Methods and compositions for identifying ligands on arrays using indexes and barcodes

Abstract

Some embodiments provided herein include methods and compositions for detecting target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the position of a bead containing the capture probe is determined within the array, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode indicates the capture probe attached to the bead, and an index indicates a subpopulation of beads. Some embodiments relate to sequencing target polynucleotides from several different nucleic acid samples on a bead array.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 909,014, filed October 1, 2019, entitled "METHODS AND COMPOSITIONS FOR SEQUENCING NUCLEIC ACIDS ON ARRAY," and U.S. Provisional Patent Application No. 62 / 903,108, filed September 20, 2019, entitled "METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT GENOTYPING ON ARRAYS USING INDEXES AND BARCODES," each of which is incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION Some embodiments provided herein include methods and compositions for detecting target ligands on an array. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the position of a bead containing the capture probe is determined within the array, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode indicates the capture probe attached to the bead, and an index indicates a subpopulation of beads. Some embodiments relate to sequencing target polynucleotides from several different nucleic acid samples on a bead array. [Background technology]

[0003] The detection of specific nucleic acid sequences present in biological samples has been used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, determining genetic susceptibility to disease, and measuring response to various types of treatment. A common technique for detecting specific nucleic acid sequences in biological samples is nucleic acid sequencing.

[0004] Nucleic acid sequencing methods have evolved significantly from the chemical degradation method used by Maxam and Gilbert and the strand extension method used by Sanger. Several sequencing methods are now in use that allow for the parallel processing of thousands of nucleic acids on a single chip. Several platforms include bead-based and microarray formats, in which silica beads are functionalized with probes amenable to the application of such formats in applications including sequencing, genotyping, and gene expression profiling.

[0005] Current methods for genotyping different samples on bead-based arrays require a gasket to physically subdivide different regions of the beadchip into multiple sectors. Individual samples are then loaded into each separate section created by the gasket. However, while such methods are useful for inputting relatively small numbers of samples, they have proven difficult and unmanageable as the density of samples per beadchip increases from 24 to 96, 384, 1536, or even more samples per beadchip. Summary of the Invention

[0006] Some embodiments are directed to a method of sequencing a target nucleic acid on an array, comprising: (a) obtaining first and second populations of beads, wherein the first population of beads comprises a first capture probe, a first barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the first barcode, and the second population of beads comprises a second capture probe, a second barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the second barcode; and (b) obtaining first and second pluralities of polynucleotides, wherein the first plurality of polynucleotides comprises a first target nucleic acid, the plurality of first polynucleotides being in solution, and the second plurality of polynucleotides being in solution. wherein the first target nucleic acid comprises a first capture probe and a plurality of second polynucleotides are in solution; (c) hybridizing the first target nucleic acid to a first capture probe to obtain hybridized first beads and hybridizing the second target nucleic acid to a second capture probe to obtain hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes; and (f) extending the first and second capture probes to obtain nucleic acid sequence data for the first and second target nucleic acids.

[0007] In some embodiments, the first plurality of polynucleotides comprises a first index and an index primer binding site adjacent to the first index, and the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index.

[0008] In some embodiments, the first or second plurality of polynucleotides is obtained by tagging a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprises a first or second index. Some embodiments also include adding an adapter to the tagged nucleic acid sample, wherein the adapter comprises the first or second index. Some embodiments also include amplifying the tagged nucleic acid sample with primers comprising the first or second index.

[0009] In some embodiments, extending the first and second capture probes incorporates the first and second indexes, and sequences complementary to the first and second index primer binding sites, into the extended capture probes.

[0010] In some embodiments, a first population of beads comprises a first index and an index primer binding site adjacent to the first index, and a second population of beads comprises a second index and an index primer binding site adjacent to the second index. In some embodiments, the oligonucleotides of the first population of beads comprise a first index and the oligonucleotides of the second population of beads comprise a second index. In some embodiments, the first index indicates a source of a first target nucleic acid and the second index indicates a source of a second target nucleic acid. In some embodiments, the first indices are the same as each other and the second indices are the same as each other.

[0011] Some embodiments also include sequencing the first and second indexes. In some embodiments, sequencing the first and second indexes includes extending a primer hybridized to the index primer binding site. In some embodiments, the index-binding primer sites (index primer binding sites) are the same.

[0012] In some embodiments, the first and second target nucleic acids are obtained from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are obtained from genomic DNA.

[0013] In some embodiments, the first and second barcodes represent the nucleic acid sequence of the first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes comprises extending a primer hybridized to the barcode primer binding site. In some embodiments, the barcode primer binding sites are the same.

[0014] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, extending the first and second capture probes comprises adding a single nucleotide to the capture probe. Some embodiments also comprise ligating a locus-specific oligonucleotide to the extended capture probe. In some embodiments, extending the first and second capture probes comprises ligating a locus-specific oligonucleotide to the capture probe.

[0015] In some embodiments, step (c) is carried out in solution.

[0016] In some embodiments, the array is located on the surface of a flow cell. In some embodiments, the first and second beads are adapted to be attached to the array. In some embodiments, the first and second beads comprise biotin, streptavidin, or derivatives thereof, and the array comprises biotin, streptavidin, or derivatives thereof. In some embodiments, the first and second beads are magnetic.

[0017] Some embodiments provide a method for sequencing a target nucleic acid on an array, comprising: (a) obtaining first and second populations of beads, wherein the first population of beads comprises an oligonucleotide comprising a first capture probe, a first barcode, and a barcode primer binding site adjacent to the first barcode, and the second population of beads comprises an oligonucleotide comprising a second capture probe, a second barcode, and a barcode primer binding site adjacent to the second barcode; and (b) obtaining first and second pluralities of polynucleotides, wherein the first pluralities of polynucleotides comprise a first target nucleic acid, a first index, and an index primer binding site adjacent to the first index, the plurality of first polynucleotides being in solution, and the second pluralities of polynucleotides comprise a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index. (c) hybridizing a first target nucleic acid to the first capture probe to obtain hybridized first beads and hybridizing a second target nucleic acid to the second capture probe to obtain hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding positions of the first and second beads on the array by sequencing the first and second barcodes; (f) extending the first and second capture probes to obtain nucleic acid sequence data for the first and second target nucleic acids; and (g) determining a source of the nucleic acid sequence data for the first and second target nucleic acids by sequencing the first and second indexes.

[0018] In some embodiments, the first plurality of polynucleotides comprises a first index and an index primer binding site adjacent to the first index, and the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index.

[0019] In some embodiments, the first or second plurality of polynucleotides is obtained by tagging a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprises a first or second index. Some embodiments also include adding an adapter to the tagged nucleic acid sample, wherein the adapter comprises the first or second index. Some embodiments also include amplifying the tagged nucleic acid sample with primers comprising the first or second index.

[0020] In some embodiments, extending the first and second capture probes incorporates the first and second indexes, and sequences complementary to the first and second index primer binding sites, into the extended capture probes.

[0021] Some embodiments also include a method of sequencing a target nucleic acid on an array, comprising: (a) obtaining first and second populations of beads, wherein the first population of beads comprises oligonucleotides comprising a first capture probe, a first barcode, a barcode primer binding site adjacent to the first barcode, a first index, and an index primer binding site adjacent to the first index; and the second population of beads comprises oligonucleotides comprising a second capture probe, a second barcode, a barcode primer binding site adjacent to the second barcode, a second index, and an index primer binding site adjacent to the second index; and (b) obtaining first and second pluralities of polynucleotides, wherein the first pluralities of polynucleotides comprise a first target nucleic acid; The method includes: (a) a plurality of first polynucleotides in a solution; (b) a second plurality of polynucleotides comprising a second target nucleic acid; and (c) hybridizing the first target nucleic acid to a first capture probe to obtain hybridized first beads and hybridizing the second target nucleic acid to a second capture probe to obtain hybridized second beads; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes; and (f) extending the first and second capture probes to obtain nucleic acid sequence data of the first and second target nucleic acids.

[0022] In some embodiments, the oligonucleotides of the first population of beads comprise a first index and the oligonucleotides of the second population of beads comprise a second index. In some embodiments, the first index indicates a source of the first target nucleic acid and the second index indicates a source of the second target nucleic acid. In some embodiments, the first indexes are the same as each other and the second indexes are the same as each other.

[0023] Some embodiments also include sequencing the first and second indexes. In some embodiments, sequencing the first and second indexes includes extending a primer hybridized to the index primer binding site. In some embodiments, the index binding primer sites are the same.

[0024] In some embodiments, the first and second target nucleic acids are obtained from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are obtained from genomic DNA.

[0025] In some embodiments, the first and second barcodes represent the nucleic acid sequence of the first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes comprises extending a primer hybridized to the barcode primer binding site. In some embodiments, the barcode primer binding sites are the same.

[0026] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, extending the first and second capture probes comprises the addition of a single nucleotide to the capture probe. Some embodiments also comprise ligating a locus-specific oligonucleotide to the extended capture probe. In some embodiments, extending the first and second capture probes comprises ligating a locus-specific oligonucleotide to the capture probe.

