo detection, such as by optical imaging in the detection system 20. Reagents may then be added by the fluid control / delivery system 18 to deblock the last added nucleotides and remove the fluorescent tag from each. The fluid control / delivery system 18 may then wash the sample again, and the sample is then prepared for a subsequent cycle of sequencing. Exemplary fluid and detection configurations that can be used in the methods and devices described herein are described in WO 07 / 123744, which is incorporated herein by reference. In some embodiments, such sequencing may continue until the quality of the data derived from the sequencing deteriorates due to cumulative loss of yield, or until a predetermined number of cycles have been completed.

[0017] In some embodiments, the quality of the sample 24 during processing, as well as the quality of the data derived by the system, and various parameters used to process the sample, are controlled by a quality / process control system 26. The quality / process control system 26 may include one or more programmed processors or general-purpose or application-specific computers that communicate with sensors and other processing systems in the fluid control / delivery system 18 and detection system 20. Some process parameters may be used for advanced quality and process control, for example, as part of a feedback loop that can change instrument operating parameters during the course of a sequencing run.

[0018] In some embodiments, the sequencer 12 also communicates with a system control / operator interface 28, and ultimately with a post-processing system 30. The system control / operator interface 28 may include a general-purpose or application-specific computer designed to monitor process parameters, acquired data, system settings, and the like. The operator interfaces may be generated by programs executed locally or by programs executed within the sequencer 12. In some embodiments, they may provide visual indications of the health of the sequencer's systems or subsystems, the quality of acquired data, and the like. The system control / operator interface 28 may also allow a human operator to interface with the system to adjust operations, start and pause sequencing, and perform any other interactions that may be desired with the system hardware or software. For example, the system control / operator interface 28 may automatically perform and / or modify steps performed in the sequencing procedure without input from a human operator. Alternatively or additionally, the system control / operator interface 28 may generate recommendations regarding steps performed in the sequencing procedure and display these recommendations to a human operator. This mode may allow input from a human operator before performing and / or modifying steps in the sequencing procedure. In addition, system control / operator interface 28 may provide the human operator with options that allow the human operator to select certain steps in the sequencing procedure that will be automatically performed by sequencer 12, while requiring input from the human operator before other steps are performed and / or modified. In any event, allowing both an automated mode and an operator interactive mode may provide increased flexibility in performing the sequencing procedure. Additionally, the combination of automation and human-controlled interaction may further enable a system capable of creating and modifying new sequencing procedures and algorithms through adaptive machine learning based on input collected from a human operator.

[0019] The post-processing system 30 may further include one or more programmed computers that receive the detected information, which may be in the form of pixelated image data, and derive sequence data from the image data. The post-processing system 30 may include image recognition algorithms that distinguish the colors (e.g., the fluorescent emission spectra of the dyes) attached to the nucleotides that bind at each site as the sequencing progresses (e.g., by analysis of the image data that encodes specific colors and / or intensities) and record the sequence of the nucleotides at the location of each site. Progressively, the post-processing system 30 can then build a sequence list for each site of the sample array, which can be further processed by various bioinformatics algorithms to establish genetic information for the extended length of material.

[0020] The sequencing system 10 can be configured to handle individual samples or can be designed for higher throughput in a manner in which multiple stations are provided for delivery of reagents and other fluids and for detection that progressively builds up a sequence of nucleotides. Further details can be found in U.S. Patent No. 9,797,012, which is incorporated herein by reference.

[0021] In particular, if the fluid control system 18 or the quality / process control system 26 detects that one or more operations have not been performed in an optimal or desired manner, the sample may be removed from processing and reprocessed, and the scheduling of such processing may be altered in real time. In embodiments where a sample is removed from a process or experiences a pause in processing that is of substantial duration, the sample may be placed in storage. Placing the sample in storage may include altering the sample's environment or composition to stabilize biomolecular reagents, biopolymers, or other components of the sample. Exemplary methods for altering the sample environment include, but are not limited to, lowering the temperature to stabilize sample components, adding an inert gas to reduce oxidation of sample components, and removing from the light source to reduce photobleaching or photodegradation of sample components. Exemplary methods for altering the sample composition include, but are not limited to, adding antioxidants, stabilizing solvents such as glycerol, changing the pH to a level that stabilizes enzymes, or removing components that degrade or alter other components. Additionally, certain steps in the sequencing procedure may be performed before removing the sample from processing. For example, if it is determined that the sample should be removed from processing, the sample may be directed to the fluid control / delivery system 18 so that the sample can be washed prior to storage. These steps may be performed to ensure that information from the sample is not lost.

[0022] Furthermore, the sequencing operation may be interrupted by the sequencer 12 whenever certain predetermined events occur. These events include, but are not limited to, unacceptable environmental factors such as undesirable temperature, humidity, vibration, stray light, etc., insufficient reagent delivery or hybridization, unacceptable changes in sample temperature, unacceptable sample site number / quality / distribution, attenuated signal-to-noise ratio, insufficient image data, etc. It should be noted that the occurrence of such events does not require the interruption of the sequencing operation. Rather, such events may be factors that are weighted by the quality / process control system 26 in determining whether the sequencing operation should continue. For example, if an image of a particular cycle is analyzed in real time and shows a low signal for that optical channel, the image may be re-exposed using a longer exposure time or a particular chemical treatment may be repeated. If the image shows an air bubble in the flow cell, the instrument may automatically flush more reagents to remove the bubble and then re-record the image. If the image shows low signal for a particular optical channel in one cycle due to fluidics issues, the instrument can automatically stop scanning and reagent delivery for that particular optical channel, thus saving analysis time and reagent consumption.

[0023] Although the present system is illustrated above with respect to a system in which the sample interacts with different stations by physical movement of the sample, it will be understood that the principles described herein are also applicable to systems in which the steps occurring at each station are accomplished by other means that do not require movement of the sample. For example, reagents present at the stations can be delivered to the sample by a fluidic system connected to reservoirs containing various reagents. Similarly, an optical system can be configured to detect a sample in fluid communication with one or more reagent stations. Thus, a detection step can occur before, during, or after delivery of any particular reagent described herein. Thus, a sample can be effectively removed from processing by ceasing one or more processing steps, be it fluidic delivery or optical detection, without necessarily physically removing the sample from its location in the device.

[0024] The disclosed system can be used to sequentially sequence nucleic acids in multiple different samples. The disclosed system can be configured to include an arrangement of samples and an arrangement of stations for performing sequencing steps. The samples in the arrangement of samples can be arranged in a fixed order and at fixed intervals relative to each other. For example, the arrangement of nucleic acid arrays can be arranged along the outer edge of a circular table. Similarly, the stations can be arranged in a fixed order and at fixed intervals relative to each other. For example, the stations can be arranged in a circular arrangement with a perimeter corresponding to the layout for the arrangement of sample arrays. Each of the stations can be configured to perform a different operation in a sequencing protocol. The arrangement of sample arrays and stations can be moved relative to each other such that the stations perform the desired steps of the reaction scheme at each reaction site. The schedule of the relative positions and relative movements of the stations can be correlated with the order and duration of the reaction steps in the sequencing reaction scheme, such that a single sequencing reaction cycle is completed when the sample array completes a cycle of interaction with the full set of stations. For example, primers hybridized to nucleic acid targets on an array can be extended, detected, and deblocked, respectively, by the addition of a single nucleotide, when the order of stations, the spacing between stations, and the rate of passage through the array correspond to the order of reagent delivery and reaction time of a complete sequencing reaction cycle.

[0025] According to the above configuration, each lap (or complete rotation in embodiments where a circular table is used) completed by an individual sample array may correspond to the determination of a single nucleotide for each of the target nucleic acids on the array (e.g., including the steps of capture, imaging, cleavage and deblocking performed in each cycle of a sequencing run). Furthermore, several sample arrays present in the system (e.g., on the circular table) move simultaneously along similar repeating laps through the system, thereby resulting in continuous sequencing by the system. Using the disclosed system or method, reagents can be actively delivered or removed from a first sample array according to a first reaction step of a sequencing cycle, while incubation, or some other reaction step in the cycle, occurs for a second sample array. Thus, the set of stations can be configured in a spatial and temporal relationship with the arrangement of the sample arrays to allow reactions to occur simultaneously in multiple sample arrays, thereby allowing continuous and simultaneous sequencing, even if the sample arrays are subjected to different steps of a sequencing cycle at any given time. Such a circular system may be used when chemical and imaging times are unbalanced. For small flow cells that take only a short time to scan, a system can have several flow cells running in parallel to optimize the time the instrument spends acquiring data. If imaging time and chemistry time are equal, a system that is sequencing samples on a single flow cell will spend half the time running the chemistry cycle rather than the imaging cycle, so a system that can process two flow cells can have one on the chemistry cycle and one on the imaging cycle. If the imaging time is one tenth of the chemistry processing time, a system can have ten flow cells in various stages of chemistry processing while continuously acquiring data.

[0026] In some embodiments, the disclosed system is configured to allow a first sample array to be replaced by a second sample array while the system is sequentially sequencing the nucleic acids of a third sample array. Thus, a first sample array can be added to or removed from the system individually without interrupting the sequencing reaction occurring on another sample array, thereby allowing sequential sequencing of a set of sample arrays. Furthermore, sequencing runs of different lengths can be performed sequentially and simultaneously in the system, since individual sample arrays can complete different numbers of wraps through the system, and sample arrays can be removed from or added to the system in an independent manner so that reactions occurring at other sites are not disturbed.

[0027] FIG. 2 illustrates an exemplary detection station 38 that can detect nucleotides added to the sites of the array and can be used in conjunction with the exemplary sequencing system of FIG. 1. As described above, the sample can be moved to two or more stations of the device located at different physical locations, or one or more steps can be performed on the sample in communication with one or more stations without necessarily moving it to a different location. Thus, a description herein of a particular station is understood to relate to the stations in various configurations, regardless of whether the sample moves between stations, whether the station moves to the sample, or whether the station and sample are stationary relative to each other. In the embodiment shown in FIG. 2, one or more light sources 46 provide a light beam that is directed to the conditioning optics 48. The light source 46 may include one or more lasers, with multiple lasers being used to detect dyes that fluoresce at different corresponding wavelengths. The light source can direct the beam to the conditioning optics 48 for filtering and shaping the beam in the conditioning optics. For example, in an embodiment contemplated by the present invention, the conditioning optics 48 combines the beams from the multiple lasers to generate a substantially linear radiation beam that is transmitted to the focusing optics 50. The laser modules may further include a measurement component that records the power output of each laser. The measurement of the power output may be used as a feedback mechanism to control the length of time that images are recorded in order to obtain a uniform exposure energy, and therefore signal, for each image. If the measurement component detects a failure of a laser module, the instrument may flush the sample in a "holding buffer" to store the sample until errors in the lasers can be corrected.

