Systems and methods for detecting nucleotide incorporation

Abstract

A system includes a fluidic system configured to flow a plurality of reagent solutions; an imaging apparatus in communication with the fluidic system, the imaging apparatus comprising an array of image sensors, an array of wells disposed over the array of image sensors, and a flow cell structure defining a flow space over the array of wells; and a data collection circuitry to receive light signal data from the imaging apparatus.

Description

Docket No. TP389002 WO 1SYSTEMS AND METHODS FOR DETECTING NUCLEOTIDEINCORPORATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 704,698 filed October 8, 2024, and is herewith incorporated by reference.FIELD OF THE INVENTION

[0002] This disclosure in general relates to systems and methods for detecting nucleotide incorporation and has application in biomolecule sequencing.BACKGROUND OF THE INVENTION

[0003] The field of genetic sequencing has seen remarkable advancements over the past few decades, driven by a growing interest in understanding the complexities of genetic information. Genetic sequencing, the process of determining the precise order of nucleotides within a DNA molecule, has become a cornerstone in various scientific and medical disciplines. The completion of the Human Genome Project in 2003 marked a pivotal moment, ushering in an era where genetic information could be leveraged for a myriad of applications.

[0004] The applications of genetic sequencing are vast and diverse. In medical diagnostics, genetic sequencing is utilized to identify genetic disorders, enabling early intervention and personalized treatment plans. It plays a critical role in oncology for identifying cancer-associated mutations and tailoring targeted therapies. In the field of infectious diseases, sequencing is employed to track pathogen evolution and outbreaks, providing vital information for public health responses. Beyond human health, genetic sequencing is extensively used in agriculture to enhance crop yield and resistance, in environmental studies to understand biodiversity and ecosystem dynamics, and in forensic science for accurate identification and solving crimes.

[0005] Despite these advancements, the demand for faster and more accurate genetic sequencing technologies continues to grow. Traditional sequencing methods, while groundbreaking, often require considerable time and resources, limiting their accessibility and scalability. The need for rapid turnaround times is particularly pronounced in clinical settings where timely diagnosis canDocket No. TP389002 WO 1 significantly impact patient outcomes. Furthermore, the accuracy of sequencing is paramount to ensure reliable data, which is critical for making informed decisions in research, clinical, and industrial applications.

[0006] In light of these considerations, there is a pressing need for innovative genetic sequencing instruments that can deliver high-speed and high-accuracy results.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.

[0008] FIG. 1 is a flow diagram illustrating an example sequencing process.

[0009] FIG. 2 illustrates an example sequencing system.

[0010] FIG. 3 includes an illustration of an example sequencing apparatus.

[0011] FIG. 4 includes an illustration of a cross-section of an example sequencing device.

[0012] FIG. 5, FIG. 6, FIG. 7, and FIG. 8 include illustrations of cross-sections of portions of example sequencing devices.

[0013] FIG. 9 includes an illustration of an example cross-section of a sequencing apparatus.

[0014] FIG. 10 and FIG. 11 include illustrations of a plan view of an example sequencing apparatus.

[0015] FIG. 12 includes an illustration of a cross-section of an example sequencing apparatus.

[0016] FIG. 13, FIG. 14, and FIG. 15, and FIG. 16 include plan view illustrations of example sequencing apparatuses.

[0017] FIG. 17 includes a block flow diagram of an example method for detecting nucleotide incorporation.

[0018] FIG. 18 includes an illustration of an example modified nucleotide.

[0019] FIG. 19 includes illustrations of example modified nucleotides.

[0020] FIG. 20 includes an illustration of an example schema for generating chemiluminescence.Docket No. TP389002 WO 1

[0021] FIG. 21 , FIG. 22, and FIG. 23 include block flow diagrams of example methods for detecting nucleotide incorporations or sequencing.

[0022] The use of the same reference symbols in different drawings indicates similar or identical items.DETAILED DESCRIPTION

[0023] A system includes a fluidic system providing reagent solutions to a sequencing apparatus including one or more die with an array of image sensors or pixels, each dies having an array of wells disposed over the image sensors. A controller in communication with the fluidic system and the sequencing apparatus controls flow of the reagent solutions. A data collection circuitry collects image data from the image sensors. In a method, supports incorporating monoclonal populations of polynucleotides are applied into or disposed in wells of the array of wells, modified nucleotides are incorporated complementary to the polynucleotides, and enzymes are attached to the modified nucleotides. Chemiluminescent substrates are provided that are reactive with the enzymes, resulting in chemical decomposition of the substrates and production of light through chemiluminescence. The chemiluminescence signal is collected by the image sensors. Data collected from the image sensors is used to determine a sequence of the polynucleotides.

[0024] FIG. 1 includes an illustration of an example process for performing sequencing of a biomolecule. As illustrated at block 10, a library of biomolecules is prepared. For example, the biomolecules can include polynucleotides, proteins, or other biomolecules. The biomolecules are templated onto supports, as illustrated at block 12. The supports can be formed on a surface or can be particles or beads. In a particular example, the supports include polymeric beads.

[0025] Supports with the biomolecules can be applied to a sequencing device and the biomolecules sequenced, as illustrated at block 14. In an example, the biomolecule can be a polynucleotide. Many copies of the polynucleotide can be formed on a bead support disposed in the sequencing device. The polynucleotide can be sequenced using sequencing-by-synthesis in which nucleotide incorporation is detected using chemiluminescence.

[0026] As illustrated at block 16, the detected chemiluminescence signals can be utilized to determine sequences and perform bioinformatics analysis on the sequenced nucleotide incorporations.Docket No. TP389002 WO 1

[0027] In a particular example, a library of target polynucleotides is prepared. The target polynucleotides of the library can be separately applied to different supports, for example, such that one target polynucleotide is applied to one support. The one target nucleotide can be amplified to form many copies of the target polynucleotide on the one support, providing monoclonal populations of polynucleotides on the supports. Example methods are described in US Publication No. 2012 / 156, 728A1 or US Publication No. 2019 / 255, 5O5A1, each of which is incorporated by reference.

[0028] In an example, the support can be a region on a surface or a particle or bead. In an example, the support can be a polymer support formed at distinct locations on a surface or in a well. In another example, the support can be a polymer bead or particle that is deposited into a well. Example polymer supports are described in US Patent No. 9,243,085, US Patent No. 9,868,826, or US Patent No. 10,066,260, each of which is incorporated herein by reference.10029] In an example, the supports include polymeric beads. The beads can be formed of a polymer such as a radically polymerizable monomer, such as a vinyl-based monomer. In particular, the monomer can include a hydrophilic monomer coupled to a hydrophobic protection group. In an example, the hydrophilic monomer can include acrylamide, vinyl acetate, hydroxyalkyl methacrylate, or any combination thereof. In a particular example, the hydrophilic monomer is an acrylamide, such as an acrylamide including hydroxyl groups, amino groups, carboxyl groups, or a combination thereof. In an example, the hydrophilic monomer is an aminoalkyl acrylamide, an acrylamide functionalized with an amine terminated polypropylene glycol (C, illustrated below), an acrylopiperazine (D, illustrated below), or a combination thereof. In another example, the acrylamide can be a hydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In particular, the hydroxyalkyl acrylamide can include N- tris(hydroxymethyl)methyl)acrylamide (A, illustrated below), N-(hydroxymethyl)acrylamide (B, illustrated below), or a combination thereof. In a further example, a mixture of monomers, such as a mixture of hydroxyalkyl acrylamide and amine functionalized acrylamide or a mixture of acrylamide and amine functionalized acrylamide, can be used. In an example, the amine functionalized acrylamide can be included in a ratio of hydroxyalkyl acrylamide:amine functionalized acrylamide or acrylamide: amine functionalized acrylamide in a range of 100:1 to 1:1, such as a range of 100:1 to 2:1, a range of 50: 1 to 3:1, a range of 50:1 to 5:1 or even a range of 50:1 to 10:1.Docket No. TP389002 WO 1|0030| The particles or beads can have a desirable particle size, such as a particle size not greater than 100 pm, not greater than 30 pm, or not greater than 3 pm. The average particle size is the mean particle diameter. For example, the average particle size may be not greater than 2 pm, such as not greater than 1.5 pm, not greater than 1.1 pm, not greater than 0.8 pm, not greater than 0.6 pm, not greater than 0.5 pm, or even not greater than 0.3 pm. In a particular example, the average particle size can be in a range of 0.1 pm to 100 pm, such as a range of 0.1 pm to 50 pm or a range of 0.1 pm to 1 . 1 pm. In some aspects, the above described method provides technical advantages for production of particles having a particle size in a range of 5 pm to 100 pm, such as a range of 20 pm to 100 pm, or a range of 30 pm to 70 pm. In other aspects, the above described method provides technical advantages for the production of particles having a particle size of not greater than 1.1 pm.