[0027] In some embodiments, step (c) is carried out in solution.

[0028] In some embodiments, the flow cell comprises an array. In some embodiments, the array comprises a plurality of wells. In some embodiments, the first and second beads are adapted to be attached to the array. In some embodiments, the first and second beads comprise biotin, streptavidin, or derivatives thereof, and the array comprises biotin, streptavidin, or derivatives thereof. In some embodiments, the first and second beads are magnetic.

[0029] Some embodiments include a kit comprising multiple populations of beads comprising oligonucleotides attached to the beads, the oligonucleotides comprising an index, an index primer binding site adjacent to the index, a capture probe, a barcode, and a barcode primer binding site adjacent to the barcode, wherein the index varies among the populations of beads. In some embodiments, the index primer binding site is the same in the multiple populations. In some embodiments, the barcode indicates the nucleic acid sequence of the capture probe. In some embodiments, the barcode varies among the populations of beads. In some embodiments, the barcode primer binding site is the same in the multiple populations. Some embodiments also include a reagent selected from a locus-specific oligonucleotide, a transposome for tagging a nucleic acid sample, a transposome comprising an index and an index primer binding site, an adapter comprising an index and an index primer binding site, a primer capable of hybridizing to the index primer binding site or its complement, and a primer capable of hybridizing to the barcode primer binding site or its complement. Some embodiments also include a flow cell.

[0030] Some embodiments include a method of preparing a population of indexed beads, the method comprising: (a) obtaining a population of beads, each bead comprising an adapter, a capture probe, and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining a plurality of index polynucleotides, each index polynucleotide comprising an index and an index primer binding site; and (c) attaching the plurality of index polynucleotides to the population of beads via the adapters, thereby obtaining the population of indexed beads.

[0031] In some embodiments, (c) comprises extending the adapter by polymerase extension.

[0032] In some embodiments, each index polynucleotide comprises an adapter binding site, and attaching comprises hybridizing the adapter binding site to the adapter.

[0033] In some embodiments, (c) comprises ligating the index polynucleotide to an adaptor.

[0034] In some embodiments, the attaching comprises hybridizing the splint polynucleotide to the adaptor and index polynucleotide.

[0035] In some embodiments, (c) comprises attaching the plurality of index polynucleotides to adaptors of the population of beads via chemically reactive moieties. In some embodiments, the attaching comprises a click chemistry reaction.

[0036] In some embodiments, the first polynucleotides of the population of beads comprise capture probes that differ from each other.

[0037] In some embodiments, the index of each index polynucleotide is the same.

[0038] In some embodiments, the first polynucleotide comprises a capture probe.

[0039] Some embodiments also include contacting the population of indexed beads with a plurality of nucleic acids, including the target nucleic acid.

[0040] Some embodiments also include mixing the population of indexed beads contacted with a plurality of nucleic acids, including the target nucleic acid, with an additional population of indexed beads, the additional population of indexed beads comprising index polynucleotides that comprise an index that differs from the index of the population of indexed beads contacted with the plurality of nucleic acids.

[0041] In some embodiments, the capture probe comprises a protein.

[0042] In some embodiments, the method is carried out on a flow cell.

[0043] Some embodiments include a method of detecting a target ligand, the method including: (a) obtaining a population of beads, each bead comprising a capture probe and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adaptor capable of binding to the barcode primer binding site; (c) specifically binding the target ligand to the capture probe; (d) hybridizing the index polynucleotide to the first polynucleotide via the adaptor; (e) detecting the target ligand on the array; and (f) determining the index and barcode of the first polynucleotide.

[0044] In some embodiments, (e) comprises distributing the population of beads onto an array.

[0045] In some embodiments, (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index.

[0046] In some embodiments, the method includes dehybridizing the index polynucleotide from the first polynucleotide, hybridizing a barcode primer to the barcode primer binding site, and extending the barcode primer to determine the sequence of the barcode.

[0047] In some embodiments, the index polynucleotide further comprises a cleavable linker positioned between the adapter and the index, and (f) comprises (i) cleaving the cleavable linker and (ii) extending the adapter to determine the sequence of the barcode.

[0048] In some embodiments, the capture probe comprises a protein.

[0049] In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, the extension comprises adding a detectable dideoxynucleotide.

[0050] In some embodiments, the method is carried out on a flow cell.

[0051] Some embodiments include a method of detecting a target ligand, the method comprising: (a) obtaining a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode and a barcode primer binding site, and a second polynucleotide; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adapter; (c) specifically binding the target ligand to the capture probe; (d) attaching the index polynucleotide to the second polynucleotide via the adapter; (e) detecting the target ligand on the array; and (f) determining the index and barcode of the first polynucleotide.

[0052] In some embodiments, the second polynucleotide comprises a barcode and a barcode primer binding site.

[0053] In some embodiments, (d) comprises adding a reactive moiety to the second polynucleotide, wherein the adaptor can be attached to the reactive moiety. In some embodiments, adding the reactive moiety comprises a click chemistry reaction.

[0054] In some embodiments, (e) comprises distributing the population of beads onto an array.

[0055] In some embodiments, (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index.

[0056] In some embodiments, (f) comprises hybridizing a barcode primer to the barcode primer binding site and determining the sequence of the index.

[0057] In some embodiments, the capture probe comprises a protein.

[0058] In some embodiments, the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, the extension comprises adding a detectable dideoxynucleotide.

[0059] In some embodiments, the method is carried out on a flow cell.

[0060] Some embodiments provide a method for detecting a target ligand on an array, comprising: (a) obtaining first and second populations of beads, each bead comprising a capture probe capable of specifically binding to a target ligand, a nucleic acid encoding a barcode and a barcode primer binding site, where the barcode indicates the capture probe, and a nucleic acid encoding an index and an index primer binding site, where the index indicates the source of the bead from the first population or the second population; and (b) contacting the first population of beads with a first sample comprising a first target ligand, where the first target ligand specifically binds to the capture probe of the first population of beads and (c) contacting the second population of beads with a second sample containing a second target ligand, whereby the second target ligand specifically binds to the capture probes of the second population of beads, thereby obtaining a second population of target-bound beads; (d) randomly distributing the first population of target-bound beads and the second population of target-bound beads onto an array; (e) detecting the positions of the beads containing the first target ligand and the second target ligand on the array; and (f) determining the index and barcode sequences of the beads containing the first target ligand and the second target ligand on the array.

[0061] In some embodiments, the capture probe comprises a polynucleotide. In some embodiments, the target ligand comprises a nucleic acid. In some embodiments, detecting the location of the bead comprising the first target ligand and the second target ligand on the array comprises extending the capture probe by polymerase extension or ligation.

[0062] In some embodiments, the capture probe comprises a protein.

[0063] In some embodiments, step (e) is performed after step (f).

[0064] In some embodiments, the barcodes of the first population of beads comprise barcodes that are different from one another, and the barcodes of the second population of beads comprise barcodes that are different from one another.

[0065] In some embodiments, the indices of the first population of beads are the same as each other and the indices of the second population of beads are the same as each other.

[0066] In some embodiments, the array is located on a surface of a flow cell. In some embodiments, the first and second populations of beads are adapted to be attached to the array. In some embodiments, the first and second populations of beads comprise biotin, streptavidin, or derivatives thereof, and the array comprises biotin, streptavidin, or derivatives thereof. In some embodiments, the first and second populations of beads are magnetic. [Brief explanation of the drawings]

[0067] [Figure 1] 1 shows an exemplary embodiment of a polynucleotide comprising a barcode, a primer binding site, and a capture probe attached to a bead via a 5′ linker.

[0068] [Figure 2]1 shows an exemplary embodiment of a polynucleotide comprising a capture probe, a barcode, and a primer binding site, attached to a bead via a 5′ linker and with a cleavable linker between the capture probe and the barcode.

[0069] [Figure 3] 1 shows an exemplary embodiment of a polynucleotide comprising a barcode, a primer binding site, and a capture probe attached to a bead via a 3′ linker.

[0070] [Figure 4] 1 shows an exemplary embodiment of a polynucleotide comprising a spacer, a barcode, a primer binding site, and a capture probe attached to a bead via a 5′ linker.

[0071] [Figure 5A] 1 shows an exemplary embodiment of a bead, wherein an attached first polynucleotide comprises a barcode, a barcode primer binding site, and a capture probe, and an attached second polynucleotide comprises an index and an index primer binding site.

[0072] [Figure 5B] 1 shows an exemplary embodiment of a target nucleic acid containing a single nucleotide polymorphism (SNP) hybridized to a capture probe attached to a bead.

[0073] [Figure 5C] 1 shows an exemplary embodiment of a capture probe extended with a detectable marker.

[0074] [Figure 5D] 1 shows an exemplary embodiment of a barcode primer hybridized to a barcode primer binding site and extension of the barcode primer.

[0075] [Figure 5E]1 shows an exemplary embodiment of an index primer hybridized to an index primer binding site and extension of the index primer.