[0028] The sample 24 is positioned on a sample positioning system 52, which can appropriately position the sample in three dimensions and move the sample for progressive imaging of sites on the sample array. In embodiments contemplated herein, focusing optics 50 confocally direct radiation to one or more surfaces of the array where the individual sites to be sequenced are located. Depending on the wavelength of light in the focused beam, a reverse beam of radiation is returned from the sample due to fluorescence of dyes attached to nucleotides at each site.

[0029] The retro beam is then passed back through retro beam optics 54, which may filter the beam, such as by separating different wavelengths in the beam and directing these separated beams to one or more cameras 56. The cameras 56 may be based on any suitable technology, such as by including charge-coupled devices that generate pixelated image data based on photons impinging on a location in the device. In some embodiments, the cameras 56 may include CMOS sensors. In some embodiments, the cameras 56 may include one or more autofocus cameras. In some embodiments, the cameras 56 may include one or more time delay and integration (TDI) cameras. The cameras generate image data, which is then transferred to image processing circuitry 58. In some embodiments, the processing circuitry 58 may perform various operations, such as analog-to-digital conversion, scaling, filtering, and correlating the data in multiple frames to properly and accurately image multiple sites at specific locations on the sample. The image processing circuitry 58 may store the image data and may ultimately transfer the image data to a post-processing system 30, where the array data may be derived from the image data. Exemplary detection devices that can be used in the detection station include those described, for example, in U.S. Patent Application Publication No. 2007 / 0114362 (U.S. Patent Application No. 11 / 286,309) and WO 07 / 123744, each of which is incorporated herein by reference.

[0030] A computer system 106, such as that shown in Figure 3, can be used to implement the system control / operator interface 28 and post-processing system 30 of the exemplary sequencing system 10 of Figure 1. As shown in Figure 3, the computer system 106 can include functionality for controlling the optical / fluidics system and for determining the nucleobase sequence of a polynucleotide.

[0031] In one embodiment, the computer system 106 includes a processor 202 in electrical communication with the memory 204, the storage device 206, and the communication interface 208. The processor 202 can be configured to execute instructions to cause the fluidic system 104 to provide reagents to the flow cell 114 during a sequencing reaction. The processor 202 can execute instructions to control the light source 120 of the optical system 102 to generate light near a predetermined wavelength. The processor 202 can execute instructions to control the detector 126 of the optical system 102 and receive data from the detector 126. The processor 202 can execute instructions to process data received from the detector 126, e.g., a fluorescent image, and determine a nucleotide sequence of a polynucleotide based on the data received from the detector 126. The memory 204 can be configured to store instructions to configure the processor 202 to perform the functions of the computer system 106 when the sequencing system 100 is powered on. The storage device 206 can store instructions to configure the processor 202 to perform the functions of the computer system 106 when the sequencing system 100 is powered off. The communications interface 208 may be configured to facilitate communications between the computer system 106 , the optical system 102 , and the fluid system 104 .

[0032] The computer system 106 can include a user interface 210 configured to communicate with a display device (not shown) for displaying the sequencing results of the sequencing system 100. The user interface 210 can be configured to receive input from a user of the sequencing system 100. The optical system interface 212 and the fluidic system interface 214 of the computer system 106 can be configured to control the optical system 102 and the fluidic system 104 through communication links 108a and 108b illustrated in FIG. 1A. For example, the optical system interface 212 can communicate with the computer interface 110 of the optical system 102 via communication link 108a.

[0033] The computer system 106 may include a nucleic acid base determiner 216 configured to determine a nucleotide sequence of a polynucleotide using data received from the detector 126. The nucleic acid base determiner 216 may include one or more of a template generator 218, a position register 220, an intensity extractor 222, an intensity corrector 224, a base caller 226, and a quality score determiner 228. The template generator 218 may be configured to generate a template of the position of the polynucleotide cluster in the flow cell 114 using the fluorescent image captured by the detector 126. The position register 220 may be configured to register the position of the polynucleotide cluster in the flow cell 114 in the fluorescent image captured by the detector 126 based on the position template generated by the template generator 218. The intensity extractor 222 may be configured to extract the intensity of the fluorescent emission from the fluorescent image to generate an extracted intensity. For example, a peak intensity value found at a diffraction-limited spot of a DNA cluster may be extracted from the image and used to represent the signal of the DNA cluster. In another example, the total intensity contained within the diffraction-limited spot of the DNA cluster can be extracted from the image and used to represent the signal of the DNA cluster. Alternatively, intensity estimation can be performed through the use of equalization and channel estimation.

[0034] The intensity corrector 224 may be configured to reduce or eliminate noise or aberrations inherent to the sequencing reaction or optical system. For example, the intensity may be affected by laser intensity variations, DNA cluster shape / size variations, non-uniform illumination, optical distortions or aberrations, and / or phasing / pre-phasing occurring within the DNA cluster. In some embodiments, the intensity corrector 224 may phase correct or pre-phase the extracted intensity. The base caller 226 may be configured to determine the nucleobase of the polynucleotide from the corrected intensity. The base of the polynucleotide determined by the base caller 226 may be associated with a quality score determined by the quality score determiner 228. Quality scoring refers to the process of assigning a quality score to each base call. To assess the quality of the base calls from the sequencing reads, an exemplary process may include calculating a set of predictor values ​​for the base calls and using the predictor values ​​to look up the quality scores in a quality table. The quality scores may be presented in any suitable format that allows a user to determine the probability of error for any given base call. In some embodiments, the quality scores are presented as a numerical value. For example, a quality score may be quoted as QXX, where XX is the score and indicates that that particular call is 10 -XX / 10 It means that the Q30 has an error probability of 1 in 1000, i.e., 0.1%, and the Q40 has an error rate of 1 in 10,000, i.e., 0.01%. The error rate can be calculated using a control nucleic acid. In addition, some metric displays can include error rates per cycle. In some embodiments, the quality table is generated using a calibration data set, where the calibration set represents run and sequence variability. Further details of the calculations that can be performed by the nucleic acid base determiner, calculation of error rate and quality score can be found in U.S. Patent No. 8,392,126, U.S. Patent Application Publication No. 2020 / 0080142 and U.S. Patent Application Publication No. 2012 / 0020537, each of which is incorporated herein by reference in its entirety.

[0035] Parallel insert and index sequencing FIG. 4 shows a schematic diagram of sequencing an indexed sample according to one embodiment of the disclosed technology. The clonal nucleic acid cluster 402 may be one of many clonal nucleic acid clusters attached to the surface of a solid support 401 (e.g., part of a flow cell). The clonal nucleic acid cluster 402 may include a clonal copy of a template polynucleotide 403. For example, the clonal nucleic acid cluster 402 may be generated by bridge PCR amplification of the template polynucleotide. In some embodiments, the template polynucleotide may be an indexed nucleic acid sample. For example, the template polynucleotide strand 403 may include an insert portion 409 derived from the nucleic acid sample and an index portion 404 that is added during the library preparation process. The added index portion may include a barcode that uniquely identifies the sample (e.g., uniquely indicates the source and / or batch number of the sample). The added index portion may be a nucleic acid of about 6, 10, 15, 20, 25, or any value therebetween nucleotides in length.

[0036] The library preparation process may also add a primer binding site next to the insert portion 409 so that the lead primer 429 can specifically hybridize next to the insert portion 409 to initiate a polymerization / primer extension reaction and allow sequencing of the insert portion 409 as fluorescently labeled nucleotides are added to the extended lead primer. Similarly, the library preparation process may add another primer binding site next to the index portion 404 so that the index primer 414 can specifically hybridize next to the index portion 404 to initiate a polymerization / primer extension reaction and allow sequencing of the index portion 404 as fluorescently labeled nucleotides are added to the extended index primer. However, since the fluorescent signal emitted by the extended lead primer and the fluorescent signal emitted by the extended index primer are co-located and cannot be optically resolved, in order to accurately determine the nucleic acid sequence of the insert portion 409 and the index portion 404, a method is needed to determine whether the fluorescent signal is associated with the extended lead primer or the extended index primer, at least when the labeled nucleotide in the extended lead primer is not the same as the labeled nucleotide in the extended index primer (e.g., when "A" is added to the lead primer and "C" is added to the index primer). One way to determine whether the fluorescent signal is associated with the extended lead primer or the extended index primer is to use a distinguishable level of signal intensity. In some embodiments, a portion of the index primer (e.g., one-quarter, one-third, half, two-thirds, etc., or any value therebetween) is chemically blocked and cannot be extended by a polymerase. The blocked index primer is labeled in FIG. 4 as reference number 415. For example, some of the index primers may have chemically blocked 3' ends, while all of the lead primers 429 may have unblocked 3' ends.As a result, the number of index primers extended in a cluster may be small compared to the number of lead primers. The number of labeled nucleotides associated with the extended index primers in a cluster may be small compared to the number of lead primers. After receiving fluorescence excitation, the signal emitted from the labeled nucleotide added to the index primer may be weak compared to the signal of the lead primer. Thus, the weaker signal and the nucleotide identity it represents can be determined as associated with the extended index primer.

[0037] In use, the lead primer, the blocked index primer and the unblocked index primer may be mixed together and provided to a flow cell as a mixture and hybridized to a template polynucleotide strand (e.g., hybridized simultaneously / in the same reaction step). A sequencing-by-synthesis reaction cycle may then be performed, for example, using fluorescently labeled nucleotide analogs. Thus, the fluorescent signal emitted from the cluster 402 may originate from both the extended lead primer 429 and the extended unblocked index primer 414. Furthermore, the intensity of the fluorescent signal emitted from the collection of extended lead primers 429 and the collection of extended unblocked index primers 414 in the cluster may be proportional to the amount of the two types of primer molecules in the cluster, or the amount of labeled nucleotide analogs on the two types of extended primer molecules. Thus, a determination of whether the signal originates from the extended lead primer 429 or the extended unblocked index primer 414 may be made based on the intensity (or brightness) of the received signal. For example, if half of the index primers are blocked, the intensity of the signal from the labeled nucleotide added to the index portion 404 is half as strong as the intensity of the signal from the labeled nucleotide added to the insert portion 409. Thus, both the nucleobases in the index portion 404 and the nucleobases in the insert portion 409 can be identified in parallel (or substantially simultaneously), provided that the signal-to-noise ratio (SNR) in the sequencing system remains high enough (e.g., above a threshold of about 8 dB, 10 dB, or 12 dB) to distinguish half-intensity signals from full-intensity signals. (i.e., the bins shown in FIG. 6 are sufficiently separated, e.g., to be resolved.) At this stage of simultaneous identification of two nucleic acid bases in a nucleic acid cluster, the SNR can be calculated as the average base call intensity divided by the standard deviation of the non-base call intensity, where the intensity is associated with the two signals coming from each cluster. In one embodiment, for example, considering the 16 bins in FIG. 6, the SNR can be calculated as the difference in average intensity between two bins divided by the standard deviation of the intensity within those bins, where the 16 bins represent the distribution of the intensity associated with the two signals coming from each cluster. Over the course of the SBS reaction cycles, the SNR in the sequencing system can decay due to problems such as chain termination, polymerase damage, etc. Therefore, a much higher SNR than is sufficient to achieve the required base call error rate (e.g., 0.01%) can be used at the beginning of the SBS reaction cycles. For example, an SNR of more than 20 dB can be used at the beginning of the sequencing run, and the SNR can gradually decay over the SBS cycles, reaching a threshold value (e.g., 8 dB, 10 dB, or 12 dB) after about 10 or 20 SBS cycles. Thus, a surplus of SNR at the beginning of a sequencing run may allow simultaneous identification of nucleobases in the index portion 404 and nucleobases in the insert portion 409 for up to about 10 or 20 SBS cycles.