[0031] As used herein, the term “templating” refers to a process of generating two or more, or a plurality or population, of substantially identical polynucleotides, or of generating a substantially monoclonal population of nucleic acids, that can be used as templates in nucleic acid analysisDocket No. TP389002 WO 1 methods, including, for example, nucleic acid sequencing, such as sequencing by synthesis, of the polynucleotides. The polynucleotides generated in a tcmplating process arc typically referred to as nucleic acid templates. In some embodiments, templating involves attachment of polynucleotide templates to a surface or support. In some embodiments, templating involves generating two or more, or a plurality, of separate surfaces or supports, or discrete sites on a surface or support, each having attached thereto two or more, or a plurality or population, of substantially identical polynucleotides, or a substantially monoclonal population of polynucleotides. In some embodiments, templating involves generating one or more surfaces or supports, or discrete sites on a surface or support, having a substantially monoclonal population of polynucleotides attached thereto. In some embodiments, templating generates one or more surfaces or supports having a substantially monoclonal population of at least 50,000, 75,000, 100,000, 125,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000 or 106or more template nucleic acid molecules attached to each templated surface or support. In some embodiments, templating generates surfaces or supports having a substantially monoclonal population of between about 50,000 and 500,000 template nucleic acid molecules attached to each templated surface or support, or, for example, between about 50,000 and 400,000 template nucleic acid molecules, between about 50,000 and 300,000 template nucleic acid molecules, between about 50,000 and 200,000 template nucleic acid molecules, or between about 50,000 and 100,000 template nucleic acid molecules attached to each templated support. In some embodiments, templating generates one or more templated surfaces or supports having a substantially monoclonal population of between about 100,000 and 400,000 template nucleic acid molecules attached to each templated surface or support, between about 100,000 and 300,000 template nucleic acid molecules, between about 100,000 and 200,000 template nucleic acid molecules, or between about 150,000 and 300,000 template nucleic acid molecules attached to each templated support. In some embodiments, templating is performed starting with one or more pre-seeded or seeded surfaces or supports. In such embodiments, templating can generate one or more templated surfaces or supports including at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times,Docket No. TP389002 WO 1 at least 1000 times, at least 2500 times, at least 5000 times, at least 10,000 times, at least 25,000 times, at least 50,000 times, at least 100,000 times, at least 250,000 times, at least 5000,000 times, or at least 106times or more as many template nucleic acid molecules on the templated surfaces or supports as were present on the pre- seeded surfaces or supports. In such embodiments, templating can generate one or more templated surfaces or supports including not greater than 1010times as many template nucleic acid molecules on the templated surfaces or supports as were present on the pre-seeded surfaces or supports. In some embodiments, only about 1 or only 1 nucleic acid molecule is present on a pre-seeded support. In some embodiments, at least 50,000, 75,000 or 100,000 substantially monoclonal template nucleic acid molecules or between about 25,000 and 1,000,000 substantially monoclonal template nucleic acid molecules are present on a pre-seeded surface or support, for example between about 25,000 and 500,000, between about 25,000 and 250,000, between about 25,000 and 125,000, or between about 25,000 and 100,000 substantially monoclonal template nucleic acid molecules are present on a pre-seeded surface or support, e.g., a solid surface or support.

[0032] In another example, the system utilizes DNA balls that include at least one polynucleotide formed to include many copies of a template polynucleotide within the same strand. Such DNA balls can, for example, be formed by rolling circle amplification.

[0033] The beads or balls can be applied to a sequencing apparatus. In an example, the sequencing apparatus includes wells into which the beads or balls can be applied. For example, the beads or balls are applied one bead or ball to a well.

[0034] Sequencing can be performed using sequencing-by-synthesis in which a terminated nucleotide flows over the wells of the sequencing apparatus. Nucleotide incorporation and the type of the incorporated nucleotide is detected by imaging circuitry of the sequencing apparatus. For example, nucleotide incorporation can produce chemiluminescence signals to be detected by the imaging circuitry. The sequence of chemiluminescence signals is indicative of the nucleotide sequence of the target polynucleotide. This sequence of signals can be utilized to determine the nucleotide sequence and can be used to perform additional bioinformatics.

[0035] FIG. 2 includes an illustration of a sequencing system 100. The sequencing system 100 includes a fluidics system, a sequencing apparatus 106, a controller 108 in communication with the fluidics system 102 and the sequencing apparatus 106, a data collection circuitry 110 inDocket No. TP389002 WO 1 communication with the sequencing apparatus 106 and a bioinformatics system 112 in communication with the data collection circuitry 110. The fluidics system 102 can include a plurality of reservoirs 104 for reagents useful sequencing process. Example reagents include modified nucleotide reagents, enzyme solutions, chemiluminescence substrate solutions, wash solutions, and catalytic solutions, among others.

[0036] The sequencing apparatus 106 can include a sequencing die in which a plurality of microwells is disposed over an imaging circuitry. The imaging circuitry is configured to detect chemiluminescence signals resulting from nucleotide incorporation.

[0037] The controller 108 can direct the sequential flow of reagent solutions, such as solutions of modified nucleotide, enzyme solutions, and chemiluminescence substrate solutions to facilitate production of chemiluminescence signals indicative of nucleotide incorporation.

[0038] Signals associated with detection of chemiluminescence can be retrieved by the data collection circuitry 110 and processed. The data collection circuitry 110 or the bioinformatics analysis system 112 can determine a nucleotide sequence and perform other bioinformatics analysis steps and processes.

[0039] FIG. 3 illustrates an expanded and cross-sectional view of a sequencing apparatus including a flow cell 200 and illustrates a portion of a flow chamber 206. A reagent flow 208 flows across a surface of a well array 202, in which the reagent flow 208 flows over the open ends of wells of the well array 202. The well array 202 and a sensor array 205 together may form an integrated unit forming a lower wall (or floor) of flow cell 200. Further, a flow cell cover 230 encapsulates flow chamber 206 to contain reagent flow 208 within a confined region.

[0040] FIG. 4 includes an illustration of a cross-section of example sequencing devices. These sequencing devices 400 can include an imaging circuitry layer 402, readout circuitry 404 and a carrier substrate 406. The imaging circuitry 402 can include photodiodes 410 to detect chemiluminescence signals. The photodiodes 410 can be provided in an array. A well structure 408 can be disposed over the imaging circuitry 402. The well structure 408 can define an array of wells 412 preferably aligned with the array of photodiodes 410. For example, the photodiodes 410 can be arranged in rows and columns, and the array of wells defined in corresponding and aligned rows and columns. Supports 414 securing biomolecules can be disposed in wells 412 of the array of wells.Docket No. TP389002 WO 1

[0041] In an example, the sequencing device can include frontside-illuminated sensors or backside-illuminated sensors. As illustrated, the sequencing device includes backside-illuminated sensors.

[0042] As illustrated in FIG. 4, the carrier substrate 406 can include vias 418 connecting the readout circuitry 404 with a connector balls 416 of a ball grid array. In an example, the carrier substrate 406 includes a silicon substrate. The vias 418 can be formed of a metal, such as copper, aluminum, tungsten, or alloys thereof.

[0043] The readout circuitry 404 can be a metallization stack including a plurality of metallization layers within an interlayer dielectric material. The metallization stack can extend between the imaging circuitry layer and vias of the carrier substrate. The interlayer dielectric material can be a low-k dielectric material. In an example, the interlayer dielectric can be an oxide, such as an oxide of silicon. The metallization layers can be formed of a metal, such as aluminum, copper, gold, tungsten, or alloys thereof.

[0044] FIG. 5 includes an illustration of a cross-section of a portion of a sequencing device. The sequencing device includes an array 502 of photodiodes 504 formed in a semiconductor substrate layer. In an example, the semiconductor substate layer can be silicon bulk substrate or a silicon- on-insulator substrate.

[0045] The photodiodes 504 can be optically isolated from each other by an isolation structure 506, such as a deep trench isolation structure. The isolation structure 506 can be formed of a dielectric, such as an oxide of silicon (e.g., SiO2) or hafnium oxide (e.g., HfD2), or a material with a refractive index less than that of silicon.

[0046] An array 508 of wells 512 are formed in a well structure 510. The well structure 510 can be formed of one or more layers of one or more materials. The materials can include polymers, metals, metal oxides, semi metal oxides or nitrides, or combinations thereof. Example polymers include photoresist polymers, such as polymethyl methacrylate, polyisoprene, novolac resin, epoxy-based resist, off- stoichiometry thiol-enese (OSTE), or hydrogen silsesquioxane, or copolymers or combinations thereof. Example metals include tungsten, copper, aluminum, or alloys thereof. In another example, the materials can include hafnium oxide or tantalum oxide. Examples of semi-metal oxides and nitrides include oxides of silicon or nitrides of silicon.Docket No. TP389002 WO 1

[0047] The well structure 510 can be coated with a layer 514. In an example, the layer 514 is a dielectric layer. For example, the layer 514 can be silicon oxide, hafnium oxide, or tantalum oxide layer.

[0048] As illustrated, each photodiode 504 of the array 502 is uniquely associated with a well 512 of the array of wells defined by the well structure 508. Alternatively, more than one photodiode 504 can be associated with a well 512. In another example, more than one well 512 can be associated with a photodiode 504.

[0049] One or more layers 516 or 518 can be formed over the array 502 of photodiodes 504, separating the array 502 from the array 508 of wells 512. Example intermediate layers include dielectric layers, such as an oxide of silicon, or antireflective layers. Example antireflective layers include layers of metal oxide, nitride, or oxynitride formed of a metal including aluminum, magnesium, calcium, hafnium, zirconium, yttrium, hafnium, tantalum, strontium, titanium, lanthanum, copper, or barium, or combinations thereof.

[0050] FIG. 6 includes an illustration of a cross-section of a portion of a sequencing device. The sequencing device includes an array 602 of photodiode 604 separated by isolation structures 606. The array 602 of photodiodes 604 and isolation structures 606 can be formed as described above.

[0051] An array 608 of optical filters 612 can be formed in an array structure 610 that defines a grid corresponding to the array 602 of the photodiodes 604. The array structure 610 can be formed of one or more layers of one or more materials. The materials can include metals, metal oxides, semi metal oxides or nitrides, or combinations thereof. Example metals include tungsten, copper, aluminum, or alloys thereof. In another example, the materials can include hafnium oxide or tantalum oxide. Examples of semi-metal oxides and nitrides include oxides of silicon or nitrides of silicon.

[0052] The optical filters 612 can be formed of a polymeric material, such as a photoresist. An example filter material includes a novolac photoresist, such as diazonapthoquione-novolac photoresist. Optionally, the polymeric material may include a dye.