[0076] [Figure 6A] Exemplary embodiments are shown of beads comprising a single polynucleotide comprising a capture probe (left panel), beads comprising a polynucleotide comprising a nucleic acid capture probe, a polynucleotide comprising a barcode indicative of the capture probe, and an index for distinguishing one subpopulation of beads from another subpopulation of beads (center panel), and beads comprising a capture probe comprising an antibody or antigen-binding fragment of an antibody, a polynucleotide comprising a barcode indicative of the capture probe, and a polynucleotide comprising an index for distinguishing one subpopulation of beads from another subpopulation of beads (right panel).

[0077] [Figure 6B] The following exemplary embodiments are shown: a bead comprising first and second capture probes hybridized to a target nucleic acid, where the first capture probe comprises a cleavable linker (left panel), and a bead comprising a protein capture probe that transiently binds to a substrate and generates a signal comprising a detectable label (right panel).

[0078] [Figure 6C] An exemplary embodiment of a dual-probe assay is shown in which a target nucleic acid is hybridized to a first and second capture probe on a bead, the first capture probe is ligated to the second capture probe, and the cleavable linker is cleaved to generate a bead containing an extended first capture probe that includes a detectable label.

[0079] [Figure 7A] Photograph of a pool of beads immobilized on a flow cell.

[0080] [Figure 7B] 1 is a bar graph of the number of replicates for a particular bead type within a particular bin.

[0081] [Figure 7C] 1 is a graph of mean C intensity versus mean T intensity, where mean C intensity is the intensity of fluorescence from labeled cytosines in a pair of nucleotides, and mean T intensity is the intensity of fluorescence from labeled thymidines in a pair of nucleotides.

[0082] [Figure 8] 10 is a histogram of the number of specific bead types within a particular bin for a representative sample.

[0083] [Figure 9-1] 1 illustrates an embodiment of a workflow for sequencing a target nucleic acid on an array, in which the polynucleotide comprising the target nucleic acid also comprises an index. [Figure 9-2] 1 illustrates an embodiment of a workflow for sequencing a target nucleic acid on an array, in which the polynucleotide comprising the target nucleic acid also comprises an index.

[0084] [Figure 10] 1 illustrates an embodiment of a workflow for sequencing target nucleic acids on an array in which beads contain indexes.

[0085] [Figure 11] 1 illustrates an embodiment of a workflow for preparing a population of indexed beads and the use of indexed beads in a multi-well plate, with each well containing a different sample.

[0086] [Figure 12A] 1 is a schematic diagram of a bead having a first and second polynucleotide attached thereto. The first polynucleotide comprises a code, such as a barcode, a primer A, such as a barcode primer binding site, and a probe, such as a capture probe. The second polynucleotide comprises an index. The second polynucleotide is attached to the bead via a spacer and a linker XY.

[0087] [Figure 12B] 1 is a schematic diagram of a bead having a first and second polynucleotide attached thereto. The first polynucleotide comprises a code, such as a barcode, a primer A, such as a barcode primer binding site, and a probe, such as a capture probe. The second polynucleotide comprises a primer B, such as an index and an index primer binding site. The second polynucleotide is attached to the bead via a splint hybridized to the second polynucleotide and an adapter. The second polynucleotide can be ligated to the adapter.

[0088] [Figure 12C] 1 is a schematic diagram of a bead having a first and second polynucleotide attached thereto. The first polynucleotide comprises a code, such as a barcode, a primer A, such as a barcode primer binding site, and a probe, such as a capture probe. The second polynucleotide comprises an index, a primer B, such as an index primer binding site, and an adapter binding site. The second polynucleotide is attached to the bead via hybridization to an adapter attached to the bead. The adapter can be extended to incorporate the complement of the index and primer B.

[0089] [Figure 13A] 1 is a schematic diagram of a bead having a first polynucleotide attached thereto, the first polynucleotide comprising a code such as a barcode, a primer such as a barcode primer binding site, and a probe such as a capture probe. The second polynucleotide is shown comprising an index, a primer 2 such as an index primer binding site, a spacer which may include an optional cleavable site such as a uridine cleavage site, and an adapter hybridized to the barcode primer binding site.

[0090] [Figure 13B]1 illustrates an embodiment of a workflow for detecting target nucleic acids on an array in which index polynucleotides hybridize to first polynucleotides attached to beads.

[0091] [Figure 14] 1 is a schematic diagram of a bead having first and second polynucleotides attached thereto. The first polynucleotide comprises a code, such as a barcode, a primer A, such as a barcode primer binding site, and a probe, such as a capture probe. The second polynucleotide comprises an index and repeats of the index primer binding site.

[0092] [Figure 15A] 1 illustrates an embodiment of a workflow for detecting target nucleic acids on an array in which index polynucleotides are attached to beads via reactive groups.

[0093] [Figure 15B] 15B illustrates an embodiment of a workflow for detecting target nucleic acids on an array, and is a continuation of the workflow of FIG. 15A. DETAILED DESCRIPTION OF THE INVENTION

[0094] Some embodiments provided herein include methods and compositions for detecting target ligands on an array. In some embodiments, the target ligand can include a nucleic acid, a protein, or another antigen. In some embodiments, a capture probe specifically binds to a target ligand from a sample, the position of a bead containing the capture probe is determined within the array, and the bead is decoded to identify the capture probe and the sample. In some embodiments, a barcode indicates the capture probe attached to the bead, and an index indicates a subpopulation of beads. In some embodiments, the barcode and index are determined by sequencing. Some embodiments also include dual-probe assays in which first and second capture probes attached to beads are ligated together in the presence of a target nucleic acid, the end of the ligation product is cleaved from the bead, and the bead contains an extended capture probe that is detected and decoded on the array.

[0095] Some embodiments provided herein relate to high-throughput genotyping on arrays. Some embodiments relate to decoding the location of microfeatures within an array. In some embodiments, the microfeatures comprise polynucleotides having barcodes and indexes. Some embodiments include sequencing the barcodes and indexes to identify the location of the polynucleotides within the array. Certain aspects that may be useful for the methods and compositions disclosed herein are disclosed in International Publication No. WO 2020 / 086746, which is incorporated by reference in its entirety.

[0096] Decoding by hybridization involves identifying the location of a capture probe within a randomly distributed array of capture probes. This method typically involves several successive cycles of hybridizing a labeled hybridization probe to one or more portions of the capture probe, imaging the hybridization event, and removing the hybridization probe. Decoding by hybridization requires specialized reagents, specialized fluidic devices, and specialized detectors. In some embodiments, decoding by hybridization can take up to 8 hours with 7-8 successive cycles.

[0097] Embodiments provided herein include randomly distributed arrays of polynucleotides that include primer binding sites and barcodes. In some embodiments, the barcodes can be easily sequenced to decode the array using high-throughput sequencing systems. Some embodiments can significantly reduce the time it takes to decode the array without additional reagents, hybridization probes, or specialized decoding equipment.

[0098] Some embodiments involve the use of next-generation sequencing (NGS) technology and bead-based microarrays, resulting in high-performance, low-cost, and high-throughput genotyping assays that can be run on common NGS sequencing platforms with minor modifications to the substrate and reagents.

[0099] Some multiplexing methods allow for parallel processing of multiple nucleic acid samples from different sources by maintaining physical separation of each sample. Some embodiments provided herein include nucleic acid indexing methods that eliminate the need for physical barriers to separate individual samples at every step by indexing each sample via an index on an associated bead. In some embodiments, index demultiplexing (demultiplexing) is performed by sequencing and can be performed at the user's site using standard sequencing-by-synthesis (SBS) chemistry. Additionally, adopting a decode-by-sequencing (DBS) approach, which can be implemented at the customer's site using standard platforms and SBS chemistry, reduces the cost, space, and time limitations associated with internal decoding of bead arrays.

[0100] Some embodiments include an indexed enrichment bead pool comprising the beads shown in Figure 5 A. In some such embodiments, the beads comprise a first polynucleotide comprising a locus-specific capture probe, a barcode for positional identification of the associated probe on the array, and a barcode primer binding site for SBS reading of the barcode, and a second polynucleotide comprising an index for sample multiplexing and an index primer binding site for SBS reading of the index.

[0101] In some embodiments, bead pool complexity is defined by the number of capture probes, or plexity (N), and by the number of samples supported (S). Thus, a bead pool with plexity N and supporting S samples consists of S x N unique bead types. In some embodiments, the capture probes and / or index primers contain an additional 3' orthogonal blocker to avoid interference during SBS with one of the two oligonucleotides.