[0038] The embodiment using a portion of blocked primers for the index portion and unblocked primers for the insert portion can help ensure that the index signal (signal from index portion 404) and the insert signal (signal from insert portion 409) are physically located at the same cluster location. Furthermore, it can ensure that the ratio of index to insert signal (i.e., signal from index portion 404 to signal from insert portion 409) is maintained for each cluster, and thus there is no expected effect on the ratio of signals from the size of the cluster. That is, regardless of whether a cluster is larger or smaller (e.g., contains more or fewer copies of the template strand), both its insert and index signals are scaled by the same amount according to the cluster size.

[0039] In some embodiments, after a certain number of primer extension steps / sequencing cycles (e.g., about 10 or 20 cycles) or when the signal to noise ratio in the sequencing system falls below a desired threshold (e.g., 8 dB, 10 dB, or 12 dB), the index primer can be removed from the cluster 402 such that sequencing occurs only from the lead primer 429. For example, the index primer can be thermally dehybridized. As another example, the index primer can be removed by contacting a locked nucleic acid (LNA) with the template polynucleotide strand to displace the index primer, which may be chemically blocked and may not polymerize. In some embodiments, the extended index primer can eventually migrate to the lead primer 429, and polymerization from the index primer 414 can be terminated. In one example, a non-strand displacing polymerase can be used such that extension of the index primer is terminated when the extension portion contacts the hybridized lead primer. In some embodiments, the extended index primer may eventually migrate to the solid support 401 and polymerization from the index primer 414 may stop (e.g., in a scenario where polymerization migrates toward the solid support 401, but the index portion 404 is closer to the solid support compared to the insert portion 409). After removal of the index primer, or after polymerization from the index primer has stopped, the reaction from the lead primer 429 may continue and the insert portion 409 may continue to be sequenced. In a stage of identifying only one nucleic acid base in a nucleic acid cluster (e.g., after about 10 or 20 cycles, sequencing only the insert portion 409), the signal-to-noise ratio (SNR) in the sequencing system may be calculated differently, for example, as the average base call intensity divided by the standard deviation of the non-base call intensity, where the intensity is related to only one signal from each cluster.In one embodiment, only four bins are considered in the scatter plot of the distribution of signals from nucleic acid clusters, as described, for example, in US Publication No. 2013 / 0079232, which is incorporated herein by reference.In this scenario, SNR can be calculated as the difference in average intensity between two bins divided by the standard deviation of the intensity in those bins, and the four bins represent the distribution of the intensity associated with only one signal from each cluster.Due to the same problem as above, the SNR during the stage of identifying only one nucleic acid base in a cluster may gradually decay over sequencing cycles, and reach a threshold value (e.g., 8 dB, 10 dB, or 12 dB, corresponding to the minimum required base call error rate) after about 500 SBS cycles.

[0040] Thus, the disclosed technology provides a more efficient sequencing workflow in that the index primer and the lead primer can be hybridized and / or dehybridized in the same operation (or substantially simultaneously) (upon completion of sequencing of the template strand), thus saving time compared to using separate operations. Furthermore, the disclosed technology provides a more efficient sequencing workflow in that the index portion can be sequenced in parallel (or simultaneously) with the insert portion, thus saving SBS read cycles compared to sequencing the index portion and the insert portion separately (or sequentially). Saving read cycles can allow the sequencing workflow to be implemented with shorter run times, for example, run time savings can be about half an hour in some examples.

[0041] In further embodiments of the disclosed technology, the ratio of index primers and lead primers bound to a cluster may be adjusted and determined prior to sequencing, and this information may be used to calibrate the fluorescent imaging data obtained during sequencing. For example, the ratio of index primers and lead primers hybridized to a cluster can be monitored by fluorescent imaging if different fluorophores are bound to the index primers and lead primers. The index primers and lead primers can be removed at different rates by controlled thermal dehybridization until the desired ratio is achieved. Alternatively, the fluorophores can be chemically removed from the index primers and lead primers prior to sequencing.

[0042] FIG. 5 illustrates an exemplary dye labeling scheme that may be used in conjunction with the embodiments of the disclosed technology illustrated in FIG. 4. As illustrated in FIG. 5, different types of nucleotide analogs may be labeled with different fluorescent labels / dyes having different absorption and / or emission spectra. For example, dGTP is unlabeled, dATP is labeled with a first label / dye, dCTP is labeled with a second label / dye, and dTTP is labeled with a third label / dye, and thus, different types of nucleotide analogs may generate different characteristics of fluorescent emission after being excited by a light source. Alternative dye labeling schemes may be contemplated. For example, dTTP may be labeled with two different dyes. In some embodiments, the absorption spectrum of the dye allows for the dye to be excited by a single light source of a given wavelength, such as a "blue" laser at about 450 nm. However, the embodiments are not limited to light sources generating this particular wavelength of light, and other wavelengths corresponding to red, green, violet, or other available wavelengths of light are contemplated. In other embodiments, two or more light sources may be used to excite the dye if the absorption spectra of the dyes are sufficiently different.

[0043] The first fluorescent label / dye may have an emission spectrum that can be captured in a first image taken in a first light channel (e.g., "IMAGE 1" in FIG. 5). The second fluorescent label / dye may have an emission spectrum that can be captured in a second image taken in a second light channel (e.g., "IMAGE 2" in FIG. 5) that is different from the first light channel. The third fluorescent dye may have an emission spectrum that can be captured in both the first and second light channels (e.g., both "IMAGE 1" and "IMAGE 2" in FIG. 5). As a result, in the example shown in FIG. 5, dTTP can be identified as being shown in both the first and second images with a sufficiently high intensity. dGTP can be identified as being shown in both images with a very low intensity (e.g., below a cutoff value). dATP can be identified as being shown in the first image with a sufficiently high intensity, but in the second image with a very low intensity (e.g., below a cutoff value). dCTP may be identified as having a sufficiently high intensity in the first image, but showing a second image with a very low intensity (e.g., below a cutoff value). The fluorescent dyes conjugated to the four types of nucleotide analogs are merely exemplary and are not intended to be limiting. In other embodiments, the nucleotide analog that is not conjugated to any fluorescent dye may be dTTP, dCTP, or dATP. In other embodiments, the nucleotide analog conjugated to the first fluorescent dye may be dGTP, dCTP, or dTTP. In other embodiments, the nucleotide analog conjugated to the second fluorescent dye may be dGTP, dTTP, or dATP. In other embodiments, the nucleotide analog conjugated to the third fluorescent dye may be dGTP, dATP, or dCTP.

[0044] In some embodiments, the nucleotide analogs used in the disclosed sequencing system can be fully functionalized nucleotides. The linker located between the nucleotide base and the fluorescent molecule can include one or more cleavage groups. Prior to a subsequent sequencing cycle, the fluorescent label can be removed from the nucleotide analog by cleavage of the linker. For example, the linker attaching the fluorescent label to the nucleotide analog can include an azide and / or alkoxy group, e.g., on the same carbon, such that the linker can be cleaved after each incorporation cycle with a phosphine reagent, thereby releasing the fluorescent label. The nucleotide triphosphates can be reversibly blocked at the 3' position so that sequencing is controlled, and only a single nucleotide analog can be added to each extendible primer-polynucleotide in each cycle. For example, the 3' ribose position of the nucleotide analog can include both an alkoxy and an azide functional group that can be removed by cleavage with a phosphine reagent, thereby generating a nucleotide that can be further extended. Prior to a subsequent sequencing cycle, the reversible 3' block can be removed and another nucleotide analog can be added to each extendible primer-polynucleotide.

[0045] In some embodiments, the fluorescent label is selected from the group consisting of polymethine derivatives, coumarin derivatives, benzopyran derivatives, chromenoquinoline derivatives, and compounds containing bis-boron heterocycles, such as BOPPY and BOPYPY. In some embodiments, the fluorescent label is attached to the nucleotide via a cleavable linker. In some further embodiments, the labeled nucleotide may have a fluorescent label attached to the C5 position of a pyrimidine base, or a fluorescent label attached to the C7 position of a 7-deazapurine base, optionally through a linker moiety. For example, the nucleobase may be 7-deazaadenine, and the dye is attached to the 7-deazaadenine at the C7 position, optionally through a cleavable linker. The nucleobase may be 7-deazaguanine, and the dye is attached to the 7-deazaguanine at the C7 position, optionally through a cleavable linker. The nucleobase may be cytosine, and the dye is attached to the cytosine at the C5 position, optionally through a cleavable linker. As another example, the nucleobase may be thymine or uracil, and the dye is attached to the thymine or uracil at the C5 position, optionally through a cleavable linker. In some further embodiments, the cleavable linker may contain a chemical moiety similar or the same as the reversible terminator 3' hydroxy blocking group, such that the 3' hydroxy blocking group and the cleavable linker may be removed under the same reaction conditions or in a single chemical reaction. Non-limiting examples of cleavable linkers include the LN3 linker, the sPA linker, and the AOL linker, each of which is exemplified below.

[0046] [ka]

[0047] [ka]

[0048] In some embodiments, the nucleotide is selected from the group consisting of an analog of dGTP, an analog of dTTP, an analog of dUTP, an analog of dCTP, and an analog of dATP. In some embodiments, the first nucleotide is a first reversibly blocked nucleotide triphosphate (rbNTP), the second nucleotide is a second rbNTP, the third nucleotide is a third rbNTP, and the fourth nucleotide is a fourth rbNTP, and each of the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are different types of nucleotides from each other. In some embodiments, the four rbNTPs are selected from the group consisting of rbATP, rbTTP, rbUTP, rbCTP, and rbGTP. In some embodiments, each of the four rbNTPs includes a modified base and a reversible terminator 3' blocking group. Non-limiting examples of 3' blocking groups include azidomethyl ( * -CH2N3), substituted azidomethyl (e.g. * -CH(CHF2)N3 or * -CH(CH2F)N3) and * -CH2-O-CH2-CH=CH2, where the asterisk * indicates a point attachment to the 3' oxygen of the ribose or deoxyribose ring of a nucleotide.