[0053] Optionally, one or more intermediate layers arc disposed between the array structure 610 and the array 602. Example intermediate layers include dielectric layers, such as an oxide of silicon, or antireflective layers.Docket No. TP389002 WO 1

[0054] An array 614 of wells 618 can be formed over the array 608 and array 602. The array 614 can be defined in or by a well structure 616. In an example, the well structure 616 is an extension of the array structure 610. In another example, the well structure 616 can be formed over the array 608.

[0055] Optionally, one or more intermediate layers are disposed between the well structure and the array 602. Example intermediate layers include dielectric layers, such as an oxide of silicon, or antireflective layers.

[0056] In further example, FIG. 7 includes an illustration of a cross-section of a portion of a sequencing device. The sequencing device includes an array 702 of photodiode 704 separated by isolation structures 706. The array 702 of photodiodes 704 and isolation structures 706 can be formed as described above.

[0057] An array 708 of optical filters 712 can be formed in an array structure 710 that defines a grid corresponding to the array 702 of the photodiodes 704 . The array structure 710 can be formed of one or more layers of one or more materials as described above. The optical filters 712 can be formed of a polymeric material, such as a photoresist. Optionally, one or more intermediate layers are disposed between the array structure 710 and the array 702.

[0058] An array of lenses 714 is defined over the filters 712. The lenses 718 can correspond with the array 708 of optical filters and the array 702 of photodiodes 704.

[0059] An array 716 of wells 720 can be formed over the lenses 718, the array 708 and the array 702. The array 716 can be defined by a well structure 718. In an example, the well structure 618 is an extension of the array structure 710. In another example, the well structure 718 can be formed over the array 708.

[0060] In an additional example, FIG. 8 includes an illustration of a cross-section of a portion of a sequencing device. The sequencing device includes an array 802 of photodiodes 804 formed in a semiconductor substrate layer. The photodiodes 804 can be optically isolated from each other by an isolation structure 806, such as a deep trench isolation structure. An array 808 of wells 812 are formed in a well structure 810. As illustrated, each photodiode 804 of the array 802 is uniquely associated with a well 812 of the array of wells defined by the well structure 808.Docket No. TP389002 WO 1Alternatively, more than one photodiode 804 can be associated with a well 812. In another example, more than one well 812 can be associated with a photodiode 804.

[0061] A hydrophobic or partially hydrophobic material 814 can be disposed at the bottom of the wells 812. In some examples, hydrophobic or partially hydrophobic materials can attract chemiluminescence species to the bottom of the wells closer to the photodiodes and optional filters or lenses. Example hydrophobic materials 814 include surface agents. In an example, the surface agents bind as a monolayer to the bottom of the well or optionally the side walls of the well. The surface agents have a surface reactive functional group. An exemplary surface reactive functional group of the surface agent can include a silane, phosphates, phosphonic acid, phosphinic acid, bisphosphonic acid, multidentate phosphates or phosphonates, polyphosphates / phosphonates, isocyanate, catechol, hydroxamate, alkoxy derivatives thereof, or any combination thereof. Exemplary alkoxy groups include methoxy, ethoxy, or combinations thereof. In another example, a combination of a clodronic acid and a functionalized primary amine can be used in place of a surface reactive functional group. In an example, silanes can functionalize many ceramic and metallic surfaces. In a particular example, silanes, isocyanates, hydroxamates, and clodronic acid can functionalize silica surfaces. In another example, phosphates, catechols, and hydroxamates can be used to functionalize titania surfaces. In further examples, particular surface reactive functional groups may preferentially deposit on one or more metal or ceramic surfaces relative to other metal or ceramic surfaces.

[0062] In an example, the surface reactive functional group is attached to an alkyl, alkoxy, or aryl, moiety, or a combination thereof. For example, an alkyl moiety can have 6 to 30 carbons, such as 8 to 24 carbons. In an example, the alkyl moiety can have 10 to 22 carbons, such as 10 to 20 carbons or 12 to 18 carbons.

[0063] In an example, the surface agent includes long-chain alkyl trialkoxy or tricholoro silanes, such as C12-C18 alkyl trialkoxy or tricholoro siloxanes. In another example, the surface agent includes long-chain alkyl phosphonic acids or multidentate phosphonates, such as C12-C18 alkyl phosphonic acid.

[0064] Optionally, the surface agent can include a distal functional group attached to the alkyl, alkoxy, or aryl group. The distal functional group can be a positively charged functional group or can be a neutral functional group. Example neutral functional groups include alkyl, branchedDocket No. TP389002 WO 1 alkyl, or cyclic aromatic groups. Example positively charged groups that lack a donor pair of electrons include salts of quaternary ammonium ions derived from secondary amines, tertiary amines or heterocyclic groups incorporating nitrogen. In another example, the distal functional group can be a nitroso functional group. Example heterocyclic groups incorporating nitrogen include quaternary amines derived from pyrrolidine, pyrrole, imidazole, piperidine, pyridine, pyrimidine, purine, triazolium, or combinations thereof. In particular, the salt can include a halide salt of the quaternary ammonium ions, such as a bromide salt. The secondary, tertiary, or quaternary amines can be conjugated to alkyl groups including methyl, ethyl, propyl, butyl, or tert-butyl alkyl groups. In another example, the distal functional group can include hindered primary, secondary or tertiary amines, such as amines hindered by proximal phosphate, phosphonate, phosphinate, or silane groups, or combinations thereof. In a particular example, the distal functional group can include biotin, streptavidin, avidin, or a derivative thereof.

[0065] In a further example, the hydrophobic or partially hydrophobic material can include a polymer. Example polymers include photoresist, such as novolac resin, polymethylmethacrylate, polyisoprene, bisphenol epoxies, or copolymers and combinations thereof. In another example, the polymer includes vinyl polymers, such as styrenic, alkylene, or norbornene polymers, derivatives thereof, copolymers thereof, and combinations thereof. In a further example, the polymer is derived from a trialkyl amine modified vinyl benzene or copolymers thereof, wherein the alkyl group has between 4 and 10 carbons. In another example, the polymer is a block copolymer with hydrophilic and hydrophobic polymers, such as hydrophilic acrylamide with hydrophobic, C12-C18 polyacrylamide, polyalkylene, or norbornene, among others.

[0066] In a further example, the material can include a positively charged moiety, such as a quaternary amine.

[0067] In an example, the material can be grown on the surface, for example, using ATRP is initiated at a surface to directly bond the polymer to the surface. In another example, the ATRP system can be used to attach a polymer to a surface of the well using a modified phosphonate, sulfonate, silicate, titanate, or zirconate compounds. In particular, an amine or hydroxyl terminated alkyl phosphonate or an alkoxy derivative thereof can be applied to a surface and initiated using an initiator. The catalyst complex and monomers can be applied, extending the surface compound.

[0068] In an exemplary method, a solution including precursors to the polymer matrix can be applied into wells of the structure defining an array of wells. The aqueous solution in the wells can be isolated byDocket No. TP389002 WO 1 providing an immiscible fluid over the wells and initiating polymerization of the polymer precursors within the solution within the wells.

[0069] In an example, anchoring compounds, such as anchoring compounds useful in Atom Transfer Radical Polymerization (ATRP), can be secured to the material layer defining a bottom surface of the wells. Alternatively, a sidewall material defined within the wells or layers of the structure expose within the wells can anchor compounds such as compounds useful in ATRP, as described above.

[0070] In such an example, a solution including polymer precursors such as monomers, crosslinkers, and optionally surface reactive additive, can be applied over the structure and within the wells. The anchoring compounds can be initiated to facilitate polymerization extending from the anchoring compound, isolating the polymerization within the wells and securing the polymer to the well. In an example, the anchoring compound has a surface reactive group and a distal radical-forming group. The surface reactive group can include a phosphonate, a silicate, a sulfonate, a zirconate, titanate or a combination thereof. The distal radical-forming group can include an amine or hydroxyl that can undergo transfer, for example, with a halogenated (e.g., bromilated) compound and subsequently form a free-radical for use in polymerizing the polymer precursors and anchoring the resulting polymer to a well surface. In another example, the anchoring compound can include an alkyl bromoacetate modified with a surface reactive group. For example, the anchoring compound can include an alkylphosphono bromoacetate compound. The alkyl group can include between 3 and 16 carbons, such as between 6 and 16 carbons, or between 9 and 13 carbons. The alkyl group can be linear or branched. In particular, the alkyl group is linear. In a further example, the bromoacetyl group can be modified to include an alkyl modified bromoacetyl group, such as an ethyl or methyl modified bromoacetyl group forming an ester with a surface functional alkyl group. In a particular example, the anchoring compound includes the following compound:

[0071] An ATRP system can be selected to terminate polymerization after a statistical average length or number of monomer additions. In such a manner, the amount polymerization within a well can be controlled. In a further example, other agents influencing chain extension or termination can be applied and added to the aqueous solution.

[0072] The sequencing device can be formed in a die cut from a semiconductor wafer. The die can be incorporated into a sequencing apparatus, such as a sequencing chip. FIG. 9 includes an illustration of a portion of a sequencing apparatus. The sequencing apparatus includes one orDocket No. TP389002 WO 1 more sequencing dies 920 or 922, which include an array of photodiodes 912 and associated circuitry, a readout circuitry layer 910, and a carrier substrate 908. The sequencing dies further include an array of wells 914 disposed over the array of photodiodes 912.

[0073] The one or more dies 920 and 922 can be secured to a through silicon via (TSV) interposer 904. The TSV interposer 904 can be secured to a printed circuit board 906 using a ball grid array or other bulk connectors 916. The printed circuit board 906 can include an array of readout and control pads 918.

[0074] The one or more dies 920 and 922 can be formed as part of the same wafer. In an example, the dies remain un-separated and form an array of un-separated dies that are secured to the printed circuit board 906. In another example, the dies can be separated and separately secured to the TSV layer 904 which is secured to the printed circuit board.