[0102] Some embodiments involve performing a genotyping assay in which S bead pools are loaded into a multiwell plate containing S wells, each bead pool having a unique sample index and each well containing N unique bead types. After generating a nucleic acid library from the sample, such as by processing the nucleic acid sample through steps including random primer amplification, subsequent enzymatic fragmentation, and cleanup, each sample library is added to an indexed well and hybridized to a capture probe. After hybridization is complete, a single-base extension assay is performed to probe for the SNPs of interest by adding an incorporation mix containing fluorescent nucleotides and an appropriate polymerase. At the end of this incorporation, all bead-captured samples in the plate are pooled and loaded into a flow cell. The flow cell can be plain or patterned, and the surface can be appropriately modified to support bead immobilization at a desired density. In some embodiments, upon bead immobilization, an SNP readout is performed, which includes one scanning cycle to read signals from fluorescent incorporation at the SNP sites. This cycle may include a SBS cycle on the instrument. A barcode readout, involving 12-20 SBS cycles, depending on the bead pool complexity, is also performed to identify the capture probe and location of a specific bead within the flow cell. In some embodiments, this step can be replaced by an additional cycle of sequencing past the identified SNPs. A sample index readout, involving 6-12 SBS cycles to read the sample index, is also performed. In some embodiments, the entire assay on the flow cell can involve less than about 30 SBS cycles and can be performed in less than 4 hours.

[0103] Some embodiments relate to methods and compositions for sequencing target polynucleotides on an array. Some embodiments relate to sequencing target polynucleotides from several different nucleic acid samples on a bead array. In some such embodiments, index sequences are associated with target nucleic acids from the nucleic acid samples.

[0104] In one embodiment of the present invention, gaskets are not used to subdivide various regions of the gene array. Instead, index sequences are added to the samples to distinguish samples hybridized to the beads and support high-plexity sample pools. In some embodiments, the index can be added to either the bead pool or the sample.

[0105] Embodiments relate to preparing polynucleotide libraries from many different nucleic acid samples and determining the presence of a particular feature, such as a single nucleotide polymorphism, insertion, deletion, etc., in the target nucleic acid of each nucleic acid sample. The polynucleotide libraries are interrogated in parallel on an array for the presence of the particular feature in the target nucleic acid.

[0106] In some embodiments, each nucleic acid sample is associated with a different index sequence such that a library of polynucleotides derived from the nucleic acid sample contains the same index. Target nucleic acids are identified in the library of polynucleotides by selectively hybridizing the target nucleic acid to a capture probe attached to a bead, extending the capture probe, and detecting the extension of the capture probe on the array. Each capture probe can include a barcode, and thus the capture probe can be identified by sequencing the barcode associated with the capture probe on the array. In some such embodiments, the oligonucleotide attached to the bead includes a capture probe and a barcode.

[0107] In some embodiments, the indexes associated with the polynucleotides can be sequenced on the array. The position of the signal on the array for capture probe extension, the sequence of the barcode, and the sequence of the index can identify the presence of a particular feature, such as a single nucleotide polymorphism, insertion, deletion, etc., in the target nucleic acid from a particular nucleic acid sample. In some embodiments, many different nucleic acid samples can be tested on a single array.

[0108] As used herein, "array" can refer to a collection of different microfeatures, such as microfeatures containing polynucleotides bound or attached to a surface such that different microfeatures can be distinguished from one another according to their relative positions. An individual feature of the array can contain a single copy of a microfeature, or multiple copies of a microfeature can be present in the population of microfeatures in an individual feature of the array. The population of microfeatures in each feature is typically homogeneous, having a single type of microfeature. Thus, multiple copies of a single nucleic acid sequence can be present in a feature, for example, on multiple nucleic acid molecules having the same sequence.

[0109] In some embodiments, a heterogeneous population of microfeatures can be present in a single feature. Thus, a feature may, but need not, include only a single microfeature type; instead, it can contain multiple different microfeature types, such as a mixture of nucleic acids with different sequences. Adjacent features of an array can be distinct from one another in that they do not overlap. Thus, features can be adjacent to one another or separated by a gap. In embodiments in which features are spaced apart, adjacent sites can be separated by, for example, a distance of less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, 1 pm, or any distance within a range of any two of the aforementioned distances. The layout of features on an array can also be understood in terms of the center-to-center distance between adjacent features. Arrays useful in the present invention can have adjacent features with a center-to-center spacing of less than about 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, 1 pm, or any distance within a range of any two of the aforementioned distances.

[0110] In some embodiments, the distance values ​​described above and elsewhere herein can represent the average distance between adjacent features of the array. Thus, unless specifically indicated otherwise, for example, by a specific statement that a distance constitutes a threshold distance between all adjacent features of the array, it is not necessary that all adjacent features be in the specified range. Embodiments can be used with arrays having features at any of a variety of densities. An exemplary range of density for certain embodiments is about 10,000,000 features / cm. 2 ~approximately 2,000,000 features / cm 2 Approximately 100,000,000 features / cm 2 ~approximately 1,000,000,000 features / cm 2 Approximately 100,000 features / cm 2~approximately 10,000,000 features / cm 2 Approximately 1,000,000 features / cm 2 ~approximately 5,000,000 features / cm 2 Approximately 10,000 features / cm 2 ~100,000 features / cm 2 Approximately 20,000 features / cm 2 ~approximately 50,000 features / cm 2 Approximately 1,000 features / cm 2 ~approximately 5,000 features / cm 2 , or any density within a range between any two of the aforementioned densities.

[0111] As used herein, a "surface" can refer to a portion of a substrate or support structure that is accessible for contact with a reagent, bead, or analyte. A surface can be substantially flat or planar. Alternatively, a surface can be rounded or contoured. Exemplary contours that can be included on a surface are wells, depressions, posts, ridges, channels, and the like. Exemplary materials that can be used as a substrate or support structure include glass, such as modified or functionalized glass; plastics, e.g., acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON®; polysaccharides or cross-linked polysaccharides, e.g., agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metals; inorganic glasses; fiber optic bundles; or various other polymers. A single material or a mixture of several different materials can form a surface useful in the present invention. In some embodiments, the surface comprises a well. In some embodiments, the support structure can comprise one or more layers. Exemplary support structures can include chips, films, multi-well plates, and flow cells.

[0112] As used herein, "beads" can refer to small objects made of rigid or semi-rigid materials. The objects can have shapes characterized as, for example, spheres, ellipsoids, microspheres, or other recognized particle shapes, whether they have regular or irregular dimensions. Exemplary materials useful for beads include glass, such as modified or functionalized glass; plastics, such as acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON®; polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metals; inorganic glasses; or various other polymers. Exemplary beads include controlled pore glass beads, paramagnetic beads, thoria sol, Sepharose beads, nanocrystals, and others known in the art. Beads can be made of biological or non-biological materials. Magnetic beads are particularly useful due to the ease of manipulation of the magnetic beads using magnets in the various steps of the methods described herein. Beads used in certain embodiments can have a diameter, width, or length of about 0.1 μm to about 100 μm, or about 0.1 nm to about 500 nm. In some embodiments, beads used in certain embodiments can have a diameter, width, or length of less than about 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, 0.5 nm, 100 pm, 50 pm, or 1 pm, or any diameter, width, or length within a range of any two of the aforementioned diameters, widths, or lengths. Bead size can be selected to have a reduced size while maintaining sufficient signal (number of template copies per feature) to analyze features, thereby allowing for more features to be obtained per unit area.

[0113] In some embodiments, the polynucleotides can be attached to beads. In some embodiments, the beads can be distributed within wells on the surface of a substrate. Exemplary bead arrays that can be used in certain embodiments include randomly ordered bead array technology (Illumina Inc., San Diego CA). Such bead arrays have been described by Michael et al., Anal Chem 70, 1242-8 (1998); Walt, Science 287, 451-2 (2000); Fan et al., Cold Spring Harb Symp Quant Biol 68:69-78 (2003); Gunderson et al., Nat Genet 37:549-54 (2005); Bibikova et al., Am J Pathol 165:1799-807 (2004); Fan et al., Genome Res 14:878-85 (2004); Kuhn et al., Genome Res 14:2347-56 (2004); Yeakley et al., Nat Biotechnol 20:353-8 (2002); and Bibikova et al., Genome Res 16:383-93 (2006), each of which is incorporated by reference in its entirety.

[0114] As used herein, "polynucleotide" and "nucleic acid" can be used interchangeably and can refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes single-, double-, or multi-stranded DNA or RNA. The term "polynucleotide" also refers to both double- and single-stranded molecules. Examples of polynucleotides include genes or gene fragments, genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, non-coding RNA (ncRNA) (such as Piwi-binding RNA (piRNA), small interfering RNA (siRNA), and long non-coding RNA (lncRNA)), short hairpin (shRNA), small nuclear RNA (snRNA), microRNA (miRNA), small nucleolar RNA (snoRNA) and viral RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the above. Polynucleotides can contain modified nucleotides, such as methylated nucleotides, and nucleotide analogs, including nucleotides with unnatural bases and nucleotides with modified natural bases, such as azapurines or deazapurines. A polynucleotide can be composed of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Uracil (U) can also be present as a natural substitute for thymine, for example, when the polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term "sequence" refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and unnatural bases.

[0115] As used herein, "target nucleic acid" or its grammatical equivalents can refer to a nucleic acid molecule or sequence that is desired to be sequenced, analyzed, and / or further manipulated. In some embodiments, the target nucleic acid can be attached to an array. In some embodiments, capture probes can be attached to the array, which can then be used to detect target nucleic acids in a sample that interact with the probes. In this regard, it will be understood that in some embodiments, the terms "target" and "probe" can be used interchangeably with respect to nucleic acid detection methods.