[0049] Further details regarding dyes and fully functionalized nucleotides can be found in U.S. Patent Application Publication Nos. 2018 / 0094140 and 2020 / 0277670, International Patent Application Publication No. 2017 / 051201, and U.S. Provisional Patent Application Nos. 63 / 057758 and 63 / 127061, the disclosures of which are incorporated by reference in their entireties.

[0050] FIG. 6 is a scatter plot showing an example of 16 distributions of signals from a nucleic acid cluster according to an embodiment of the disclosed technology shown in FIG. 4, which in one example may be implemented using the dye labeling scheme shown in FIG. 5. As described in connection with FIG. 4, in one embodiment, the fluorescent signal from the collection of extended lead primers 429 is brighter than the fluorescent signal from the collection of extended unblocked index primers 414 in the same cluster. The scatter plot in FIG. 6 shows 16 distributions (or bins) of intensity values ​​from the combination of the brighter and dimmer signals of the cluster. The two signals may be co-localized and may not be optically resolved. The intensity values ​​shown in FIG. 6 may be up to a scale factor and the units of the intensity values ​​may be arbitrary. The sum of the brighter signal from the extended lead primer 429 and the dimmer signal from the extended unblocked index primer 414 results in a combined signal. The combined signal may be captured by a first optical channel and a second optical channel (e.g., the "IMAGE1" and "IMAGE2" channels in FIG. 5). Because the brighter signal can be A, T, C or G and the dimmer signal can be A, T, C or G, there are 16 possibilities for the combined signal, corresponding to 16 distinguishable patterns when optically captured according to the embodiment described in connection with Figure 5. That is, each of the 16 possibilities corresponds to a bin as shown in Figure 6. The computer system can map the combined signal from the cluster into one of the 16 bins and thus determine the additional nucleobases in the extended lead primer 429 and the extended unblocked index primer 414, respectively.

[0051] For example, if the composite signal is mapped to bin 612 for a base calling cycle, the computer processor base calls both the added nucleobase in the extended read primer 429 and the added nucleobase in the extended unblocked index primer 414 as a C. If the combined signal is mapped to bin 614 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a C and the added nucleobase in the extended unblocked index primer 414 as a T. If the combined signal is mapped to bin 616 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a C and the added nucleobase in the extended unblocked index primer 414 as a G. If the combined signal is mapped to bin 618 for the base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a C and the added nucleobase in the extended unblocked index primer 414 as an A.

[0052] If the combined signal is mapped to bin 622 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a T and the added nucleobase in the extended unblocked index primer 414 as a C. If the combined signal is mapped to bin 624 for a base calling cycle, the processor base calls both the added nucleobase in the extended read primer 429 and the added nucleobase in the extended unblocked index primer 414 as a T. If the combined signal is mapped to bin 626 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a T and the added nucleobase in the extended unblocked index primer 414 as a G. If the combined signal is mapped to bin 628 for the base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a T and the added nucleobase in the extended unblocked index primer 414 as an A.

[0053] If the combined signal is mapped to bin 632 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as G and the added nucleobase in the extended unblocked index primer 414 as C. If the combined signal is mapped to bin 634 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as G and the added nucleobase in the extended unblocked index primer 414 as T. If the combined signal is mapped to bin 636 for a base calling cycle, the processor base calls both the added nucleobase in the extended read primer 429 and the added nucleobase in the extended unblocked index primer 414 as G. If the combined signal is mapped to bin 638 for the base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as a G and the added nucleobase in the extended unblocked index primer 414 as an A.

[0054] If the combined signal is mapped to bin 642 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as A and the added nucleobase in the extended unblocked index primer 414 as C. If the combined signal is mapped to bin 644 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as A and the added nucleobase in the extended unblocked index primer 414 as T. If the combined signal is mapped to bin 646 for a base calling cycle, the processor base calls the added nucleobase in the extended read primer 429 as A and the added nucleobase in the extended unblocked index primer 414 as G. If the combined signal is mapped to bin 648 for the base calling cycle, the processor base calls both the added nucleobase in the extended read primer 429 and the added nucleobase in the extended unblocked index primer 414 as an A.

[0055] Simplified Sequencing Workflow Figure 7 is a flow diagram showing a simplified method for sequencing polynucleotides that may utilize an embodiment of the disclosed technology according to Figure 4. The simplified method may require shorter sequencing time because index sequencing preparation and insert sequencing preparation (e.g., hybridizing primers) are combined into a single reaction step for both forward and reverse template sequencing. Furthermore, the index and insert may be sequenced simultaneously in the first few sequencing cycles (e.g., the first 10 cycles), thus saving time for several sequencing cycles for both forward and reverse template sequencing.

[0056] As shown in FIG. 7, the disclosed method may begin with block 701, a pre-run check of sequencer and reagent conditions. The method may then move to block 702 (seeding and cluster generation), which may include attachment of a forward template polynucleotide strand to a planar, optically transparent surface to which an oligonucleotide anchor is attached. The forward template polynucleotide includes an index portion and an insert portion, as described in FIG. 4. For example, the forward template polynucleotide may be end-repaired to generate a 5' phosphorylated blunt end, and a single A base may be added to the 3' end of the blunt phosphorylated nucleic acid fragment using the polymerase activity of the Klenow fragment. This addition prepares the nucleic acid fragments for ligation to oligonucleotide adaptors, which have a single T base overhang at their 3' end to enhance ligation efficiency. The adaptor oligonucleotide is complementary to the flow cell anchor oligo. Under limiting dilution conditions, an adaptor-modified single-stranded forward template polynucleotide is added to the flow cell and immobilized by hybridization to the anchor oligo. The attached nucleic acid fragments are extended and the bridge is amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing about 1,000 copies of the same template.Details on enriching nucleic acids using cluster amplification can be found in Kozarewa et al., Nature Methods 6:291-295 (2009), which is incorporated herein by reference.

[0057] After cluster generation, the method may move to block 703, which is sequencing preparation for both the index and insert portions of the forward template strand, which includes providing a mixture of primers to a flow cell and hybridizing / annealing both the index and read primers to the DNA clusters. The method may then move to block 704 to sequence both the index and insert portions of the forward template strand. Sequencing proceeds by extending the primers to generate nucleic acid base reads. In each cycle, fluorescently labeled nucleotides compete to add to the growing strand of the extended primer. Only one will be incorporated at the primer position based on the sequence of the forward template. After addition of the nucleotide, the cluster is excited by a light source and a characteristic fluorescent signal is emitted. The emission spectrum and signal intensity determine the base call. Hundreds of millions of nucleic acid clusters, or thousands to tens of thousands of clusters, may be sequenced in a massively parallel manner. Once primer extension of the index primers has stopped, the method may move to block 705, where sequencing of only the insert portion of the forward template continues.

[0058] After completion of forward template strand sequencing (i.e., blocks 703-705), the method may move to block 706, paired-end turn. In the paired-end turn process, the primer extension products synthesized during forward template strand sequencing may be dehybridized and washed away. The disclosed method may further save sequencing time because both the extended lead primer and the extended index primer may be dehybridized and washed away in the same step. The 3' end of the forward template strand may then be deprotected. The forward template strand may then fold over and bind to the second oligo on the flow cell. The second flow cell oligo may then be extended using a polymerase to form a double-stranded bridge. This double-stranded nucleic acid bridge may then be denatured and the 3' end may be blocked. The original forward template strand may be cleaved and washed away, leaving behind the reverse template strand complementary to the forward template strand.

[0059] After the paired-end turn, the method may proceed to reverse template sequencing. The method may move to block 707, where sequencing preparation for both the index and insert portions of the reverse template strand is performed, which includes providing a mixture of (reverse) primers to a flow cell and hybridizing / annealing both the (reverse) index primer and the (reverse) read primer to the nucleic acid cluster. The method may then move to block 708, where both the index and insert portions of the reverse template strand are sequenced. Sequencing proceeds by extending the primers to generate nucleic acid base reads. Once primer extension of the (reverse) index primer has stopped, the method may move to block 709, where sequencing of only the insert portion of the reverse strand continues.

[0060] sample Thus, in some embodiments, the sample comprises or consists of purified or isolated polynucleotides derived from tissue samples, biological fluid samples, cell samples, and the like. Suitable biological fluid samples include, but are not limited to, blood, plasma, serum, sweat, tears, phlegm, urine, sputum, ear fluid, lymph, saliva, cerebrospinal fluid, ravage, bone marrow suspension, vaginal flow, cervical lavage, brain fluid, peritoneal fluid, milk, secretions of the respiratory, intestinal and genitourinary tracts, amniotic fluid, milk, and leukocytosis samples. In some embodiments, the sample is a sample that can be easily obtained by a non-invasive procedure, such as, for example, blood, plasma, serum, sweat, tears, phlegm, urine, sputum, ear fluid, saliva, or feces. In certain embodiments, the sample is a peripheral blood sample, or the plasma and / or serum fraction of a peripheral blood sample. In other embodiments, the biological sample is a swab or smear, a biopsy specimen, or a cell culture. In another embodiment, the sample is a mixture of two or more biological samples, for example, the biological sample can include two or more of a biological fluid sample, a tissue sample, and a cell culture sample. As used herein, the terms "blood," "plasma," and "serum" expressly include fractions thereof or processed portions thereof. Similarly, if a sample is taken from a biopsy, swab, smear, etc., the "sample" expressly includes processed fractions or portions thereof obtained from the biopsy, swab, smear, etc.

[0061] In certain embodiments, samples include, but are not limited to, samples from different individuals, samples from different developmental stages of the same individual or different individuals, samples from different diseased individuals (e.g., individuals having cancer or suspected of having a genetic disease), normal individuals, samples obtained at different stages of a disease in an individual, samples obtained from individuals receiving different treatments for a disease, samples from individuals exposed to different environmental factors, samples from individuals predisposed to a disease condition, samples from individuals exposed to an infectious agent, etc.

[0062] In one exemplary but non-limiting embodiment, the sample is a maternal sample obtained from a pregnant woman, e.g., a pregnant woman. The maternal sample can be a tissue sample, a biological fluid sample, or a cell sample. In another exemplary but non-limiting embodiment, the maternal sample is a mixture of two or more biological samples, e.g., the biological sample can include two or more of a biological fluid sample, a tissue sample, and a cell culture sample.