[0075] For example, FIG. 10 illustrates an array of dies 1000 that are un-separated and secured to a TSV layer. In another example illustrated in FIG. 11, an array 1100 of dies 1102 are separated and secured to a TSV interposer separately. Optionally, interstitial material 1104 can be applied between the separate dies to prevent fluid from contacting the electric circuitry or connections to the TSV layer. In an example, the interstitial material 1104 can be an epoxy material.

[0076] A sequencing apparatus 1200 is illustrated in FIG. 12. The sequencing apparatus 1200 includes the printed circuit board 1202 to which one or more dies 1204 are attached. Encapsulant 1206 is disposed around the dies. A flow cell cover or structure 1208 is applied over the dies 1204 and optionally secured to the encapsulant 1206 or an edge of the dies 1204.

[0077] The flow cell cover 1208 defines at least one inlet 1210 and at least one outlet 1212. The flow cell cover 1208 further defines a flow space 1214 or flow chamber providing for fluid flow from the inlet 1210 to the outlet 1212 over the exposed wells of the dies 1204. In an example, reagent solutions can be applied to the inlet 1210, flow over and into the wells of the dies 1204, and out of the outlet 1212.

[0078] FIG. 13 includes an illustration of an example sequencing apparatus 1300. The sequencing apparatus 1300 includes a printed circuit board 1302 to which a plurality of sequencing die 1304 is attached. A flow cell structure 1306 is secured over the sequencing diesDocket No. TP389002 WO 11304. The flow cell structure 1306 defines openings 1308 and 1310 with access or fluidic access to a flow cell space defined above exposed wells of the sequencing die 1304. As illustrated in FIG. 13, the sequencing die arc un-separated from each other and cut as a set from the original wafer.

[0079] In another example, a sequencing apparatus 1400 illustrated in FIG. 14 includes a printed circuit board 1402 to which a plurality of separated die 1404 are attached. The separated die 1404 can be secured with an interstitial material 1412, such as an epoxy. A flow cell structure 1406 is secured over the dies 1404 and includes openings 1408 and 1410 that have access to a flow space defined over the separate die 1404.

[0080] In a further example, illustrated in FIG. 15, a sequencing apparatus 1500 includes a printed circuit board 1502 to which a plurality of die 1504 is attached. As illustrated, the dies are separated from each other and from the original wafer. In another example, the die can be unseparated when cut as a group from the original wafer. A flow cell structure 1506 is attached over the die 1504. The flow cell structure 1506 can separate the flow cell space into different space configurations over each die. Each separate flow space can be accessed through openings 1508 and 1510. In an example, each flow space is isolated from the other flow spaces, and each isolated flow space has individual ports 1508 and 1510 to access that flow space.

[0081] While FIG. 15 illustrates six dies each with a separate flow space, optionally more than one flow space can access more than one die. For example, a flow space may define a lane over three dies. In another example, the flow space may define a lane over two dies.

[0082] Further, a plurality of dies can be applied to the printed circuit board for example, four dies, six dies, eight dies, nine dies, twelve dies or more can be applied over the printed circuit board and a flow cell structure configured to define access to a flow space over the dies in various configurations. For example, as illustrated in FIG. 16, nine dies 1604 can be applied over the printed circuit board 1602 to form the sequencing apparatus 1600. A flow cell structure 1606 can be applied over the nine dies 1604.

[0083] FIG. 17 includes a block flow diagram of an example method 1700 for detecting a nucleotide incorporation. As illustrated at block 1702, the system can flow a modified nucleotide through the flow space defined over wells including supports having copies of target polynucleotides. The modified nucleotide may be reversibly terminated at the 3’ position toDocket No. TP389002 WO 1 prevent further incorporations. In addition, the modified nucleotide may include capture moieties or attached enzymes. For example, the nucleotide may include capture moieties secured to the reversible terminator at the 3’ position. In another example, the modified nucleotide may include a capture moiety reversibly connected to the base of the nucleotide. When the nucleotide is complementary to the next nucleotide of the target polynucleotide, the nucleotide is incorporated. The 3’ terminal group on the nucleotide prevents further incorporations until the terminal group is removed. Optionally, a polymerase is provided with the modified nucleotide or can be provided in a separate flow. Example polymerases include BST, RB69, or terminator families.

[0084] As illustrated at block 1704, the system can flow a modified enzyme. For example, the enzyme can include a complementary moiety that attaches to the capture moiety of the modified polynucleotide nucleotide. As such, the enzyme may be secured to the nucleotide when it is incorporated. In particular, the enzyme is reactive with a chemiluminescent substrate that facilitates chemiluminescence. For example, the enzyme can include a phosphatase, luciferase, galactosidase, amidase, esterase, or peptide cleavage enzyme.

[0085] The system can flow the chemiluminescent substrate, as illustrated at block 1706. The chemiluminescent substrate can react with the enzyme secured to the nucleotide to cause chemiluminescence. In an example, the enzyme is a phosphatase that reacts with 3-(2'- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-l,2-dioxetane (AMPPD) to facilitate decomposition of the dioxetane and cause chemiluminescence. In another example, the enzyme is a luciferase that reacts with luciferin to cause chemiluminescence. As long as a solution including chemiluminescent substrates flows, chemiluminescence is generated in those wells in which the nucleotide was incorporated onto the target polynucleotide. As such, sufficient chemiluminescence can be generated based on concentration and time the substrate solution flows.

[0086] As illustrated at block 1708, chemiluminescence signals can be detected by the photodiodes associated with the wells containing supports that include polynucleotides incorporating the nucleotide. Chemiluminescence signals can be utilized to determine the sequence of the target polynucleotides which can be used for various bioinformatics purposes.

[0087] The system can further provide reagents to cleave the terminator and modified enzyme from the modified nucleotide, as illustrated at block 1710. A subsequent modified nucleotideDocket No. TP389002 WO 1 solution can then be applied to the sequencing apparatus and the process repeated. For example, the process can be repeated for each type of modified nucleotide, including a modified adenine (A), modified cytosine (C), modified guanine (G), modified thymine (T), or modified uracil (U).

[0088] FIG. 18 includes an illustration of an example modified nucleotide. For example, the modified nucleotide includes a ribose 1802 to which a base 1804 is secured at the one position. Example bases include purine or pyrimidine bases.

[0089] One or more phosphates 1806 are attached at the 5’ position of the ribose 1802. For example, three phosphates can be attached to the 5’ position. In another example, one phosphate can be attached to the 5’ position. In a further example, six phosphates can be applied to the 5’ position.

[0090] A reversible terminator 1808 can be attached to the 3’ position of the ribose 1802. The reversible terminator 1808 prevents further extension or incorporation of additional nucleotides until the terminator is removed. Example terminators include 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl or benzyl moieties, among others. Other example terminators include methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxytrityl, or nitrophenyl propyloxycarbonyl, among others.

[0091] Optionally, the capture moiety or an enzyme 1810 can be secured to the reversible terminator 1808. In another example, a cleavable moiety 1812 can be secured to the base 1804. A capture moiety or an enzyme 1814 can be secured to the cleavable moiety 1812.10092] Example capture moieties include biotin, digoxigenin, 4-nitrophenol, or fluorescein, among others. An example enzyme includes phosphatase, luciferase, galactosidase, amidase, esterase, or peptide cleavage enzyme. In an example, the phosphatase is an alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, or Antarctic phosphatase, or combinations or modified versions thereof, among others.

[0093] In an example illustrated in FIG. 19, various cleavable moieties and reversible terminators can be applied to the ribose or the base. A biotin capture moiety is attached either to the reversible terminator at the 3’ position or the cleavable moiety attached to the base.Docket No. TP389002 WO 1

[0094] For example, Nucleotide 1 includes an azide reversible terminator at the 3’ position and the clcavablc moiety attached to biotin attached to the base.

[0095] In another example, modified Nucleotide 2 includes a reversible terminator azide at the 3’ position. The azide can be modified to couple with a capture moiety. For example, modified Nucleotide 3 includes an azide coupled to biotin at the 3’ position. In other examples (4), reversible terminators L can be attached at the 3’ position of the ribose and secure a biotin capture moiety.

[0096] Using a capture moiety, such as biotin, a modified enzyme including a complementary moiety, such as streptavidin, can be applied and secured to the modified nucleotide. Such an enzyme may react with a substrate, destabilizing the substrate resulting in chemiluminescence.

[0097] As illustrated at FIG. 20, an example substrate includes a modified 1, 2 dioxetane. For example, the 1, 2 dioxetane can be modified with a reporter group Rl. Example reporter groups (Rl) include phosphate, galactosyl, ester, amide, or peptide groups on the ortho, meta, or para positions of the aromatic ring. Stability and decomposition of the 1, 2 dioxetane can be modified or tuned with steric factors, such as introducing a steric group R2, for example, including alkyl, aryl, halide, alkoxy, dialkylamino groups into o, m and p-positions of the aromatic ring. An enzyme can be selected that cleaves the report group or steric group of the modified dioxetane, resulting in a metastable molecule that decomposes to provide a chemiluminescence light signal that can be detected by imaging circuitry of a sequencing apparatus.

[0098] In an example, each type of nucleotide (A, T, C, or G) can be modified with the same reversible terminator and capture moiety. In such an example, each type of modified nucleotide is provided separately through the flow space, followed by the flow of enzymes and substrates. The reversible terminator and enzyme can be cleaved from the modified nucleotides before the next modified nucleotide is applied.