[0116] As used herein, a "capture probe" can refer to a polynucleotide that has sufficient complementarity to specifically hybridize to a target nucleic acid. A capture probe can function as an affinity binding molecule to isolate a target nucleic acid from other nucleic acids and / or components in a mixture. In some embodiments, a target nucleic acid can be specifically bound by a capture probe via an intervening molecule. Examples of intervening molecules include linkers, adapters, and other bridging nucleic acids that have sufficient complementarity to specifically hybridize to both the target sequence and the capture probe.

[0117] As used herein, "hybridization," "hybridize," or grammatical equivalents thereof, can refer to a reaction in which one or more polynucleotides react to form a complex formed, at least in part, by hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific method. The complex can have two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. Strands can also be cross-linked or bonded by forces in addition to hydrogen bonding.

[0118] As used herein, "extending," "extension," or any grammatical equivalent thereof, can refer to the addition of dNTPs to a primer, polynucleotide, or other nucleic acid molecule by an extension enzyme, such as a polymerase. For example, in some methods disclosed herein, the resulting extended primer contains RNA sequence information. Although some embodiments are discussed as performing extension using a polymerase, such as a DNA polymerase, or a reverse transcriptase, extension can be performed by any other method known in the art. For example, extension can be performed by ligating together short fragments of random oligonucleotides, such as oligonucleotides hybridized to the strand of interest.

[0119] As used herein, "ligation" or "ligate" or other grammatical equivalents can refer to the joining of two nucleotide strands by a phosphodiester bond. Such a reaction can be catalyzed by a ligase. Ligases refer to a class of enzymes that catalyze this reaction with the hydrolysis of ATP or a similar triphosphate. Decoding by sequencing

[0120] Some embodiments of the methods and compositions provided herein include using high-throughput sequencing to decode the locations of microfeatures of an array. In some embodiments, the microfeatures of the array comprise polynucleotides. In some embodiments, the polynucleotides can be randomly distributed on the surface of the substrate. In some embodiments, the polynucleotides can comprise a primer binding site and a barcode. In some embodiments, the polynucleotides can comprise a capture probe, a primer binding site, and a barcode.

[0121] Some embodiments of decoding the position of a polynucleotide within an array can include (a) obtaining a substrate having an array of polynucleotides distributed on its surface, each polynucleotide comprising a barcode and a primer binding site; (b) hybridizing a plurality of primers to the primer binding sites; and (c) determining the sequence of the barcode by extending the hybridized primers. In some such embodiments, the sequence of each barcode can indicate the position of the polynucleotide within the array. For example, in some embodiments, an array can be prepared with polynucleotides whose barcodes are known to be associated with specific capture probes, such that identifying the position of a barcode on the array can indicate the position of the associated capture probe. In such embodiments, each polynucleotide can be associated with the capture probe via a common element. For example, the polynucleotide and the capture probe can each be bound to the same microfeature, such as a bead. In more such embodiments, each polynucleotide can include a capture probe.

[0122] In some embodiments, a barcode can comprise a nucleic acid sequence that can be used to identify a polynucleotide within an array. A barcode can comprise a unique nucleotide sequence that can be distinguished from other barcodes. It can also be distinguished from other nucleotide sequences within a polynucleotide and a target nucleic acid by the sequence of the barcode and also by its location within the polynucleotide, e.g., its location 5' of a primer binding site. For example, in some embodiments, a barcode sequence can occur more than once within a plurality of nucleic acids, but a barcode located 5' of a primer binding site can be detected. Barcodes can be of any desired sequence length sufficient to be unique nucleotide sequences within the plurality of barcodes within a population and / or within the plurality of polynucleotides and target nucleic acids being analyzed or studied. In some embodiments, a barcode is a nucleic acid or region within a polynucleotide ranging from about 6 to 30 nucleotides. Barcodes can be, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or longer. For example, barcodes can be 35, 40, 45, or 50 nucleotides or longer. Suitable barcodes for some embodiments are disclosed in U.S. Patent No. 8,053,192, which is incorporated by reference in its entirety. In some embodiments, barcodes can distinguish one polynucleotide from another polynucleotide in an array, such that each barcode is different from another barcode. In some embodiments, barcodes can distinguish one population of polynucleotides from another population of polynucleotides in an array, such that a set of barcodes is different from another set of barcodes. Some aspects useful in the methods and compositions provided herein are disclosed in U.S. Patent Application Publication Nos. 20180334711(A1) and 20190085384(A1), each of which is incorporated by reference in its entirety. In some embodiments, the barcode can include a unique molecular identifier (UMI).

[0123] In some embodiments, the primer binding site can be 3' of the barcode such that a primer hybridized to the primer binding site can be extended to provide the complement of the barcode. For example, the primer can be extended to obtain the sequence of the barcode. In some embodiments, the primer binding site can be directly adjacent to the barcode within the polynucleotide. In some embodiments, each primer binding site within a population of polynucleotides can have the same sequence. In some embodiments, a subpopulation of polynucleotides can include a primer binding site with a first sequence, and another subpopulation of polynucleotides can include a primer binding site with a second sequence. In some embodiments, hybridizing different primers to multiple different primer binding sites can be done simultaneously, sequentially, or iteratively.

[0124] Some embodiments include polynucleotides comprising capture probes. In some embodiments, the capture probes comprise sequences capable of hybridizing to a target nucleic acid. In some embodiments, the population of polynucleotides comprises capture probes that are different from one another. In some embodiments, each capture probe may be different from another. In some embodiments, the capture probes may be similar to one another, e.g., they may have similar sequences and / or similar lengths. In some embodiments, the capture probes may differ from one another by less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides, which may be consecutive, non-consecutive, inserted, or deleted nucleotides within the capture probe. In some embodiments, a capture probe may differ from another capture probe by a single nucleotide. In some embodiments, the primer binding site and barcode may be 5' of the capture probe. In some embodiments, the primer binding site and barcode may be 3' of the capture probe.

[0125] Some embodiments include a polynucleotide comprising a cleavable linker. In some such embodiments, the cleavable linker can be positioned such that cleavage of the linker separates the capture probe from the primer binding site and barcode. In some embodiments, the cleavable linker can be positioned within the polynucleotide between the capture probe and the primer binding site and barcode. In some embodiments, the cleavable linker can remove the polynucleotide comprising the primer binding site and barcode linked to the capture probe. For example, both the polynucleotide and the capture probe can be bound to a bead. Cleavage of the cleavable linker can remove the polynucleotide comprising the primer binding site and barcode from the bead.

[0126] In some embodiments, the cleavable linker can have a length corresponding to at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 500 nucleotides, or a length within a range of any two of the foregoing lengths. In some embodiments, the cleavable linker is susceptible to cleavage by an agent such as light, base, acid, and / or an enzyme such as a sequence-specific restriction enzyme or protease. The cleavable linker can include a specific sequence of nucleotides, such as a recognition site for an enzyme, and / or can include specific modified nucleotides that are susceptible to cleavage by an agent. In some embodiments, the cleavable linker can include uracil, which is cleavable by an exogenous base-cleaving agent such as DNA glycosylase (UDG). In some embodiments, the cleavable linker can include 8-hydroxyguanine, which can be cleaved by 8-hydroxyguanine DNA glycosylase (FPG protein). Many examples of cleavable linkers are described in U.S. Patent Application Publication No. 2005 / 0181394, incorporated by reference in its entirety.

[0127] In some embodiments, the polynucleotide is attached to a substrate. In some embodiments, the substrate can comprise beads. The polynucleotide can be immobilized to a substrate, such as a bead or other surface, by single-point covalent attachment to the substrate surface at or near the 5' or 3' end of the polynucleotide. In some embodiments, the polynucleotide can comprise a spacer attached to the substrate. In some embodiments, the spacer can have a length corresponding to at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, or 500 nucleotides, or a length within the range of any two of the aforementioned lengths. Any suitable covalent attachment means known in the art can be used for this purpose. The attachment chemistry selected will depend on the nature of the solid support and any derivatization or functionalization applied thereto. The polynucleotide may contain a moiety that facilitates attachment, which may be a non-nucleotide chemical modification. In some embodiments, the polynucleotide can contain a sulfur-containing nucleophile, such as a phosphorothioate or thiophosphate, located at the 5' end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile binds to bromoacetamide groups present in the hydrogel. An exemplary means of attaching polynucleotides to a solid support is via a 5' phosphorothioate linkage to a hydrogel composed of polymerized acrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), which is disclosed in U.S. Pat. No. 8,168,388, which is incorporated by reference in its entirety.

[0128] In some embodiments, the location of a polynucleotide in an array can be decoded by sequencing the polynucleotide's barcode. For example, the sequence of the barcode can be associated with a site on the array, and the site can be associated with a specific capture probe. Some embodiments include next-generation sequencing (NGS), which can refer to a sequencing method that enables massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules. Examples of NGS include sequencing-by-synthesis (SBS) using reversible dye terminators and sequencing-by-ligation. In SBS, the extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization. In certain polymerase-based SBS embodiments, fluorescently labeled nucleotides are added to extend the primer in a template-dependent manner, such that the sequence of the template can be determined using detection of the order and type of nucleotides added to the primer.