[0063] In certain embodiments, samples can also be obtained from in vitro cultured tissues, cells, or other polynucleotide-containing sources. Cultured samples can be taken from sources including, but not limited to, cultures (e.g., tissues or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), cultures (e.g., tissues or cells) maintained for different time periods, cultures (e.g., tissues or cells) treated with different factors or reagents (e.g., drug candidates, or modulators), or cultures of different types of tissues and / or cells.

[0064] In some embodiments, the use of the disclosed sequencing techniques does not involve the preparation of a sequencing library. In other embodiments, the sequencing techniques contemplated herein include the preparation of a sequencing library. In one exemplary approach, the preparation of a sequencing library includes the production of a random collection of adaptor-modified DNA fragments (e.g., polynucleotides) that are ready to be sequenced.

[0065] A polynucleotide sequencing library can be prepared from DNA or RNA, including equivalents, analogs of either DNA or cDNA, such as DNA or cDNA that is a complementary or copy DNA produced from an RNA template, for example, by the action of reverse transcriptase. Polynucleotides can occur in double-stranded form (e.g., genomic DNA fragments, cDNA, dsDNA such as PCR amplification products, etc.), or in certain embodiments, polynucleotides can occur in single-stranded form (e.g., ssDNA, RNA, etc.) and have been converted to dsDNA form. By way of illustration, in certain embodiments, single-stranded mRNA molecules can be copied into double-stranded cDNA suitable for use in preparing a sequencing library. The exact sequence of the primary polynucleotide molecule is generally not critical to the method of library preparation and may be known or unknown. In one embodiment, the polynucleotide molecules are DNA molecules. More specifically, in certain embodiments, the polynucleotide molecule represents the entire genetic complement of an organism or substantially the entire genetic complement of an organism, and is a genomic DNA molecule (e.g., cellular DNA, cell-free DNA (cfDNA), etc.), but typically includes intronic and exonic sequences (coding sequences), as well as non-coding regulatory sequences such as promoter and enhancer sequences. In certain embodiments, the primary polynucleotide molecule comprises a human genomic DNA molecule, e.g., a cfDNA molecule present in the peripheral blood of a pregnant subject.

[0066] Methods for isolating nucleic acids from biological sources may vary depending on the nature of the source. One skilled in the art can easily separate nucleic acids from sources as required for the methods described herein. In some cases, it may be advantageous to fragment large nucleic acid molecules (e.g., cellular genomic DNA) in a nucleic acid sample to obtain polynucleotides of a desired size range. Fragmentation may be random or specific, for example, as achieved using restriction endonuclease digestion. Methods for random fragmentation may include, for example, limited DNase digestion, alkaline treatment, and physical shearing. Fragmentation may also be achieved by any of several methods known to those skilled in the art. For example, fragmentation may be achieved by mechanical means, including, but not limited to, nebulization, sonication, and water shearing.

[0067] In some embodiments, sample nucleic acid is obtained from unfragmented cfDNA.For example, cfDNA typically exists as fragments of less than about 300 base pairs, and therefore fragmentation is typically not required to use cfDNA sample to generate sequencing library.

[0068] Typically, whether polynucleotides are forcibly fragmented (e.g., fragmented in vitro) or naturally present as fragments, they are converted to blunt-ended DNA with a 5'-phosphate and a 3'-hydroxyl. Standard protocols, such as those for sequencing using the Illumina platform, instruct users to purify the end-repaired product prior to dA-tailing for end-repaired sample DNA, and to purify the dA-tailed product prior to the adapter-ligating step of library preparation.

[0069] In various embodiments, verification of sample integrity and sample tracking can be achieved by sequencing a mixture of sample genomic nucleic acid, e.g., cfDNA, and associated marker nucleic acid that has been introduced into the sample, e.g., prior to processing.

[0070] Computer Systems In some embodiments, the disclosed systems and methods may involve approaches to shift or distribute certain sequence data analysis functions and sequence data storage to a cloud computing environment or cloud-based network. User interaction with sequencing data, genomic data, or other types of biological data may be mediated through a central hub that stores the data and controls access to various interactions with the data. In some embodiments, the cloud computing environment may also provide sharing of protocols, analysis methods, libraries, sequence data, and distributed processing for sequencing, analysis, and reporting. In some embodiments, the cloud computing environment facilitates correction or annotation of sequence data by users. In some embodiments, the systems and methods may be implemented in a computer browser, on-demand, or online.

[0071] In some embodiments, software written to perform the methods described herein is stored on some form of computer readable medium, such as memory, a CD-ROM, a DVD-ROM, a memory stick, a flash drive, a hard drive, an SSD hard drive, a server, a mainframe storage system, or the like.

[0072] In some embodiments, the method may be written in any of a variety of suitable programming languages, for example, compiled languages ​​such as C, C#, C, Fortran, and Java. Other programming languages ​​may include scripting languages ​​such as Perl, MatLab, SAS, SPSS, Python, Ruby, Pascal, Delphi, R, and PHP. In some embodiments, the method is written in C, C#, C++, Fortran, Java, Perl, R, Java, or Python. In some embodiments, the method may be a separate application having data entry and data display modules. Alternatively, the method may be a computer software product, and distributed objects may include classes that include applications that include the computational methods described herein.

[0073] In some embodiments, the method may be incorporated into existing data analysis software such as that found on sequencing equipment. Software including the computer-implemented methods described herein may be installed directly on a computer system or indirectly held on a computer-readable medium and loaded onto the computer system as needed. Additionally, the method may be located on a computer that is remote to where the data is generated, such as software found on a server or the like that is maintained at a separate location to where the data is generated, such as that provided by a third-party service provider.

[0074] The assay instrument, desktop computer, laptop computer, or server may include a processor in operative communication with an accessible memory that includes instructions for implementation of the system and method. In some embodiments, the desktop computer or laptop computer is in operative communication with one or more computer-readable storage media or devices and / or output devices. The assay instrument, desktop computer, and laptop computer may operate under many different computer-based operating languages, such as those utilized by Apple-based computer systems or PC-based computer systems. The assay instrument, desktop, and / or laptop computer, and / or server system may further provide a computer interface for creating or modifying experimental definitions and / or conditions, viewing data results, and monitoring experimental progress. In some embodiments, the output device may be a graphic user interface, such as a computer monitor or computer screen, a printer, a portable device such as a portable digital assistant (i.e., PDA, Blackberry, iPhone), a tablet computer (e.g., iPAD), a hard drive, a server, a memory stick, a flash drive, etc.

[0075] The computer readable storage device or medium may be any device, such as a server, mainframe, supercomputer, magnetic tape system, etc. In some embodiments, the storage device may be located in close proximity to the assay device, e.g., adjacent or proximate to the assay device. For example, the storage device may be located in relation to the assay device, in the same room, in the same building, in an adjacent building, on the same floor in a building, on a different floor in a building, etc. In some embodiments, the storage device may be located outside or distal to the assay device. For example, the storage device may be located in a different part of a city, in a different city, in a different state, in a different country, relative to the assay device. In embodiments where the storage device is located distal to the assay device, communication between the assay device and one or more of a desktop, laptop, or server is typically via an internet connection, either wirelessly or by network cable via an access point. In some embodiments, the storage device may be maintained and managed by an individual or entity directly associated with the assay device, while in other embodiments, the storage device may be maintained and managed by a third party, typically in a distal location relative to an individual or entity associated with the assay device. In the embodiments described herein, the output device can be any device for visualizing data.

[0076] The assay instrument, desktop, laptop, and / or server system may be used to store and / or retrieve computer-implemented software programs incorporating computer code for executing and implementing the computational methods described herein, data for use in implementing the computational methods, and the like. One or more of the assay instrument, desktop, laptop, and / or server may include one or more computer-readable storage media for storing and / or retrieving software programs incorporating computer code for executing and implementing the computational methods described herein, data for use in implementing the computational methods, and the like. The computer-readable storage media may include, but are not limited to, one or more of a hard drive, SSD hard drive, CD-ROM drive, DVD-ROM drive, floppy disk, tape, flash memory stick, or card. Additionally, a network, including the Internet, may be a computer-readable storage medium. In some embodiments, the computer-readable storage medium refers to a computational resource storage accessible by a computer network over the Internet or a corporate network provided by a service provider, rather than, for example, a local desktop or laptop computer from a local desktop or laptop computer to the assay instrument.

[0077] In some embodiments, computer-implemented software programs incorporating computer code for executing and implementing the computational methods described herein, computer readable storage media for storing and / or retrieving data used in implementing the computational methods, etc. are operated and maintained by a service provider in operative communication with the assay instruments, desktops, laptops, and / or server systems via an internet connection or a network connection.

[0078] In some embodiments, a hardware platform for providing a computing environment includes a processor (i.e., CPU) where processor time and memory layout, such as random access memory (i.e., RAM), are system considerations. For example, smaller computer systems offer cheaper, faster processors and greater memory and storage capabilities. In some embodiments, a graphics processing unit (GPU) can be used. In some embodiments, a hardware platform for performing the computational methods described herein includes one or more computer systems having one or more processors. In some embodiments, smaller computers are clustered together to provide a supercomputer network.

[0079] In some embodiments, the computational methods described herein are implemented on a collection of inter- or intra-connected computer systems (i.e., grid technologies) that may cooperatively run a variety of operating systems. For example, the CONDOR framework (University of Wisconsin-Madison) and systems available from United Devices are illustrative of the cooperation of multiple independent computer systems for the purpose of handling large amounts of data. These systems may provide a Perl interface for submitting, monitoring, and managing large sequence analysis jobs on a cluster in a serial or parallel configuration.

[0080] definition Unless otherwise defined, technical and scientific terms used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989).For purposes of this disclosure, the following terms are defined below.

[0081] As used herein, the term "cluster" or "clamp" refers to a group of molecules (e.g., a group of DNA, or a group of signals). In some embodiments, the signals of a cluster are derived from different features. In some embodiments, a signal clump represents a physical area covered by one amplified oligonucleotide. Each signal clump can ideally be observed as several signals. Thus, overlapping signals can be detected from the same signal clump. In some embodiments, a cluster or clump of signals may include one or more signals or spots that correspond to a particular feature. When used in connection with a microarray device or other molecular analysis device, a cluster may include one or more signals that together occupy a physical area occupied by an amplified oligonucleotide (or other polynucleotide or polypeptide with the same or similar sequence). For example, if the feature is an amplified oligonucleotide, the cluster may be a physical area covered by one amplified oligonucleotide. In other embodiments, a cluster or clump of signals does not have to correspond strictly to a feature. For example, spurious noise signals may be included in a signal cluster, but not necessarily within a feature area. For example, a cluster of signals from four cycles of a sequencing reaction may include at least four signals.