[0099] Alternatively, one or more of the types of nucleotides can be modified with a different capture moiety. Each capture moiety may be associated with a different modified enzyme having a complementary moiety to the capture moiety. As such, different enzymes couple with different types of modified nucleotides. For example, a first type of enzyme can be configured to couple with a modified T nucleotide, while a second type of enzyme can be configured couple with a modified G nucleotide. In a further example, one type of modified nucleotide may be configuredDocket No. TP389002 WO 1 with two different types of capture moieties providing the ability to capture two different modified enzymes.

[0100] In an example illustrated in FIG. 21, a method 2100 includes flowing a labeled T, as illustrated at block 2102. The method further includes flowing a modified enzyme, as illustrated at block 2104. The modified enzyme attaches to any incorporated labeled T nucleotides.

[0101] As illustrated at block 2106, a chemiluminescence substrate is provided which reacts with the modified enzyme to cause chemiluminescence light emissions. Such chemiluminescence signal is collected by the image sensor, as illustrated at block 2108, indicating incorporation of the labeled T nucleotide.

[0102] Subsequent nucleotides can be provided by the system. For example, as illustrated at block 2110, a labeled A nucleotide can flow. The labeled A nucleotide can incorporate where it is complementary to target polynucleotides. As illustrated at block 2112, the modified enzyme can flow, and as illustrated at block 2114, the chemiluminescence substrate can flow, resulting in chemiluminescence that is collected by the image sensor, as illustrated at block 2116.

[0103] In further example, the system can flow a labeled C nucleotide, as illustrated at block 2118. The label C nucleotide incorporates where it is complementary to the target polynucleotides. As illustrated at block 2120, an enzyme can flow that couples with the labeled C nucleotide. The chemiluminescence substrate is provided, as illustrated at block 2122, interacting with the modified enzyme to cause chemiluminescence, which is collected by the image sensor, as illustrated at block 2124.|00104| In an additional example, the system can flow a labeled G nucleotide, as illustrated at block 2126. The system can flow a modified enzyme, as illustrated at block 2128. The system can flow a chemiluminescence substrate, as illustrated at block 2130, resulting in chemiluminescence that is collected by the image sensor, as illustrated at block 2132.

[0105] As illustrated at block 2134, reversible terminators and incorporated enzymes can be cleaved and the process started again. The modified nucleotides can be applied in the same sequence in series in the same order. In another example, the modified nucleotides can be applied in series in different orders.Docket No. TP389002 WO 1

[0106] In an example, each of the labeled nucleotides are modified with the same capture moiety. The same modified enzyme that has a complementary moiety to attach to the capture moiety is used. The same modified chemiluminescence substrate can be used, which interacts with the enzyme to provide chemiluminescence signal. In such an example, which nucleotide was incorporated can be determined based on the step in which the chemiluminescence signal is first detected. For example, those wells in which the labeled T nucleotide was incorporated provide chemiluminescence at each of the steps in which a substrate flows. Whereas those wells in which a labeled G nucleotide was incorporated chemiluminesce only at the final flow of the substrate.

[0107] Alternatively, each labeled nucleotide can be labeled with a different capture moiety or combination of capture moieties. Different modified enzymes or combinations of modified enzymes can be used at different steps in which a modified enzyme flows. In an example in which each labeled nucleotide is modified with different capture moiety, different modified enzymes are used at each enzyme flow step. Associated chemiluminescence substrates flow at different flow steps, chemiluminescence and image detection only occur during the flow of the substrate reactive with the enzyme modified to attach to be labeled nucleotide. In another example, one or more of the modified nucleotides can be modified with two capture moieties. In such an example, the nucleotide modified with the two capture moieties chemiluminesce at two of the substrate flow steps. As such, detection or identification of the incorporation of that nucleotide is based on two separate chemiluminescence events. Alternatively, one of the nucleotides can be free of a capture moiety or enzyme. While other incorporated nucleotides may cause chemiluminescence, the type of nucleotide that is free of a capture moiety or enzyme remains dark.

[0108] In a further example illustrated in FIG. 22, a method 2200 includes flowing four labeled nucleotides simultaneously or consecutively, as illustrated at block 2202. Each of the four labeled nucleotides can be modified with a different capture moiety or different enzyme. In the case of modification with different capture moieties, the modified enzymes can flow with the label nucleotides or can be provided in a separate step.

[0109] Upon incorporation of the label nucleotides and their associated enzymes, the system can flow a substrate A, as illustrated at block 2204. The substrate A interacts with a first type ofDocket No. TP389002 WO 1 enzyme attached to a first type of nucleotide, resulting in chemiluminescence. An image can be collected, as illustrated at block 2206. An image is a collection of signals of an array of image sensors.

[0110] As illustrated at block 2208, a substrate B can flow and interact with an associated enzyme coupled to an associated nucleotide. The interaction results in chemiluminescence detected by an image sensor, as illustrated at block 2210.

[0111] Subsequently, a substrate C can flow, as illustrated at block 2212, resulting in chemiluminescence in association with the incorporation of one type of nucleotide. The chemiluminescence can be detected by collecting images with the image sensor, as illustrated at block 2214.

[0112] As illustrated at block 2216, the terminators and all of the enzymes associated with the modified nucleotides can be cleaved in the same step or in two or more steps at the end of the method.

[0113] Nucleotides can be further modified such that two types of nucleotides associate with two different enzymes, a third type of nucleotide associates with both of the two different enzymes, and a fourth type of modified nucleotide does not associate with an enzyme. As such, the first two types of modified nucleotides only chemiluminesce in association with one substrate of two different substrates, whereas the third modified nucleotide is associated with chemiluminescence when both different types of substrates flow. The fourth modified nucleotide is not associated with chemiluminescence and remains dark.|001141 For example, in a method 2300 illustrated in FIG. 23, all four nucleotides can flow, as illustrated at block 2302. A first type of enzyme A can flow, as illustrated at block 2304, and a substrate A can flow, as illustrated at block 2306. The chemiluminescence signal can be detected by collecting images with the image sensor, as illustrated at block 2308.

[0115] Subsequently, an enzyme B can flow, as illustrated at block 2310, and a substrate B can flow, as illustrated at block 2312. Chemiluminescence signals can be collected by collecting images with an image sensor, as illustrated at block 2314.

[0116] In such an example, a first type of nucleotide is associated with chemiluminescence when substrate A flows, a second type of nucleotide is associated with chemiluminescence whenDocket No. TP389002 WO 1 substrate B flows, a third type of nucleotide is associated with chemiluminescence when both substrate A flows and when substrate B flows, and a fourth type of nucleotide is not associated with chemiluminescence.

[0117] As illustrated at block 2316, terminators and enzymes can be cleaved and the process repeated to detect the next nucleotide in the sequence.

[0118] In view of the above described methods, various steps can be performed consecutively or concurrently. In an example, modified enzymes of different types can How concurrently.Additional reagent solutions and wash solutions can flow before, after, or between each of the above steps. Polymerase may flow before or with nucleotides. Further variations can be envisaged in view of the above disclosure.EXAMPLES

[0119] Example 1

[0120] Chemiluminescent light signals are generated in glass cuvettes using shrimp alkaline phosphatase (SAP) and AMPPD. A bottom of one cuvette includes a polymeric coating derived from a tripentyl amine modified vinyl benzene. Another cuvette is free of a polymer coating. A solution comprising SAP is provided to both cuvettes. AMPPD is added to the cuvettes, causing chemiluminescence. A peak of detected light signal from the cuvette free of polymer coating is in the range of 1000 units, whereas a peak of detected light signal from the cuvette with the polymer coating is in the range of 140000 units.

[0121] Example 2

[0122] Chemiluminescent light signals are generated in glass cuvettes using shrimp alkaline phosphatase (SAP) and AMPPD. A bottom of one cuvette includes a polymeric coating derived from a tripentyl amine modified vinyl benzene. Another cuvette is free of a polymer coating. A solution comprising SAP is provided to both cuvettes. AMPPD is added to the cuvettes, causing chemiluminescence, followed by an acid solution. A peak of detected light signal from the cuvette free of polymer coating is in the range of 1000 units, whereas a peak of detected light signal from the cuvette with the polymer coating is over 200000 units.Docket No. TP389002 WO 1

[0123] In a first aspect, a system comprising: a fluidic system configured to flow a plurality of reagent solutions; an imaging apparatus in communication with the fluidic system, the imaging apparatus comprising an array of image sensors, an array of wells disposed over the array of image sensors, and a flow cell structure defining a flow space over the array of wells; and a data collection circuitry to receive light signal data from the imaging apparatus.

[0124] In an example of the first aspect and the above examples, the image sensors are back side illuminated image sensors.

[0125] In an example of the first aspect and the above examples, further comprising a controller in communication with the fluidic system and the imaging apparatus.

[0126] In an example of the first aspect and the above examples, further comprising an array of filters disposed between the array of image sensors and the array of wells.

[0127] In an example of the first aspect and the above examples, the array of filters comprises optical filters formed of a polymeric material.

[0128] In an example of the first aspect and the above examples, the polymeric material comprises a novolac photoresist.

[0129] In an example of the first aspect and the above examples, further comprising a hydrophobic or partially hydrophobic material disposed in wells of the array of wells.

[0130] In an example of the first aspect and the above examples, the hydrophobic or partially hydrophobic material comprises surface agents that bind as a monolayer to a bottom of the wells.

[0131] In an example of the first aspect and the above examples, the surface agents have a surface reactive functional group selected from the group consisting of silane, phosphates, phosphonic acid, phosphinic acid, bisphosphonic acid, multidentate phosphates, multidentate phosphonates, polyphosphates, polyphosphonates, isocyanate, catechol, hydroxamate, alkoxy derivatives thereof, and combinations thereof.

[0132] In an example of the first aspect and the above examples, the surface reactive functional group is attached to an alkyl moiety having 6 to 30 carbons.