[0129] One or more amplified nucleic acids can be subjected to SBS or other detection techniques involving repeated delivery of reagents in cycles. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc. can be flowed through hydrogel beads containing one or more amplified nucleic acid molecules. The site at which the labeled nucleotide is incorporated by primer extension can be detected. Optionally, the nucleotide can further comprise a reversible termination feature that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer to prevent further extension until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell before or after detection. Washing can be performed between the various delivery steps. This cycle is then repeated n times to extend the primer by n nucleotides, thereby allowing a sequence of length n to be detected.

[0130] Some SBS embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use commercially available electronic detectors and related technologies. Examples of such sequencing systems include pyrosequencing, such as the commercially available platform from 454Life Sciences, a subsidiary of Roche; sequencing using γ-phosphite-labeled nucleotides, such as the commercially available platform from Pacific Biosciences; and sequencing using proton detection, such as the commercially available platform from Ion Torrent, a subsidiary of Life Technologies. Some embodiments include pyrosequencing, as described in U.S. Patent Application Publication Nos. 2005 / 0130173 and 2006 / 0134633, and U.S. Patent Nos. 4,971,903, 6,258,568, and 6,210,891, each of which is incorporated by reference in its entirety. Some embodiments include sequencing by ligation as disclosed in US Pat. No. 5,599,675 and US Pat. No. 5,750,341, each of which is incorporated by reference in its entirety.

[0131] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and a γ-phosphate-labeled nucleotide, or using a zero-mode waveguide (ZMW). Another useful sequencing technique is nanopore sequencing. In some nanopore embodiments, a target nucleic acid or individual nucleotides removed from the target nucleic acid pass through a nanopore. As the nucleic acid or nucleotide passes through the nanopore, each nucleotide type can be identified by measuring the fluctuations in the electrical conductance of the pore.

[0132] As shown in FIG. 1 , in some embodiments, a microfeature of an array can comprise a polynucleotide attached to a bead 10 via a 5′ linker 20. The polynucleotide can comprise a barcode 30, a primer binding site 40, and a capture probe 50. A primer 60 hybridizes to the primer binding site and is extended to obtain the sequence of the barcode for decoding the location of the microfeature within the array. In some embodiments, the capture probe can hybridize to a target nucleic acid, and the capture probe can be extended, for example, by a polymerase or by a ligase. In some embodiments, the capture probe can participate in bridge amplification. Methods of bridge amplification are disclosed in U.S. Pat. Nos. 7,985,565 and 7,115,400, each of which is incorporated by reference in its entirety.

[0133] As shown in FIG. 2 , in some embodiments, a microfeature of the array can include a polynucleotide comprising a capture probe 50, a barcode 30, and a primer binding site 40, where the polynucleotide is attached to a bead 10 via a 5′ linker 20. In some embodiments, the polynucleotide can include a cleavable linker 70 between the capture probe and the barcode. In some embodiments, a primer 60 can be hybridized to the primer binding site, and the barcode sequence can be determined, thereby decoding the location of the microfeature within the array. In some embodiments, the polynucleotide can be cleaved, and the barcode and primer binding site removed from the bead and the microfeature comprising the capture probe. Some such embodiments provide a decoded array prior to hybridizing the target nucleic acid with the capture probe. In some embodiments, the target nucleic acid can hybridize to the capture probe at a decoded position within the array. In some embodiments, the hybridized capture probe can be extended, for example, by a polymerase or by a ligase. In some embodiments, the capture probe can participate in bridge amplification.

[0134] As shown in FIG. 3 , in some embodiments, the array microfeatures can include a polynucleotide comprising a barcode 30, a primer binding site 40, and a capture probe 50 attached to a bead 10 via a 3′ linker 25. In some embodiments, the primer binding site can abut the linker attached to the bead. Some embodiments can include the use of such microfeatures in assays to screen and develop specific polymerases. For example, polymerase activity can be screened for primer sites attached to beads without spacers compared to primer sites attached to beads with spacers. In some embodiments, a target nucleic acid can hybridize to the capture probe. In some embodiments, the hybridized target nucleic acid is extended, for example, by a polymerase or by a ligase.

[0135] As shown in FIG. 4, an embodiment of the microfeatures of the array can include a polynucleotide comprising a spacer 80, a barcode 30, a primer binding site 40, and a capture probe 50 attached to a bead 10 via a 5′ linker 20. Sequencing of multiple target nucleic acids

[0136] Some embodiments include sequencing multiple target nucleic acids. In some embodiments, the target nucleic acids can be derived from different sources, such as different subjects, e.g., genomic DNA from different subjects. In some embodiments, different target nucleic acids can be associated with different indexes, such that the indexes can identify particular populations of target nucleic acids, such as a population derived from a single source.

[0137] Some embodiments include obtaining at least first and second subpopulations of beads, each bead comprising a capture probe, a barcode indicative of the capture probe of the same bead, and a first polynucleotide comprising a barcode primer binding site 3' of the barcode, and a second polynucleotide comprising an index and an index primer binding site 3' of the index, wherein the index of the first subpopulation is different from the index of the second subpopulation.

[0138] In some embodiments, the nucleotide sequences of the indexes of a first subpopulation of beads comprise the same nucleotide sequence and the nucleotide sequences of the indexes of a second subpopulation of beads comprise the same nucleotide sequence, hi some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0139] In some embodiments, the capture probes of the first and / or second subpopulations of beads each comprise a different nucleotide sequence from one another. For example, the capture probes of the first subpopulation may be different from one another and / or the capture probes of the first subpopulation may be different from one another. In some embodiments, the capture probes of the first subpopulation of beads each comprise a capture probe having the same nucleotide sequence as the capture probes of the second subpopulation of beads. In some embodiments, the capture probes comprise a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement. In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence.

[0140] Some embodiments also include hybridizing a first target nucleic acid to the capture probes of a first subpopulation of beads and hybridizing a second target nucleic acid to the capture probes of a second subpopulation of beads. In some such embodiments, hybridizing the first target nucleic acid to the capture probes of the first subpopulation of beads and hybridizing the second target nucleic acid to the capture probes of the second subpopulation of beads are performed at different locations. For example, the different locations include different reaction volumes, such as different wells, in a multi-layer plate. For example, in a 96-well plate, 96 different subpopulations of beads can be hybridized with 96 different target nucleic acids, with each different subpopulation of beads hybridizing to a different target nucleic acid in a different well.

[0141] In some embodiments, different subpopulations of beads comprising hybridized capture probes and target nucleic acids are distributed on a substrate, such as a planar substrate. In some embodiments, the distributed subpopulations of beads comprise an array. In some embodiments, the substrate comprises a plurality of distinct sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, a flow cell comprises the substrate.

[0142] In some embodiments, different subpopulations of beads comprising hybridized capture probes and target nucleic acids are combined before being distributed onto the substrate. In some embodiments, different subpopulations of beads comprising hybridized capture probes and target nucleic acids are distributed onto the substrate sequentially. For example, a first subpopulation of beads comprising hybridized capture probes and target nucleic acids is distributed onto the substrate before a second subpopulation of beads comprising hybridized capture probes and target nucleic acids is distributed onto the substrate.

[0143] In some embodiments, the hybridized capture probes are extended. In some embodiments, the hybridized capture probes are extended before different subpopulations of beads comprising hybridized capture probes and target nucleic acids are distributed onto the substrate. In some embodiments, the hybridized capture probes are extended after the beads comprising hybridized capture probes and target nucleic acids are distributed onto the substrate. In some embodiments, extending the hybridized capture probes can comprise polymerase extension. In some embodiments, extending the hybridized capture probes can comprise ligase-based extension, such as ligating an extension probe to a capture probe in the presence of a ligase. In some embodiments, the extension step can add a detectable marker to the extended capture probe. In some embodiments, the detectable marker can comprise a fluorescent marker.

[0144] Some embodiments include decoding beads on the substrate. Some embodiments include decoding beads by identifying the locations of the extended capture probe, barcode, and index on the substrate. In some embodiments, the presence of a particular barcode at a location on the substrate indicates a particular capture probe at that location. In some embodiments, the presence of a particular index at a location on the substrate indicates that a target nucleic acid of a particular subpopulation of target nucleic acids is associated with that location on the substrate. In some embodiments, the presence of an extended capture probe at a location on the substrate indicates the presence of a particular target nucleic acid within the subpopulation of target nucleic acids. In some embodiments, the identity of the barcode, the identity of the index, and the presence of the extended capture probe at a single location on the surface indicates the presence of a particular target nucleic acid within a particular subpopulation of target nucleic acids.

[0145] In some embodiments, decoding the beads on the substrate comprises detecting the position of the hybridized or extended capture probe. In some embodiments, detecting the position of the hybridized or extended capture probe comprises extending the hybridized capture probe with a detectable marker. In some embodiments, detecting the position of the hybridized or extended capture probe comprises at least one cycle of sequencing-by-synthesis.