[0082] As used herein, the term "spot radius" or "cluster radius" refers to a defined radius that encompasses a diffraction-limited spot or cluster of signals. Thus, by defining a cluster radius larger or smaller, a larger number of signals can fall within the radius for subsequent ordering and selection. The cluster radius can be defined by any distance measure, such as pixels, meters, millimeters, or any other useful measure of distance.

[0083] As used herein, a "signal" refers to a detectable event, such as, for example, a light emission, a light emission, etc., in an image. Thus, in some embodiments, a signal can represent any detectable light emission (i.e., a "spot") captured in an image. Thus, as used herein, a "signal" can refer to an actual emission from a feature of an analyte, or a spurious emission that does not correlate with an actual feature. Thus, a signal may result from noise and can be subsequently discarded as not representative of an actual analyte feature of the test strip.

[0084] As used herein, the "intensity" of emitted light refers to the intensity of light delivered per unit area, where the area is measured on a plane perpendicular to the direction of propagation of the light beam, and the intensity is the amount of energy delivered per unit time. In some embodiments, the signal "intensity", "amplitude", "magnitude", or "level" may be used synonymously with signal intensity. In some embodiments, the image taken by the detector approximates or is proportional to an intensity map integrated over a certain amount of time. In some embodiments, the signal of a diffraction-limited spot of DNA clusters is extracted from the image as the total intensity contained in the spot up to a factor of the integration time. For example, the signal of a DNA cluster may be defined as the intensity contained within the spot radius of the DNA cluster up to a factor of the integration time. In other embodiments, the peak intensity value found within the spot radius may be used to represent the signal of the DNA cluster up to a factor of the integration time.

[0085] As used herein, the process of aligning a template of signal locations onto a given image is referred to as “registration,” and the process of determining the intensity or amplitude values ​​of each signal in the template for a given image is referred to as “intensity extraction.” For registration, the methods and systems provided herein can take advantage of the random nature of the signal clamp locations by using image correlation to align the template to the image.

[0086] As used herein, a "nucleotide" comprises a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. A nucleotide is a monomeric unit of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In a ribonucleotide (RNA), the sugar is ribose, and in a deoxyribonucleotide (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present at the 2' position of the ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is attached to the N-1 of a pyrimidine or the N-9 of a purine. The phosphate group can be in mono-, di-, or triphosphate form. These nucleotides may be naturally occurring nucleotides, however it should be further understood that non-naturally occurring nucleotides, modified nucleotides or analogs of the aforementioned nucleotides may also be used.

[0087] As used herein, a "nucleobase" is a heterocyclic base, such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. Nucleobases can be naturally occurring or synthetic. Non-limiting examples of nucleobases include adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purine substituted with methyl or bromine at the 8-position, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorocytosine, 5-fluoro-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluoro-2,6-diaminopurine, 5-fluoro-2,6-diaminopurine, 5-methyl-2,6-diaminopurine, 5-methyl-1,1-dimethyl ... Uracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine, and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510, and PCT Application Nos. 92 / 002258, 93 / 10820, 94 / 22892, and 94 / 24144, and by Fasman (Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, 1989, CRC Press, Boca Raton, LO), all of which are incorporated herein by reference in their entireties.

[0088] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleotide or ribonucleotide polymers in single- or double-stranded form and includes, unless specifically limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acid (PNA) and phosphorothioate DNA. Unless otherwise specified, a particular nucleic acid sequence includes its complementary sequence. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrolo-pyrimidine triphosphate, and 2-thiocytidine, as well as alpha thiotriphosphate for all of the above, and 2'-O-methyl-ribonucleotide triphosphate for all of the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

[0089] The polymerase used is generally an enzyme for joining 3'-OH 5'-triphosphate nucleotides, oligomers and their analogs. Polymerases include DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase I, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, VentR (registered trademark) DNA polymerase (New England Biolabs), Deep VentR (registered trademark) DNA polymerase (New England Biolabs), Bst DNA polymerase large fragment, Stoeffel fragment, 90N DNA polymerase, 90N DNA polymerase, Pfu DNA polymerase, TfI DNA polymerase, Tth DNA polymerase, RepliPHI Phi29 polymerase, TIi Examples of suitable polymerases include, but are not limited to, DNA polymerases, eukaryotic DNA polymerase beta, telomerase, Therminator™ polymerase (New England Biolabs), KOD HiFi™ DNA polymerase (Novagen), KOD1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered by bioprospecting, and polymerases cited in US Patent Application Publication No. 2007 / 0048748, US Patent Nos. 6,329,178, 6,602,695, and 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant isoforms, and engineered variants. "Encode" or "parse" are verbs referring to transferring from one form to another, and refer to transferring the genetic information of the target template sequence into the reporter configuration.

[0090] Nucleosides and nucleotides may be labeled at sites on the sugar or nucleobase. Dyes may be attached at any position on the nucleotide base, for example, via a linker. In certain embodiments, Watson-Crick base pairing may still be performed on the resulting analog. Specific nucleobase labeling sites include the C5 position of pyrimidine bases, or the C7 position of 7-deazapurine bases. A linker group may be used to covalently attach the dye to the nucleoside or nucleotide. As used herein, the term "covalently attached" or "covalently bonded" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate, as compared to attachment to the surface by other means, for example, adhesion or electrostatic interactions. It will be understood that the polymer covalently attached to the surface may be attached by means in addition to covalent bonds.

[0091] A variety of different types of linkers with different lengths and chemical properties can be used. The term "linker" encompasses any moiety useful for connecting one or more molecules or compounds to each other, to other components of a reaction mixture, and / or to a reaction site. For example, a linker can attach a reporter molecule or "label" (e.g., a fluorescent dye) to a reaction component. In certain embodiments, the linker is a member selected from substituted or unsubstituted alkyl (e.g., 2-5 carbon chains), substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. In one example, the linker moiety is selected from straight and branched carbon chains, optionally containing at least one heteroatom (e.g., at least one functional group such as ether, thioether, amide, sulfonamide, carbonate, carbamate, urea, and thiourea), and optionally containing at least one aromatic, heteroaromatic, or non-aromatic ring structure (e.g., cycloalkyl, phenyl). In certain embodiments, molecules with trifunctional linking capabilities are used, including, but not limited to, cynuric chloride, mealamine, diaminopropanoic acid, aspartic acid, cysteine, glutamic acid, pyroglutamic acid, S-acetylmercaptosuccinic anhydride, carbobenzoxylidine, histidine, lysine, serine, homoserine, tyrosine, piperidinyl 1,1-amino carboxylic acid, diaminobenzoic acid, etc. In certain specific embodiments, hydrophilic PEG (polyethylene glycol) linkers are used.

[0092] In certain embodiments, the linker is derived from a molecule that includes at least two reactive functional groups (e.g., one at each end), which can react with complementary reactive functional groups on various reactive components or can be used to immobilize one or more reactive components at a reaction site. "Reactive functional group," as used herein, includes olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketones, olefins, acetylenes, aryls, aryl groups ... Reactive functional groups include, but are not limited to, aryls, anhydrides, sulfates, sulfenic acids, isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, allenes, orthoesters, sulfites, enamines, ynamines, ureas, isoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups also include those used to prepare bioconjugates, such as N-hydroxysuccinimide esters, maleimides, and the like.

[0093] The cleavable linker may be, by way of non-limiting example, an electrophilically cleavable linker, a nucleophilically cleavable linker, a photocleavable linker, a linker cleavable under reducing conditions (e.g., a disulfide or azide-containing linker), a linker cleavable under oxidative conditions, a linker cleavable by use of a safety lock linker, or a linker that is cleavable by a removal mechanism. By using a cleavable linker to attach the dye compound to the substrate moiety, the label can be removed after detection, if necessary, to avoid any interfering signals in downstream processes.

[0094] In some embodiments, one or more dye molecules or labeling molecules may be attached to the nucleotide base by non-covalent interactions or a combination of covalent and non-covalent interactions via multiple intervening molecules. In one example, the nucleotide or nucleotide analog newly incorporated by the polymerase synthesizing from the target polynucleotide is initially unlabeled. One or more fluorescent labels can be introduced to the nucleotide or nucleotide analog by binding to a labeled affinity reagent containing one or more fluorescent dyes. The use of unlabeled nucleotides and affinity reagents in sequencing by synthesis is disclosed in US Publication No. 2013 / 0079232, which is incorporated herein by reference. For example, one, two, three, or each of the four different types of nucleotides (e.g., dATP, dCTP, dGTP, and dTTP or dUTP) in the reaction mixture may be initially unlabeled. Each of the four types of nucleotides (e.g., dNTPs) has a 3' hydroxy blocking group to ensure that only a single base can be added by the polymerase to the 3' end of the copy polynucleotide being synthesized from the target polynucleotide. After incorporation of unlabeled nucleotides, an affinity reagent that specifically binds to the incorporated dNTP can then be introduced to provide a labeled extension product that includes the incorporated dNTP. The affinity reagent can be designed to specifically bind to the incorporated dNTP via, for example, antibody-antigen interaction or ligand-receptor interaction. The dNTP can be modified to include a specific antigen that pairs with the specific antibody included in the corresponding affinity reagent. Thus, one, two, three, or each of the four different types of nucleotides can be specifically labeled via their corresponding affinity reagent.In some embodiments, the affinity reagent may comprise a small molecule or protein tag that may bind to a hapten moiety of the nucleotide (e.g., streptavidin-biotin, anti-DIG and DIG, anti-DNP and DNP), an antibody (including but not limited to, a binding fragment of an antibody, a single chain antibody, a bispecific antibody, etc.), an aptamer, a knottin, an affimer, or any other known agent that binds to the incorporated nucleotide with appropriate specificity and affinity. In some embodiments, the hapten moiety of the unlabeled nucleotide may be attached to the nucleobase via a cleavable linker, which may be cleaved under the same reaction conditions as those for removing the 3' blocking group. In some embodiments, one affinity reagent may be labeled with multiple copies of the same fluorescent dye, for example, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15 copies of the same dye. In further embodiments, each affinity reagent may be labeled with a different number of copies of the same fluorescent dye. In some embodiments, the first affinity reagent may be labeled with a first number of first fluorescent dyes, the second affinity reagent may be labeled with a second number of second fluorescent dyes, the third affinity reagent may be labeled with a third number of third fluorescent dyes, and the fourth affinity reagent may be labeled with a fourth number of fourth fluorescent dyes. In some embodiments, each affinity reagent may be labeled with a different combination of one or more types of dyes, where each type of dye has a certain copy number. In some embodiments, different affinity reagents may be labeled with different dyes that can be excited by the same light source, but each dye has a distinguishable fluorescence intensity or a distinguishable emission spectrum. In some embodiments, different affinity reagents may be labeled with the same dyes in different molar ratios to produce measurable differences in their fluorescence intensities.