[0133] In an example of the first aspect and the above examples, the alkyl moiety has 10 to 22 carbons.Docket No. TP389002 WO 1

[0134] In an example of the first aspect and the above examples, the hydrophobic or partially hydrophobic material comprises a polymer selected from the group consisting of photoresist, novolac resin, polymethylmethacrylate, polyisoprene, bisphenol epoxies, vinyl polymers, styrenic polymers, alkylene polymers, norbomene polymers, and combinations thereof.

[0135] In an example of the first aspect and the above examples, further comprising an array of lenses disposed between the array of image sensors and the array of wells.

[0136] In an example of the first aspect and the above examples, the array of lenses is disposed between the array of filters and the array of wells.

[0137] In an example of the first aspect and the above examples, image sensors of the array of image sensors arc photodiodes.

[0138] In an example of the first aspect and the above examples, the photodiodes are optically isolated from each other by isolation structures.

[0139] In an example of the first aspect and the above examples, the isolation structures comprise deep trench isolation structures formed of a dielectric material.

[0140] In an example of the first aspect and the above examples, the dielectric material is selected from the group consisting of silicon oxide, hafnium oxide, and materials with a refractive index less than that of silicon.

[0141] In an example of the first aspect and the above examples, the array of image sensors is disposed on a die, and wherein the die is one of a plurality of undiced dies forming the imaging apparatus.

[0142] In an example of the first aspect and the above examples, the array of image sensors is disposed on a die, and wherein the die is one of a plurality of diced dies forming the imaging apparatus.

[0143] In an example of the first aspect and the above examples, the die is one of a plurality of dies disposed on an interposer.

[0144] In an example of the first aspect and the above examples, the interposer is connected to a printed circuit board.Docket No. TP389002 WO 1

[0145] In an example of the first aspect and the above examples, the plurality of reagent solutions comprises modified nucleotide reagents, enzyme solutions, chemiluminescence substrate solutions, wash solutions, and catalytic solutions.

[0146] In an example of the first aspect and the above examples, the flow cell structure defines at least one inlet and at least one outlet providing fluid communication with the flow space.

[0147] In an example of the first aspect and the above examples, the array of wells is configured to receive supports incorporating monoclonal populations of polynucleotides.

[0148] In a second aspect, a method of detecting a nucleotide incorporation, the method comprising: flowing at least one type of modified nucleotide over an array of wells comprising a support having a monoclonal population of target polynucleotides, the at least one type of modified nucleotide including a reversible terminator and a capture moiety, modified nucleotides incorporating when complementary to a next nucleotide of the target polynucleotide; flowing modified enzyme with a complementary moiety complementary to the capture moiety, the modified enzyme coupling to an incorporated modified nucleotide; flowing a chemiluminescent substrate reactive with the modified enzyme, the chemiluminescent substrate generating a light signal in response to the modified enzyme; and detecting the light signal with an image sensor.

[0149] In an example of the second aspect and the above examples, further comprising a step of cleaving the reversible terminator and the capture moiety.

[0150] In an example of the second aspect and the above examples, the support comprises polymeric beads.

[0151] In an example of the second aspect and the above examples, the polymeric beads are formed of a radically polymerizable monomer comprising a hydrophilic monomer coupled to a hydrophobic protection group.

[0152] In an example of the second aspect and the above examples, the hydrophilic monomer is selected from the group consisting of acrylamide, vinyl acetate, hydroxyalkyl methacrylate, and combinations thereof.

[0153] In an example of the second aspect and the above examples, the hydrophilic monomer comprises an acrylamide including hydroxyl groups, amino groups, carboxyl groups, or a combination thereof.Docket No. TP389002 WO 1

[0154] In an example of the second aspect and the above examples, the acrylamide is selected from the group consisting of aminoalkyl acrylamide, acrylamide functionalized with an amine terminated polypropylene glycol, acrylopiperazine, and combinations thereof.

[0155] In an example of the second aspect and the above examples, the acrylamide functionalized with an amine terminated polypropylene glycol has the structure .

[0156] In an example of the second aspect and the above examples, the acrylopiperazine has the structure .

[0157] In an example of the second aspect and the above examples, the polymeric beads have an average particle size not greater than 100 pm.

[0158] In an example of the second aspect and the above examples, the average particle size is in a range of 0.1 pm to 1.1 pm.

[0159] In an example of the second aspect and the above examples, the reversible terminator is selected from the group consisting of 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxytrityl, and nitrophenyl propyloxycarbonyl moieties.

[0160] In an example of the second aspect and the above examples, the capture moiety is selected from the group consisting of biotin, digoxigenin, 4-nitrophcnol, and fluorescein.

[0161] In an example of the second aspect and the above examples, the capture moiety is biotin.

[0162] In an example of the second aspect and the above examples, the modified enzyme is selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.

[0163] In an example of the second aspect and the above examples, the modified enzyme is a phosphatase selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

[0164] In an example of the second aspect and the above examples, the complementary moiety comprises streptavidin.Docket No. TP389002 WO 1

[0165] In an example of the second aspect and the above examples, the chemiluminescent substrate comprises luciferin or a modified 1,2-dioxctanc.

[0166] In an example of the second aspect and the above examples, the modified 1 ,2-dioxetane comprises 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-l,2-dioxetane (AMPPD).

[0167] In an example of the second aspect and the above examples, the at least one type of modified nucleotide comprises four types of modified nucleotides, each type corresponding to adenine, cytosine, guanine, and thymine.

[0168] In an example of the second aspect and the above examples, each of the four types of modified nucleotides includes the same capture moiety.

[0169] In an example of the second aspect and the above examples, at least three of the four types of modified nucleotides each include a different capture moiety.

[0170] In an example of the second aspect and the above examples, further comprising a step of flowing a polymerase with the at least one type of modified nucleotide.

[0171] In an example of the second aspect and the above examples, the polymerase is selected from the group consisting of BST, RB69, and terminator families.

[0172] In an example of the second aspect and the above examples, the image sensor comprises a photodiode disposed in an array of photodiodes, and wherein the array of wells is aligned with the array of photodiodes.

[0173] In a third aspect, a method of detecting a nucleotide incorporation, the method comprising: flowing four types of modified nucleotides, each type of modified nucleotide modified with a reversible terminator; at least three types of modified nucleotides each modified with a different capture moiety; flowing a first enzyme type modified with a first complementary moiety complementary to one of the different capture moieties; flowing a first chemiluminescent substrate reactive with the first enzyme type to generate a first light signal; detecting the first light signal; flowing a second enzyme type modified with a second complementary moiety complementary to a second of the different capture moieties; flowing a second chemiluminescent substrate reactive with the second enzyme type to generate a second light signal; detecting the second light signal; flowing a third enzyme type modified with a third complementary moietyDocket No. TP389002 WO 1 complementary to a third of the different capture moieties; flowing a third chemiluminescent substrate reactive with the third enzyme type to generate a third light signal; detecting the third light signal; and cleaving the reversible terminator and different capture moieties.

[0174] In an example of the third aspect and the above examples, the four types of modified nucleotides correspond to adenine, cytosine, guanine, and thymine.

[0175] In an example of the third aspect and the above examples, a fourth type of modified nucleotide is free of a capture moiety.

[0176] In an example of the third aspect and the above examples, the fourth type of modified nucleotide remains dark during detection of the light signals.

[0177] In an example of the third aspect and the above examples, the different capture moieties are selected from the group consisting of biotin, digoxigenin, 4-nitrophenol, and fluorescein.

[0178] In an example of the third aspect and the above examples, at least one of the different capture moieties is biotin.

[0179] In an example of the third aspect and the above examples, the first, second, and third enzyme types are each selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.

[0180] In an example of the third aspect and the above examples, at least one of the first, second, and third enzyme types is a phosphatase.

[0181] In an example of the third aspect and the above examples, the phosphatase is selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

[0182] In an example of the third aspect and the above examples, the first, second, and third complementary moieties each comprise streptavidin.

[0183] In an example of the third aspect and the above examples, the first, second, and third chemiluminescent substrates each comprise a luciferin or modified 1,2-dioxetane.Docket No. TP389002 WO 1

[0184] In an example of the third aspect and the above examples, at least one of the modified 1 ,2-dioxctancs comprises 3-(2'-spiroadamantanc)-4-mcthoxy-4-(3''-phosphoryloxy)phcnyl-l,2- dioxetane (AMPPD).

[0185] In an example of the third aspect and the above examples, the reversible terminator is selected from the group consisting of 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxy trityl, and nitrophenyl propyloxycarbonyl moieties.

[0186] In an example of the third aspect and the above examples, the reversible terminator is 3'- azidomethyl.

[0187] In an example of the third aspect and the above examples, further comprising a step of flowing a polymerase with the four types of modified nucleotides.

[0188] In an example of the third aspect and the above examples, the polymerase is selected from the group consisting of BST, RB69, and terminator families.

[0189] In an example of the third aspect and the above examples, the four types of modified nucleotides are flowed simultaneously.

[0190] In an example of the third aspect and the above examples, the four types of modified nucleotides arc flowed consecutively.

[0191] In an example of the third aspect and the above examples, detecting the first, second, and third light signals comprises collecting images with an image sensor.

[0192] In an example of the third aspect and the above examples, the image sensor comprises a photodiode disposed in an array of photodiodes.

[0193] In an example of the third aspect and the above examples, the array of wells is aligned with the array of photodiodes.

[0194] In an example of the third aspect and the above examples, the array of wells comprises supports having monoclonal populations of target polynucleotides.

[0195] In an example of the third aspect and the above examples, the supports comprise polymeric beads.Docket No. TP389002 WO 1

[0196] In an example of the third aspect and the above examples, the polymeric beads have an average particle size in a range of 0.1 pm to 1.1 pm.