[0146] In some embodiments, decoding the beads on the substrate comprises decoding the index position of the beads comprising the extended capture probe. Some embodiments comprise hybridizing a plurality of index primers to the index primer sites and extending the hybridized index primers. In some embodiments, extending the hybridized index primers comprises at least one cycle of sequencing-by-synthesis. In some embodiments, decoding the index position of the beads comprises sequencing the index on the substrate.

[0147] In some embodiments, decoding the beads on the substrate comprises decoding the position of the barcodes of the beads comprising the extended capture probes. Some embodiments comprise hybridizing a plurality of barcode primers to the barcode primer sites and extending the hybridized barcode primers. In some embodiments, decoding the beads on the substrate comprises extending the hybridized barcode primers, comprising at least one cycle of sequencing-by-synthesis. In some embodiments, decoding the beads on the substrate comprises decoding the position of the barcodes of the beads, comprising sequencing the barcodes on the substrate.

[0148] In some embodiments, the first and second subpopulations of beads each comprise at least 50 capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 100, 200, 300, 400, or 500 or more capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 5,000 capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 50,000 capture probes comprising different nucleotide sequences.

[0149] Some embodiments include at least 10 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation. Some embodiments include at least 100 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation. Some embodiments include at least 1000 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation. Some embodiments include at least 10,000 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation.

[0150] Aspects of some embodiments are shown in Figures 5A-5E. As shown in Figure 5A, subpopulations of beads include beads having an attached first polynucleotide that includes a barcode, a barcode primer binding site, and a capture probe, and an attached second polynucleotide that includes an index and an index primer binding site. Different subpopulations of beads can include different indexes. Different subpopulations of beads, each subpopulation having a particular index, can be distributed into wells of a 96-well plate such that each well contains a single subpopulation of beads.

[0151] As shown in Figure 5B, a target nucleic acid from a population of target nucleic acids is hybridized to a capture probe, and the target nucleic acid contains a SNP. In some embodiments, a subpopulation of target nucleic acids is added to each well containing a subpopulation of beads. Each subpopulation of target nucleic acids can be derived from a different source, such as a different subject. For example, the subpopulations of target nucleic acids can be obtained by preparing a library of nucleic acids from a single nucleic acid source, such as a single nucleic acid sample from a subject. In some embodiments, the target nucleic acid does not require an adapter to be sequenced.

[0152] As shown in Figure 5C, a capture probe hybridized to a target nucleic acid containing a SNP can be extended. In some embodiments, extension can be performed by adding an incorporation mix containing fluorescent nucleotides and an appropriate polymerase. In some embodiments, extension is a single-base extension.

[0153] In some embodiments, all the beads are combined and distributed onto the surface of a flow cell, which can have a patterned surface, which can be modified to support immobilization of the beads at a desired density.

[0154] In some embodiments, a SNP readout is performed. In some embodiments, a scanning cycle is performed to read the signal from the capture probe incorporation at the SNP site. In some embodiments, this cycle can include at least one cycle of synthetic sequencing. In some embodiments, the 3' end of the extended capture probe is cleaved and blocked.

[0155] As shown in Figure 5D, a barcode primer is hybridized to the barcode primer binding site and the barcode primer is extended. In some embodiments, the extension of the barcode primer includes a barcode readout. In some embodiments, the extension can include at least one cycle of sequencing by synthesis. In some embodiments, the number of sequencing by synthesis cycles can depend on the number of different barcodes in the bead subpopulation. In some embodiments, the extension can include 12 to 20 sequencing by synthesis cycles. In some embodiments, the extension identifies the capture probe and location of a particular bead within the flow cell.

[0156] As shown in Figure 5E, an index primer is hybridized to the index primer binding site and the index primer is extended. In some embodiments, the extension of the index primer includes an index readout. In some embodiments, the extension can include at least one cycle of sequencing by synthesis. In some embodiments, the number of cycles of sequencing by synthesis can depend on the number of different indexes in the multiple subpopulations of beads. In some embodiments, the extension can include 6 to 12 cycles of sequencing by synthesis. Specific methods for detecting target ligands

[0157] Some embodiments include methods of detecting target ligands. In some embodiments, the target ligands can include nucleic acids, proteins, or other antigens. In some embodiments, the target ligands are obtained from different sources, for example, from different samples, different individual subjects, or different populations of subjects.

[0158] In some embodiments, a method for detecting a target ligand can include obtaining a population of beads, each bead comprising a capture probe that specifically binds to a target ligand. For example, the capture probe can comprise a nucleic acid, an antibody, or an antigen-binding fragment of an antibody. In some embodiments, the beads comprise a first polynucleotide comprising a barcode indicative of the capture probe of the same bead and a barcode primer binding site 3' of the barcode. In some embodiments, each bead also comprises a second polynucleotide comprising an index and an index primer binding site 3' of the index. In some embodiments, the population of beads comprises first and second subpopulations of beads. In some embodiments, the index of the first subpopulation of beads is different from the index of the second subpopulation of beads. For example, the index of the first subpopulation of beads can be used to distinguish the first subpopulation of beads from the index of the second subpopulation of beads.

[0159] Some embodiments include contacting a first target ligand with the capture probes of a first subpopulation of beads and a second target ligand with the capture probes of a second subpopulation of beads. For example, the first target ligand can be obtained from a first sample of ligands, and the second target ligand can be obtained from a second sample of ligands. Some embodiments also include distributing the first and second subpopulations of beads containing the specifically bound first and second target ligands onto a substrate. Some embodiments also include detecting the capture probes distributed on the substrate that are specifically bound to the first and second target ligands. Some embodiments also include decoding the positions of the beads containing the detected capture probes on the substrate.

[0160] In some embodiments, the capture probe comprises a nucleic acid and the target ligand comprises a nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In other embodiments, the capture probe is distinct from the first polynucleotide. In some embodiments, the capture probes of subpopulations of beads comprise different nucleotide sequences from each other. In some embodiments, different subpopulations of beads can comprise the same different capture probe. In some embodiments, the capture probe comprises a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0161] In some such embodiments, detecting a capture probe specifically bound to a target ligand comprises extending the capture probe specifically bound to the target ligand. In some embodiments, the extending comprises polymerase extension and / or ligase extension. In some embodiments, the extension comprises a detectable nucleotide, such as a fluorescently labeled nucleotide. In some embodiments, the extension comprises a single nucleotide extension of the capture probe. In some embodiments, the extension comprises extension of the capture probe by multiple nucleotides.

[0162] In some such embodiments, the capture probe comprises an antibody or antigen-binding fragment thereof. In some embodiments, the capture probes of subpopulations of beads specifically bind to different target ligands from each other. In some embodiments, different subpopulations of beads can comprise the same different capture probes. In some such embodiments, different capture probes of subpopulations of beads specifically bind to the same target ligand.

[0163] In some embodiments, detecting capture probes specifically bound to target ligands comprises an immunoassay, for example, where the target ligands specifically bound to the capture probes are contacted with a secondary antibody or antigen-binding fragment thereof, and the secondary antibody or antigen-binding fragment thereof comprises a detectable label, such as a fluorescent label.

[0164] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence.

[0165] In some embodiments, the nucleotide sequences of the indices of a subpopulation of beads comprise the same nucleotide sequence, allowing the subpopulation of beads to be distinguished from another subpopulation of beads, for example, the nucleotide sequence of the indices of a first subpopulation of beads and the nucleotide sequence of the indices of a second subpopulation of beads comprise the same nucleotide sequence.

[0166] In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0167] In some embodiments, contacting a first target ligand with capture probes of a first subpopulation of beads and contacting a second target ligand with capture probes of a second subpopulation of beads are performed at different locations, e.g., the different locations may comprise different reaction volumes, such as different volumes in different wells of a microtiter plate.

[0168] Some embodiments also include combining the first and second subpopulations of beads prior to distributing the first and second subpopulations of beads onto the substrate. In other embodiments, the first subpopulation of beads is distributed onto the substrate before the second subpopulation of beads is distributed onto the substrate. In some embodiments, the subpopulations of beads are dispersed onto the substrate before capture probes specifically bound to ligands are detected.

[0169] In some embodiments, detecting the capture probe specifically bound to the target ligand can also include determining the location of the capture probe specifically bound to the target ligand on the substrate.

[0170] In some embodiments, decoding the position of the detected capture probe comprises decoding the position of the index of the bead containing the detected capture probe. Some embodiments also comprise hybridizing a plurality of index primers to the index primer sites and extending the hybridized index primers. Some embodiments also comprise extending the hybridized index primers, including at least one cycle of sequencing-by-synthesis. In some embodiments, decoding the position of the bead index comprises sequencing the index on the substrate.

[0171] In some embodiments, decoding the position of the detected capture probe comprises decoding the position of a barcode on a bead comprising the detected capture probe. Some such embodiments comprise hybridizing a plurality of barcode primers to the barcode primer sites and extending the hybridized barcode primers. In some embodiments, extending the hybridized barcode primers comprises at least one cycle of sequencing-by-synthesis. In some embodiments, decoding the position of the barcode on the bead comprises sequencing the barcode on the substrate.