[0095] The nucleotide analogues may be conjugated or associated with one or more optically detectable labels to provide a detectable signal. In some embodiments, the optically detectable labels may be fluorescent compounds, such as small molecule fluorescent labels. Suitable fluorescent molecules (fluorophores) as fluorescent labels include 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); fluorescein amidite (FAM); 5-carboxyfluorescein; tetrachloro-6-carboxyfluorescein (TET); hexachloro-6-carboxyfluorescein (HEX); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE); VIC®; NED®; tetramethylrhodamine (TMR); 5-carboxytetramethylrhodamine (5-TAMRA); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-JOE; Light Cycler® Red 610; Light Cycler® Red 640;Light Cycler® Red 670;Light Cycler® Red 705;7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin;9-Amino-6-chloro-2-methoxyacridine;6-Methoxy-N-(4-aminoalkyl)quinolinium bromide hydrochloride (ABQ);Acid fuchsin;ACMA (9-amino-6-chloro-2-methoxyacridine);Acridine orange;Acridine red;Acridine yellow;Acriflavine;Acriflavine Feulgen SITSA;AFP-Autofluorescent protein- (Quantum Biotechnologies);Texas Red;Texas Red-X conjugate;Thiadicarbocyanine (DiSC3);Thiazine red R;Thiazole orange;Thioflavin 5;Thioflavin S;Thioflavin TCN;Thiolite;Thiozole orange;Tinopol CBS (Calcofluor White);TMR;TO-PRO-1;TO-PRO-3;TO-PRO-5;TOTO-1;TOTO-3;TriColor (PE-Cy5);TRITC (tetramethylrhodamine-isothiocyanate);True Blue;TruRed;Ultralight;Uranine B;Uvitex SFC;WW 781;X-Rhodamine;X-Rhodamine-5-(and-6)-isothiocyanate (5(6)-XRITC);Xylene Orange;Y66F;Y66H;Y66W;YO-PRO-1;YO-PRO-3;YOYO-1;YOYO-3, interchelating dyes such as Sybr Green, thiazole orange;wide spectrum coverage,Alexa Fluor 350,Alexa Fluor Members of the Alexa Fluor® dye series (Molecular Probes / Invitrogen) that match the primary output wavelengths of common excitation sources, such as 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; members of the Cy Dye fluorophore series (GE Healthcare) that also cover a broad spectrum, such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7; and Oyster® dye fluorophores (Denovo), such as Oyster-500, -550, -556, 645, 650, and 656. members of the DY-Labels series (Dyomics) having an absorption maximum in the range of 418 nm (DY-415) to 844 nm (DY-831), such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, - 634, -635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700, -701, -730, -731 , -732, -734, -750, -751, -752, -776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL;members of the ATTO series of fluorescent labels (ATTO-TEC GmbH), such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; dyes of the CAL Fluor® series or Quasar® series (Biosearch Technologies), such as CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® Red 635, Quasar® 570, and Quasar® 670. In some embodiments, a first optically detectable label interacts with a second optically detectable moiety to alter the detectable signal, for example, via fluorescence resonance energy transfer ("FRET"; also known as Förster resonance energy transfer);

[0096] Fluorescent labels utilized by the systems and methods disclosed herein can have different peak absorption wavelengths, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak absorption wavelength of the fluorescent label can be, or can be approximately, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or range between any two of these values. In some embodiments, the peak absorption wavelength of the fluorescent label can be at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0097] Fluorescent labels can have different peak emission wavelengths, for example ranging from 400 nm to 800 nm. In some embodiments, the peak emission wavelength of the fluorescent label can be, or can be approximately, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or range between any two of these values. In some embodiments, the peak emission wavelength of the fluorescent label can be at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0098] Fluorescent labels can have different Stokes shifts, for example ranging from 10 nm to 200 nm. In some embodiments, the Stokes shift can be, or can be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or range between any two of these values. In some embodiments, the Stokes shift can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0099] In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can vary, for example, from 10 nm to 200 nm. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be, or can be approximately, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or range between any two of these values. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0100] A "light source" may be any device capable of emitting energy along the electromagnetic spectrum. The light source may be a source of visible light (VIS), ultraviolet light (UV) and / or infrared light (IR). "Visible light" (VIS) generally refers to a band of electromagnetic radiation having wavelengths of about 400 nm to about 750 nm. "Ultraviolet (UV) light" generally refers to electromagnetic radiation having wavelengths shorter than those of visible light, or in the range of about 10 nm to about 400 nm. "Infrared light" or infrared radiation (IR) generally refers to electromagnetic radiation having wavelengths greater than the VIS range, or from about 750 nm to about 50,000 nm. The light source may also provide full spectrum light. The light source may output light from a selected wavelength or range of wavelengths. In some embodiments of the invention, the light source may be configured to provide light above or below a predetermined wavelength, or may provide light within a predetermined range. The light source may be used in combination with a filter to selectively transmit or block selected wavelengths of light from the light source. The light source may be connected to a power source by one or more electrical connectors. The array of light sources may be connected in series or parallel to a power source. The power source may be a battery, or a vehicle electrical system or a building electrical system. The light sources may be connected to the power source through control electronics (control circuitry). The control electronics may comprise one or more switches. The one or more switches may be automated, controlled by a sensor, timer, or other input, or controlled by a user, or a combination thereof. For example, a user may operate a switch to turn on a UV light source. The light source may be applied on a constant basis until it is turned off, or may be pulsed (repeated on / off cycles) until it is turned off. In some embodiments, the light source may be switched from a continuous on state to a pulsed state or vice versa. In some embodiments, the light source may be configured to get brighter or dimmer over time.

[0101] For operation, the light source may be connected to a power source capable of providing sufficient intensity to illuminate the sample. The control electronics may be used to switch the intensity on or off based on input from a user or some other input, and may also be used to modulate the intensity to a suitable level (e.g., to control the brightness of the output light). The control electronics may be configured to turn the light source on and off as needed. The control electronics may include switches for manual, automatic, or semi-automatic operation of the light source. The one or more switches may be, for example, transistors, relays, or electromechanical switches. In some embodiments, the control circuitry may further comprise an AC-DC and / or DC-DC converter for converting a voltage from a voltage source to a suitable voltage for the light source. The control circuitry may comprise a DC-DC regulator for regulating the voltage. The control circuitry may further comprise a timer and / or other circuit elements for applying a voltage to the optical filter for a fixed period following receipt of the input. The switch may be activated manually, automatically in response to a predetermined condition, or with a timer. For example, the control electronics may process information such as user input, stored instructions, etc.

[0102] One or more of a plurality of light sources may be provided. In some embodiments, each of the plurality of light sources may be the same. Alternatively, one or more of the light sources may vary. The light characteristics of the light emitted by the light sources may be the same or different. The plurality of light sources may or may not be independently controllable. One or more characteristics of the light sources may or may not be controlled, including but not limited to whether the light source is on or off, the brightness of the light source, the wavelength of the light, the intensity of the light, the angle of illumination, the position of the light source, or any combination thereof.

[0103] In some embodiments, the light output from the light source may be from about 350 to about 750 nm, or any amount or range therebetween, such as from about 350 nm to about 360, 370, 380, 390, 400, 410, 420, 430, or about 450 nm, or any amount or range therebetween. In other embodiments, the light from the light source may be from about 550 to about 700 nm, or any amount or range therebetween, such as from about 550 to about 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or about 700 nm, or any amount or range therebetween. In some embodiments, the wavelength of the light generated by the light source may vary, for example, in the range of 400 nm to 800 nm. In some embodiments, the wavelength of the light produced by the light source can be, or can be approximately, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or range between any two of these values. In some embodiments, the wavelength of the light generated by the light source may be at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The light source may be capable of emitting electromagnetic waves in any spectrum. In some embodiments, the light source may have a wavelength between 10 nm and 100 μm. In some embodiments, the wavelength of the light can be between 100 nm and 5000 nm, between 300 nm and 1000 nm, or between 400 nm and 800 nm.In some embodiments, the wavelength of the light may be less than and / or equal to 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1500 nm, 1750 nm, 2000 nm, 2500 nm, 3000 nm, 4000 nm, or 5000 nm.

[0104] In one example, the light source may be a light-emitting diode (LED) (e.g., a gallium arsenide (GaAs) LED, an aluminum gallium arsenide (AlGaAs) LED, a gallium arsenide phosphide (GaAsP) LED, an aluminum gallium indium phosphide (AlGaInP) LED, a gallium (III) phosphide (GaP) LED, an indium gallium nitride (InGaN) / gallium (III) nitride (GaN) LED, or an aluminum gallium phosphide (AlGaP) LED). In another example, the light source may be a laser, e.g., a vertical cavity surface emitting laser (VCSEL), or other suitable light emitter, such as an indium-gallium-aluminum-phosphide (InGaAIP) laser, a gallium-arsenide phosphide / gallium phosphide (GaAsP / GaP) laser, or a gallium-aluminum-arsenide / gallium-aluminum-arsenide (GaAlAs / GaAs) laser. Other examples of light sources may include, but are not limited to, electronically excited light sources (e.g., cathodoluminescence, electronically stimulated luminescence (ESL bulbs), cathode ray tubes (CRT monitors), Nixie tubes), incandescent light sources (e.g., carbon button lamps, conventional incandescent light bulbs, halogen lamps, glow bars, Nernst lamps), electroluminescent (EL) light sources (e.g., light emitting diodes, organic light emitting diodes, polymer light emitting diodes, solid state lighting, LED lamps, electroluminescent sheets, electroluminescent wires), gas discharge light sources (e.g., fluorescent lamps, induction lighting, hollow cathode lamps, neon and argon lamps, plasma lamps, xenon flash lamps), or high intensity discharge light sources (e.g., carbon arc lamps, ceramic discharge metal halide lamps, hydrogen iodide arc lamps, mercury vapor lamps, metal halide lamps, sodium vapor lamps, xenon arc lamps). Alternatively, the light source may be a bioluminescent, chemiluminescent, phosphorescent, or fluorescent light source.

[0105] As used herein, an "optical channel" is a predefined profile of optical frequencies (or equivalently wavelengths). For example, a first optical channel may have wavelengths from 500 nm to 600 nm. To capture an image in the first optical channel, a detector responsive only to light from 500 nm to 600 nm may be used, or a bandpass filter with a transmission window of 500 nm to 600 nm may be used to filter the light incident on a detector responsive to light from 300 nm to 800 nm. A second optical channel may have wavelengths from 300 nm to 450 nm and 850 nm to 900 nm. To capture an image in the second optical channel, a detector responsive to light from 300 nm to 450 nm and another detector responsive to light from 850 nm to 900 nm may be used, and the detection signals of the two detectors may then be combined. Alternatively, to capture an image in the second optical channel, a bandstop filter that rejects light from 300 nm to 900 nm may be used in front of a detector responsive to light from 451 nm to 849 nm.