[0197] In an example of the third aspect and the above examples, further comprising a step of flowing wash solutions between flowing the enzyme types and the chemiluminescent substrates.

[0198] In a fourth aspect, a method of detecting a nucleotide incorporation, the method comprising: flowing four types of modified nucleotides, each type of modified nucleotide modified with a reversible terminator; first and second types of modified nucleotides each modified with one of two different capture moiety, a third type of modified nucleotides modified with both of the two different capture moieties; flowing a first enzyme type modified with a first complementary moiety complementary to one of the two different capture moieties; flowing a first chemiluminescent substrate reactive with the first enzyme type to generate a first light signal; detecting the first light signal; flowing a second enzyme type modified with a second complementary moiety complementary to a second of the two different capture moieties; flowing a second chemiluminescent substrate reactive with the second enzyme type to generate a second light signal; detecting the second light signal; and cleaving the reversible terminator and different capture moieties.

[0199] In an example of the fourth aspect and the above examples, the four types of modified nucleotides correspond to adenine, cytosine, guanine, and thymine.

[0200] In an example of the fourth aspect and the above examples, a fourth type of modified nucleotide is free of a capture moiety.1002011 In an example of the fourth aspect and the above examples, the fourth type of modified nucleotide remains dark during detection of the light signals.

[0202] In an example of the fourth aspect and the above examples, the two different capture moieties are selected from the group consisting of biotin, digoxigenin, 4-nitrophenol, and fluorescein.

[0203] In an example of the fourth aspect and the above examples, one of the two different capture moieties is biotin.Docket No. TP389002 WO 1

[0204] In an example of the fourth aspect and the above examples, the first and second enzyme types arc each selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.

[0205] In an example of the fourth aspect and the above examples, at least one of the first and second enzyme types is a phosphatase.

[0206] In an example of the fourth aspect and the above examples, the phosphatase is selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

[0207] In an example of the fourth aspect and the above examples, the first and second complementary moieties each comprise streptavidin.

[0208] In an example of the fourth aspect and the above examples, the first and second chemiluminescent substrates each comprise luciferin or a modified 1,2-dioxetane.

[0209] In an example of the fourth aspect and the above examples, at least one of the modified 1 ,2-dioxetanes comprises 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl- 1,2- dioxetane (AMPPD).

[0210] In an example of the fourth aspect and the above examples, the reversible terminator is selected from the group consisting of 3'-azidomcthyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxy trityl, and nitrophenyl propyloxycarbonyl moieties.

[0211] In an example of the fourth aspect and the above examples, the reversible terminator is 3'- azidomethyl.

[0212] In an example of the fourth aspect and the above examples, further comprising a step of flowing a polymerase with the four types of modified nucleotides.

[0213] In an example of the fourth aspect and the above examples, the polymerase is selected from the group consisting of BST, RB69, and terminator families.Docket No. TP389002 WO 1

[0214] In an example of the fourth aspect and the above examples, the four types of modified nucleotides arc flowed simultaneously.

[0215] In an example of the fourth aspect and the above examples, the four types of modified nucleotides are flowed consecutively.

[0216] In an example of the fourth aspect and the above examples, detecting the first and second light signals comprises collecting images with an image sensor.

[0217] In an example of the fourth aspect and the above examples, the image sensor comprises a photodiode disposed in an array of photodiodes.

[0218] In an example of the fourth aspect and the above examples, an array of wells is aligned with the array of photodiodes.

[0219] In an example of the fourth aspect and the above examples, the array of wells comprises supports having monoclonal populations of target polynucleotides.

[0220] In an example of the fourth aspect and the above examples, the supports comprise polymeric beads.

[0221] In an example of the fourth aspect and the above examples, the polymeric beads have an average particle size in a range of 0.1 pm to 1.1 pm.

[0222] In an example of the fourth aspect and the above examples, further comprising a step of flowing wash solutions between flowing the enzyme types and the chemiluminescent substrates.

[0223] Note that not all of the activities described above in the general description or the examples are required, that a portion of a specific activity may not be required, and that one or more further activities may be performed in addition to those described. Still further, the order in which activities are listed are not necessarily the order in which they are performed.

[0224] In the foregoing specification, the concepts have been described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.Docket No. TP389002 WO 1

[0225] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, arc intended to cover a non-cxclusivc inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive-or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0226] Also, the use of “a” or “an” are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.1002271 Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any feature(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature of any or all the claims.

[0228] After reading the specification, skilled artisans will appreciate that certain features are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, references to values stated in ranges include each and every value within that range.

Claims

Docket No. TP389002 WO 1CLAIMSWHAT IS CLAIMED IS:

1. A system comprising: a fluidic system configured to flow a plurality of reagent solutions; an imaging apparatus in communication with the fluidic system, the imaging apparatus comprising an array of image sensors, an array of wells disposed over the array of image sensors, and a flow cell structure defining a flow space over the array of wells; and a data collection circuitry to receive light signal data from the imaging apparatus.

2. The system of claim 1, wherein the image sensors are back side illuminated image sensors.

3. The system of claim 1 or 2, further comprising a controller in communication with the fluidic system and the imaging apparatus.

4. The system of any one of claims 1-3, further comprising an array of filters disposed between the array of image sensors and the array of wells.

5. The system of claim 4, wherein the array of filters comprises optical filters formed of a polymeric material.

6. The system of claim 5, wherein the polymeric material comprises a novolac photoresist.

7. The system of any one of claims 1-6, further comprising a hydrophobic or partially hydrophobic material disposed in wells of the array of wells.

8. The system of claim 7, wherein the hydrophobic or partially hydrophobic material comprises surface agents that bind as a monolayer to a bottom of the wells.

9. The system of claim 8, wherein the surface agents have a surface reactive functional group selected from the group consisting of silane, phosphates, phosphonic acid, phosphinic acid,Docket No. TP389002 WO 1 bisphosphonic acid, multidentate phosphates, multidentate phosphonates, polyphosphates, polyphosphonates, isocyanate, catechol, hydroxamate, alkoxy derivatives thereof, and combinations thereof.

10. The system of claim 9, wherein the surface reactive functional group is attached to an alkyl moiety having 6 to 30 carbons.

11. The system of claim 10, wherein the alkyl moiety has 10 to 22 carbons.

12. The system of claim 7, wherein the hydrophobic or partially hydrophobic material comprises a polymer selected from the group consisting of photoresist, novolac resin, polymethylmethacrylate, polyisoprene, bisphenol epoxies, vinyl polymers, styrenic polymers, alkylene polymers, norbornene polymers, and combinations thereof.

13. The system of any one of claims 1-12, further comprising an array of lenses disposed between the array of image sensors and the array of wells.

14. The system of claims 4 and 13, wherein the array of lenses is disposed between the array of filters and the array of wells.

15. The system of any one of claims 1-14, wherein image sensors of the array of image sensors are photodiodes.

16. The system of claim 15, wherein the photodiodes are optically isolated from each other by isolation structures.

17. The system of claim 16, wherein the isolation structures comprise deep trench isolation structures formed of a dielectric material.

18. The system of claim 17, wherein the dielectric material is selected from the group consisting of silicon oxide, hafnium oxide, and materials with a refractive index less than that of silicon.Docket No. TP389002 WO 119. The system of any one of claims 1-18, wherein the array of image sensors is disposed on a die, and wherein the die is one of a plurality of undiced dies forming the imaging apparatus.

20. The system of any one of claims 1-18, wherein the array of image sensors is disposed on a die, and wherein the die is one of a plurality of diced dies forming the imaging apparatus.

21. The system of any one of claims 19-20, wherein the die is one of a plurality of dies disposed on an interposer.

22. The system of claim 21, wherein the interposer is connected to a printed circuit board.

23. The system of any one of claims 1-22, wherein the plurality of reagent solutions comprises modified nucleotide reagents, enzyme solutions, chemiluminescence substrate solutions, wash solutions, and catalytic solutions.

24. The system of any one of claims 1-23, wherein the flow cell structure defines at least one inlet and at least one outlet providing fluid communication with the flow space.

25. The system of any one of claims 1-24, wherein the array of wells is configured to receive supports incorporating monoclonal populations of polynucleotides.

26. A method of detecting a nucleotide incorporation, the method comprising: flowing at least one type of modified nucleotide over an array of wells comprising a support having a monoclonal population of target polynucleotides, the at least one type of modified nucleotide including a reversible terminator and a capture moiety, modified nucleotides incorporating when complementary to a next nucleotide of the target polynucleotide; flowing modified enzyme with a complementary moiety complementary to the capture moiety, the modified enzyme coupling to an incorporated modified nucleotide;Docket No. TP389002 WO 1 flowing a chemiluminescent substrate reactive with the modified enzyme, the chemiluminescent substrate generating a light signal in response to the modified enzyme; and detecting the light signal with an image sensor.

27. The method of claim 26, further comprising a step of cleaving the reversible terminator and the capture moiety.

28. The method of claim 26 or 27, wherein the support comprises polymeric beads.

29. The method of claim 28, wherein the polymeric beads are formed of a radically polymerizable monomer comprising a hydrophilic monomer coupled to a hydrophobic protection group.

30. The method of claim 29, wherein the hydrophilic monomer is selected from the group consisting of acrylamide, vinyl acetate, hydroxyalkyl methacrylate, and combinations thereof.

31. The method of claim 30, wherein the hydrophilic monomer comprises an acrylamide including hydroxyl groups, amino groups, carboxyl groups, or a combination thereof.

32. The method of claim 31, wherein the acrylamide is selected from the group consisting of aminoalkyl acrylamide, acrylamide functionalized with an amine terminated polypropylene glycol, acrylopiperazine, and combinations thereof.