[0106] Additional Notes The embodiments described herein are exemplary. Modifications, rearrangements, alternative processes, etc. may be made to these embodiments and still fall within the teachings described herein. One or more of the steps, processes, or methods described herein may be performed by one or more suitably programmed processing and / or digital devices.

[0107] The various exemplary imaging or data processing techniques described in connection with the embodiments disclosed herein can be implemented as electronic hardware, computer software, or a combination of both. To illustrate this interchangeability of hardware and software, various exemplary components, blocks, modules, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends on the particular application and design constraints imposed on the overall system. The described functionality may be implemented in various ways for each particular application, and such implementation decisions should not be interpreted as causing a departure from the scope of the present disclosure.

[0108] Various exemplary detection systems described in connection with the embodiments disclosed herein may be implemented or performed by a machine such as a processor configured with specific instructions, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. The processor may be a microprocessor, but alternatively the processor may be a controller, a microcontroller, or a state machine, combinations thereof, and the like. A processor may also be implemented as a combination of computing devices, such as a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in association with a DSP core, or any other such configuration. For example, the systems described herein may be implemented using discrete memory chips, a portion of memory within a microprocessor, flash, EPROM, or other types of memory.

[0109] Elements of the methods, processes, or algorithms described in connection with the embodiments disclosed herein may be embodied directly in hardware, in software modules executed by a processor, or in a combination of the two. The software modules may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, removable disk, CD-ROM, or any other form of computer-readable storage medium known in the art. An exemplary storage medium may be coupled to the processor such that the processor can read information from, and write information to, the storage medium. Alternatively, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The software modules may include computer-executable instructions that cause a hardware processor to execute the computer-executable instructions.

[0110] Unless otherwise indicated, conditional language used in this specification, such as "can," "might," "may," "eg," and the like, unless otherwise indicated and as otherwise understood within the context in which it is used, is intended to generally convey that certain embodiments are included, and other embodiments do not include certain features, elements, and / or steps. Thus, such conditional language does not generally imply that features, elements, and / or conditions are in any manner required for one or more embodiments, or that one or more embodiments necessarily include logic for determining or prompting, with or without author input, whether those features, elements, and / or conditions are included or performed in any particular embodiment. Terms such as "comprising," "including," "having," "involving," and the like, are synonymous and used in an inclusive, open-ended manner and do not exclude additional elements, features, acts, operations, and the like. Also, the term "or," when used, for example, to connect a list of elements, is used in its inclusive sense (and not its exclusive sense), so as to mean one, some, or all of the elements in the list.

[0111] Disjunctive language, such as the phrase "at least one of X, Y, or Z," is otherwise understood in context as it is generally used to indicate that an item, term, etc., can be either X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z), unless specifically stated otherwise. Thus, such disjunctive language is generally not intended to, and should not, imply that a particular embodiment requires that at least one of X, at least one of Y, or at least one of Z, respectively, be present.

[0112] Terms such as "about" or "approximately" are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, which may be ±20%, ±15%, ±10%, ±5%, or ±1%. The term "substantially" is used to indicate that a result (e.g., a measurement) is close to a target value, where close may mean, for example, that the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0113] Unless otherwise noted, articles such as "a" or "an" should generally be construed to include one or more of the listed items. Thus, phrases such as "a device configured to" or "a device for" are intended to include one or more of the listed devices. Such one or more listed devices may also be collectively configured to perform the described detailed descriptions. For example, "a processor configured to perform detailed descriptions A, B, and C" may include a first processor to perform operations in conjunction with a second processor configured to perform detailed descriptions A and B and C.

[0114] While the above detailed description has illustrated, described, and pointed out novel features applied to the exemplary embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the devices or algorithms shown can be made without departing from the spirit of the present disclosure. As will be recognized, certain embodiments described herein may be embodied in forms that do not provide all of the features and advantages described herein, since some features may be used or practiced separately from others. All changes that come within the meaning and range of equivalency of the claims are intended to be embraced within their scope.

[0115] It is to be understood that all combinations of the foregoing concepts (provided that such concepts are not mutually inconsistent) are intended to be part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated to be part of the inventive subject matter disclosed herein.

Claims

1. 1. A method for determining a sequence of a template polynucleotide comprising a barcode index, the method comprising: hybridizing an index primer and a lead primer to the template polynucleotide; extending the index primer with a first labeled nucleotide comprising a first label; extending the lead primer with a second labeled nucleotide comprising a second label; generating light to stimulate fluorescent emission from the first and second labeled nucleotides; determining a sequence of nucleotides in the barcode index and the template polynucleotide by capturing the fluorescent emissions.

2. 2. The method of claim 1, wherein the template polynucleotide is part of a cluster of identical copies of the template polynucleotide, the index primer is part of a first population of index primers, and the lead primer is part of a second population of lead primers.

3. determining a sequence of nucleotides in the barcode index and the template polynucleotide; receiving a first signal emitted at a first intensity from the first population of index primers; receiving a second signal emitted at a second intensity from the second population of lead primers; and identifying the nucleic acid bases hybridized to the barcode index and the nucleic acid bases hybridized to the template polynucleotide based on a combination of the first signal and the second signal.

4. The method of claim 2 , wherein a portion of the first population of index primers have a blocked 3′ end.

5. 3. The method of claim 2, wherein the second population of lead primers have unblocked 3' ends.

6. 2. The method of claim 1, wherein the first labeled nucleotide and the second labeled nucleotide are hybridized to the template polynucleotide in the same reaction step.

7. 2. The method of claim 1, wherein extending the index primer is catalyzed by a non-strand displacing polymerase.

8. 2. The method of claim 1, wherein the index primer is thermally dehybridized after a predetermined number of nucleic acid polymerization cycles.

9. 2. The method of claim 1, wherein the index primer is removed after a predetermined number of nucleic acid polymerization cycles by contacting a locked nucleic acid with the template polynucleotide.

10. 10. The method of any one of claims 7 to 9, comprising further extending the lead primer and determining a sequence of nucleotides in the template polynucleotide after extension of the index primer is captured.

11. 3. The method of claim 2, comprising adjusting a ratio of the first population of index primers and the second population of lead primers prior to extending the index primers and the lead primers.

12. monitoring the ratio of the first population of index primers and the second population of lead primers that hybridize to clusters of identical copies of the template polynucleotide by stimulating and detecting fluorescent emissions from fluorophores attached to the first population of index primers and the second population of lead primers; and removing the first population of index primers and the second population of lead primers at different rates by controlled thermal dehybridization until a predetermined ratio is achieved.

13. 13. The method of claim 12, further comprising removing the fluorophores attached to the first population of index primers and the second population of lead primers that remain hybridized to the clusters.

14. 1. A system for identifying a nucleobase in a template polynucleotide, comprising: a substrate comprising a plurality of said template polynucleotides in clusters; 1. A processor configured to perform a method, the method comprising: generating light to stimulate fluorescent emission from said clusters; receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogues hybridized to the plurality of template polynucleotides at a first site; receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogues hybridized to the plurality of template polynucleotides at a second site; and identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.

15. 31. The system of claim 30, wherein the processor is configured to identify the nucleobase in the template polynucleotide based on a difference between the first intensity of the first signal and the second intensity of the second signal.

16. 1. A method for identifying a nucleobase in a template polynucleotide, comprising: providing a substrate comprising a plurality of said template polynucleotides in clusters; generating light to stimulate fluorescent emission from said clusters; receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogues hybridized to the plurality of template polynucleotides at a first site; receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogues hybridized to the plurality of template polynucleotides at a second site; and identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.

17. 17. The method of claim 16, wherein the first site is within a barcode index segment of the template polynucleotide and the second site is within a non-barcode index segment of the template polynucleotide.

18. 17. The method of claim 16, comprising identifying the nucleobase in the template polynucleotide based on a difference between the first intensity of the first signal and the second intensity of the second signal.

19. 17. The method of claim 16, wherein the nucleotide analogues in the first plurality of nucleotide analogues are part of an index primer hybridized to a site adjacent to a barcode index portion of the template polynucleotides in the cluster.

20. 20. The method of Claim 19, wherein the nucleotide analogues in the second plurality of nucleotide analogues are part of a read primer hybridized to a site adjacent to a non-barcode index portion of the template polynucleotide in the cluster.

21. 21. The method of claim 20, wherein identifying the nucleobase comprises comparing the first signal at the first intensity from the nucleotide analogue on the index primer to the second signal at the second intensity from a nucleotide analogue on the lead primer.

22. 22. The method of claim 21, wherein the first signal has an intensity that is lower than an intensity of the second signal.

23. 21. The method of claim 20, wherein the intensities of the first and second signals correspond to concentrations of the first plurality of nucleotide analogues in the index primer and the second plurality of nucleotide analogues in the lead primer.

24. 21. The method of claim 20, wherein a portion of the index primer has a blocked 3' end and the lead primer has an unblocked 3' end.

25. detecting the first signal at a first range of optical frequencies and at a second range of optical frequencies; detecting the second signal at the first range of optical frequencies and at the second range of optical frequencies; The method of claim 16 , wherein the first range of optical frequencies and the second range of optical frequencies are not the same.

26. acquiring a first fluorescence image of the cluster at a first range of optical frequencies; acquiring a second fluorescence image of the cluster at a second range of optical frequencies, the first range of optical frequencies and the second range of optical frequencies being not the same; extracting fluorescence intensities from the first and second fluorescence images of the cluster; and identifying the nucleobases at the first and second sites based on the extracted fluorescence intensities.

27. 27. The method of claim 26, wherein the nucleobase combination at the first and second sites is classified as one of 16 combinations of nucleobase types based on the extracted fluorescence intensity combination from the first and second fluorescence images and a predetermined fluorescence intensity distribution for 16 combinations of nucleobase types.

28. normalizing the extracted fluorescence intensities; and and classifying the nucleobase combination at the first and second sites as one of 16 combinations of nucleobase types based on the normalized extracted fluorescence intensity combination and a predetermined normalized fluorescence intensity distribution for 16 combinations of nucleobase types.

29. 27. The method of claim 26, comprising stimulating light emission from the clusters with light of a predetermined light frequency.

30. 27. The method of claim 26, comprising stimulating light emission from the clusters with two predetermined optical frequencies of light.

31. 17. The method of claim 16, further comprising substantially simultaneously identifying nucleobases in additional polynucleotides that are complementary to the template polynucleotide.

Images