33. The method of claim 32, wherein the acrylamide functionalized with an amine terminated polypropylene glycol has the structure34. The method of claim 32, wherein the acrylopiperazine has the structureDocket No. TP389002 WO 135. The method of any one of claims 28-34, wherein the polymeric beads have an average particle size not greater than 100 pm.

36. The method of claim 35, wherein the average particle size is in a range of 0.1 pm to 1.1 pm.

37. The method of any one of claims 26-36, wherein the reversible terminator is selected from the group consisting of 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophcnyl, cyanocthyl, tctrahydrofuranyl, tctrahydropyranyl, dimcthoxytrityl, and nitrophcnyl propyloxycarbonyl moieties.

38. The method of any one of claims 26-37, wherein the capture moiety is selected from the group consisting of biotin, digoxigenin, 4-nitrophenol, and fluorescein.

39. The method of claim 38, wherein the capture moiety is biotin.

40. The method of any one of claims 26-39, wherein the modified enzyme is selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.

41. The method of claim 40, wherein the modified enzyme is a phosphatase selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

42. The method of any one of claims 26-41, wherein the complementary moiety comprises streptavidin.

43. The method of any one of claims 26-42, wherein the chemiluminescent substrate comprises luciferin or a modified 1,2-dioxetane.Docket No. TP389002 WO 144. The method of claim 43, wherein the modified 1 ,2-dioxetane comprises 3-(2'- spiroadamantane)-4-methoxy-4-(3 "-phosphoryloxy )phenyl-l,2-dioxetane (AMPPD).

45. The method of any one of claims 26-44, wherein the at least one type of modified nucleotide comprises four types of modified nucleotides, each type corresponding to adenine, cytosine, guanine, and thymine.

46. The method of claim 45, wherein each of the four types of modified nucleotides includes the same capture moiety.

47. The method of claim 45, wherein at least three of the four types of modified nucleotides each include a different capture moiety.

48. The method of any one of claims 26-47, further comprising a step of flowing a polymerase with the at least one type of modified nucleotide.

49. The method of claim 48, wherein the polymerase is selected from the group consisting of BST, RB69, and terminator families.

50. The method of any one of claims 26-49, wherein the image sensor comprises a photodiode disposed in an array of photodiodes, and wherein the array of wells is aligned with the array of photodiodes.

51. A method of detecting a nucleotide incorporation, the method comprising: flowing four types of modified nucleotides, each type of modified nucleotide modified with a reversible terminator; at least three types of modified nucleotides each modified with a different capture moiety; flowing a first enzyme type modified with a first complementary moiety complementary to one of the different capture moieties; flowing a first chemiluminescent substrate reactive with the first enzyme type to generate a first light signal;Docket No. TP389002 WO 1 detecting the first light signal; flowing a second enzyme type modified with a second complementary moiety complementary to a second of the different capture moieties; flowing a second chemiluminescent substrate reactive with the second enzyme type to generate a second light signal; detecting the second light signal; flowing a third enzyme type modified with a third complementary moiety complementary to a third of the different capture moieties; flowing a third chemiluminescent substrate reactive with the third enzyme type to generate a third light signal; detecting the third light signal; and cleaving the reversible terminator and different capture moieties.

52. The method of claim 51, wherein the four types of modified nucleotides correspond to adenine, cytosine, guanine, and thymine.

53. The method of claim 51 or 52, wherein a fourth type of modified nucleotide is free of a capture moiety.

54. The method of claim 53, wherein the fourth type of modified nucleotide remains dark during detection of the light signals.

55. The method of any one of claims 51-54, wherein the different capture moieties are selected from the group consisting of biotin, digoxigenin, 4-nitrophenol, and fluorescein.

56. The method of claim 55, wherein at least one of the different capture moieties is biotin.

57. The method of any one of claims 51-56, wherein the first, second, and third enzyme types are each selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.Docket No. TP389002 WO 158. The method of claim 57, wherein at least one of the first, second, and third enzyme types is a phosphatase.

59. The method of claim 58, wherein the phosphatase is selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

60. The method of any one of claims 51-59, wherein the first, second, and third complementary moictics each comprise streptavidin.

61. The method of any one of claims 51-60, wherein the first, second, and third chemiluminescent substrates each comprise luciferin or a modified 1,2-dioxetane.

62. The method of claim 61, wherein at least one of the modified 1 ,2-dioxetanes comprises 3-(2'- spiroadamantane)-4-methoxy-4-(3 "-phosphoryloxy )phenyl- 1,2-dioxetane (AMPPD).

63. The method of any one of claims 51-62, wherein the reversible terminator is selected from the group consisting of 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxytrityl, and nitrophenyl propyloxycarbonyl moieties.

64. The method of claim 63, wherein the reversible terminator is 3'-azidomethyl.

65. The method of any one of claims 51-64, further comprising a step of flowing a polymerase with the four types of modified nucleotides.

66. The method of claim 65, wherein the polymerase is selected from the group consisting of BST, RB69, and terminator families.Docket No. TP389002 WO 167. The method of any one of claims 51-66, wherein the four types of modified nucleotides are flowed simultaneously.

68. The method of any one of claims 51-66, wherein the four types of modified nucleotides are flowed consecutively.

69. The method of any one of claims 51-68, wherein detecting the first, second, and third light signals comprises collecting images with an image sensor.

70. The method of claim 69, wherein the image sensor comprises a photodiode disposed in an array of photodiodes.

71. The method of claim 70, wherein the array of wells is aligned with the array of photodiodes.

72. The method of any one of claims 51-71, wherein the array of wells comprises supports having monoclonal populations of target polynucleotides.

73. The method of claim 72, wherein the supports comprise polymeric beads.

74. The method of claim 73, wherein the polymeric beads have an average particle size in a range of 0.1 pm to 1.1 pm.

75. The method of any one of claims 51-74, further comprising a step of flowing wash solutions between flowing the enzyme types and the chemiluminescent substrates.

76. A method of detecting a nucleotide incorporation, the method comprising: flowing four types of modified nucleotides, each type of modified nucleotide modified with a reversible terminator; first and second types of modified nucleotides each modified with one of two different capture moiety, a third type of modified nucleotides modified with both of the two different capture moieties;Docket No. TP389002 WO 1 flowing a first enzyme type modified with a first complementary moiety complementary to one of the two different capture moictics; flowing a first chemiluminescent substrate reactive with the first enzyme type to generate a first light signal; detecting the first light signal; flowing a second enzyme type modified with a second complementary moiety complementary to a second of the two different capture moieties; flowing a second chemiluminescent substrate reactive with the second enzyme type to generate a second light signal; detecting the second light signal; and cleaving the reversible terminator and different capture moieties.

77. The method of claim 76, wherein the four types of modified nucleotides correspond to adenine, cytosine, guanine, and thymine.

78. The method of claim 76 or 77, wherein a fourth type of modified nucleotide is free of a capture moiety.

79. The method of claim 78, wherein the fourth type of modified nucleotide remains dark during detection of the light signals.

80. The method of any one of claims 76-79, wherein the two different capture moieties are selected from the group consisting of biotin, digoxigenin, 4-nitrophenol, and fluorescein.

81. The method of claim 80, wherein one of the two different capture moieties is biotin.

82. The method of any one of claims 76-81, wherein the first and second enzyme types are each selected from the group consisting of phosphatase, luciferase, galactosidase, amidase, esterase, and peptide cleavage enzyme.

83. The method of claim 82, wherein at least one of the first and second enzyme types is a phosphatase.Docket No. TP389002 WO 184. The method of claim 83, wherein the phosphatase is selected from the group consisting of alkaline phosphatase, shrimp phosphatase, horseradish phosphatase, T-cell protein tyrosine phosphatase, lambda protein phosphatase, protein phosphatase 1, and Antarctic phosphatase.

85. The method of any one of claims 76-84, wherein the first and second complementary moieties each comprise streptavidin.

86. The method of any one of claims 76-85, wherein the first and second chemiluminescent substrates each comprise luciferin or a modified 1 ,2-dioxctanc.

87. The method of claim 86, wherein at least one of the modified 1 ,2-dioxetanes comprises 3-(2'- spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-l,2-dioxetane (AMPPD).

88. The method of any one of claims 76-87, wherein the reversible terminator is selected from the group consisting of 3'-azidomethyl, allyl carbonate, allyl carbamate, allyl thiocarbonate, allyl dithionate, azidomethyl, allyl, propargyl, benzyl, methyl, nitrobenzyl, nitrobenzylthio, nitrophenyl, cyanoethyl, tetrahydrofuranyl, tetrahydropyranyl, dimethoxytrityl, and nitrophenyl propyloxycarbonyl moieties.

89. The method of claim 88, wherein the reversible terminator is 3'-azidomethyl.

90. The method of any one of claims 76-89, further comprising a step of flowing a polymerase with the four types of modified nucleotides.

91. The method of claim 90, wherein the polymerase is selected from the group consisting of BST, RB69, and terminator families.

92. The method of any one of claims 76-91, wherein the four types of modified nucleotides are flowed simultaneously.Docket No. TP389002 WO 193. The method of any one of claims 76-91, wherein the four types of modified nucleotides are flowed consecutively.

94. The method of any one of claims 76-93, wherein detecting the first and second light signals comprises collecting images with an image sensor.

95. The method of claim 94, wherein the image sensor comprises a photodiode disposed in an array of photodiodes.

96. The method of claim 95, wherein an array of wells is aligned with the array of photodiodes.

97. The method of any one of claims 76-96, wherein the array of wells comprises supports having monoclonal populations of target polynucleotides.

98. The method of claim 97, wherein the supports comprise polymeric beads.

99. The method of claim 98, wherein the polymeric beads have an average particle size in a range of 0.1 pm to 1.1 pm.

100. The method of any one of claims 76-99, further comprising a step of flowing wash solutions between flowing the enzyme types and the chemiluminescent substrates.

Images