[0172] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, a flow cell comprises the substrate. In some embodiments, the distributed first and second subpopulations of beads comprise an array.

[0173] In some embodiments, the first and second subpopulations of beads each comprise at least 50, 100, 500, 1000, or 5000 capture probes that are different from one another, or any number between any two of the foregoing numbers. Some embodiments also comprise at least 5, 10, 20, 50, 100, 200, 500, 1000 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation, or any number between any two of the foregoing numbers.

[0174] An embodiment including beads 200 having attached polynucleotides 210 is shown in the left panel of FIG. 6A. The polynucleotide includes a capture probe hybridized to a target nucleic acid 220. The capture probe is extended with a nucleotide comprising a detectable label 230. In some embodiments, the polynucleotide includes a barcode that indicates the capture probe and a barcode primer binding site useful for sequencing and identifying the barcode. In some embodiments, the polynucleotide can also include an index that distinguishes a subpopulation of beads from another subpopulation of beads and an index primer binding site useful for sequencing and identifying the index. In some embodiments, the beads can be distributed in an array on a substrate and decoded. Decoding can include determining the location of detectable labels on the array, determining barcodes attached to beads on the array, and / or determining indices attached to beads on the array.

[0175] An embodiment including beads 200 having attached capture probes 240 is shown in the center panel of FIG. 6A. The capture probes are hybridized to target nucleic acids 220. Also attached to the beads are first polynucleotides 260 that include a barcode representing the capture probe and a barcode primer binding site useful for sequencing and identifying the barcode. Also attached to the beads are second polynucleotides 250 that include an index that distinguishes a subpopulation of beads from another subpopulation of beads. The capture probes are extended with nucleotides that include a detectable label 230. In some embodiments, the beads can be distributed in an array on a substrate and decoded. Decoding can include determining the location of the detectable labels on the array, determining the barcodes attached to the beads on the array, and / or determining the indexes attached to the beads on the array.

[0176] An embodiment is shown in the right panel of Figure 6A, which includes beads 200 having attached thereto a capture probe 270, where the capture probe is an antibody or an antigen-binding fragment of an antibody. The capture probe is specifically bound to a ligand 280. The ligand is also bound to a secondary antibody 290 that includes a detectable label 230. A first polynucleotide 260 is also attached to the beads, which includes a barcode that indicates the capture probe and a barcode primer binding site useful for sequencing and identifying the barcode. A second polynucleotide 250 is also attached to the beads, which includes an index that distinguishes a subpopulation of beads from another subpopulation of beads. In some embodiments, the beads can be distributed in an array on a substrate and decoded. Decoding can include determining the location of the detectable label on the array, determining the barcode attached to the beads on the array, and / or determining the index attached to the beads on the array.

[0177] An embodiment including beads 200 having attached capture probes 330 comprising proteins is shown in the right panel of FIG. 6B. In some embodiments, the beads can be distributed in an array on a substrate. A protein substrate 340 contacts the proteins to generate a signal comprising a detectable label 230. The location of the signal on the array can be determined. In some embodiments, the beads can include a first polynucleotide comprising a barcode indicative of the capture probe, and a barcode primer binding site useful for sequencing and identifying the barcode is also attached to the beads. In some embodiments, the beads can include a second polynucleotide also attached to the beads that includes an index that distinguishes a subpopulation of beads from another subpopulation of beads. In some embodiments, the beads are decoded on the array. Decoding can include determining the location of the detectable label on the array, determining the barcode attached to the beads on the array, and / or determining the index attached to the beads on the array.

[0178] Some embodiments provide a method for detecting a target ligand on an array, comprising: (a) obtaining first and second populations of beads, each bead comprising a capture probe capable of specifically binding to a target ligand, a nucleic acid encoding a barcode and a barcode primer binding site, where the barcode indicates the capture probe, and a nucleic acid encoding an index and an index primer binding site, where the index indicates the source of the bead from the first population or the second population; and (b) contacting the first population of beads with a first sample comprising a first target ligand, where the first target ligand specifically binds to the capture probe of the first population of beads and (c) contacting the second population of beads with a second sample containing a second target ligand, whereby the second target ligand specifically binds to the capture probe of the second population of beads, thereby obtaining a second population of target-bound beads; (d) randomly distributing the first population of target-bound beads and the second population of target-bound beads onto an array; (e) detecting the positions of the beads containing the first and second target ligands on the array; and (f) determining the index and barcode sequences of the beads containing the first and second target ligands on the array. In some embodiments, the capture probes comprise polynucleotides. In some embodiments, the target ligands comprise nucleic acids. In some embodiments, detecting the positions of the beads containing the first and second target ligands on the array comprises extending the capture probes by polymerase extension or ligation. In some embodiments, the capture probes comprise proteins. In some embodiments, step (e) is performed after step (f). In some embodiments, the barcodes of the first population of beads comprise barcodes that are different from one another and the barcodes of the second population of beads comprise barcodes that are different from one another, hi some embodiments, the indices of the first population of beads are the same as one another and the indices of the second population of beads are the same as one another.In some embodiments, the array is located on a surface of a flow cell. In some embodiments, the first and second populations of beads are adapted to be attached to the array. In some embodiments, the first and second populations of beads comprise biotin, streptavidin, or derivatives thereof, and the array comprises biotin, streptavidin, or derivatives thereof. In some embodiments, the first and second populations of beads are magnetic. Sequencing and Analysis of Target Nucleic Acids

[0179] Some embodiments include sequencing and / or analysis of target nucleic acids. Some embodiments include decoding the position of a polynucleotide in an array according to the methods provided herein, hybridizing the target nucleic acid to a capture probe, extending the capture probe, and detecting extension of the capture probe hybridized to the target nucleic acid at the position on the array. In some embodiments, the position of a polynucleotide on the array can be decoded before hybridizing the target nucleic acid to the polynucleotide. In some embodiments, the position of a polynucleotide on the array can be decoded after detecting extension of the capture probe hybridized to the target nucleic acid. In some such embodiments, each polynucleotide can be associated with a capture probe via a common element. For example, the polynucleotide and the capture probe can each be attached to the same microfeature, such as a bead. In more such embodiments, each polynucleotide can include a capture probe.

[0180] Some embodiments include single-base extension (SBE) of a capture probe. In some embodiments, SBE can be used to detect alleles, mutations, or other features in a target nucleic acid. Briefly, SBE utilizes a capture probe that hybridizes to a target genome fragment at a position proximal or adjacent to a detection position, which indicates a specific genetic locus. A polymerase can be used to extend the 3' end of the capture probe with a nucleotide analog labeled with a detection label. Based on the fidelity of the enzyme, a nucleotide is incorporated into the capture probe only if it is complementary to the detection position in the target nucleic acid. Optionally, the nucleotide can be derivatized with a blocking group (including a reversible blocking group) so that further extension cannot occur, thus adding only a single nucleotide. The presence of the labeled nucleotide in the extended capture probe can be detected, for example, at a specific position in an array, and the added nucleotide can be identified to determine the identity of the locus or allele. SBE can be carried out under known conditions, such as those described in US Pat. Nos. 9,441,267 and 9,045,796, each of which is incorporated by reference in its entirety.

[0181] Some embodiments include allele-specific primer extension (ASPE). In some embodiments, ASPE can involve the extension of capture probes that differ in nucleotide composition at their 3' ends. The ASPE method can be performed using nucleosides or nucleotides containing cleavable linkers, so that the label can be removed after the probe is detected. This allows for further use of the probe or verification that the detected signal was due to the label that was just removed. Briefly, ASPE can be performed by hybridizing a target nucleic acid to a capture probe having a 3' sequence portion complementary to the detection position and a 5' portion complementary to a sequence adjacent to the detection position. Template-directed modification of the 3' portion of the capture probe, for example, by addition of a labeled nucleotide by a polymerase, produces a labeled extension product, but only if the template contains the target sequence. The presence of such a labeled primer extension product can then be detected based on its location within an array, for example, to indicate the presence of a particular allele. In some embodiments, ASPE can be performed using multiple capture probes with similar 5' ends that anneal adjacent to the same detection position in the target nucleic acid, but with different 3' ends, so that only the capture probe with a 3' end that is complementary to the detection position is modified by the polymerase. A capture probe with a 3'-terminal base that is complementary to a specific detection position is called a perfect match (PM) probe for the position, while a capture probe with a mismatched 3'-terminal base that cannot be extended in the ASPE reaction is called a mismatch (MM) probe for the position. The presence of a labeled nucleotide in the PM probe can be detected, and the 3' sequence of the capture probe can be determined to identify the specific allele at the detection position.

[0182] Some embodiments include methods for decoding the position of a polynucleotide within an array. Some such methods include: (a) obtaining a substrate having an array of polynucleotides distributed on its surface, each polynucleotide comprising a primer binding site 3' of a barcode, each polynucleotide linked to a capture probe; (b) hybridizing a plurality of primers to the primer binding sites; and (c) determining the sequence of the barcodes by extending the hybridized primers, wherein the sequence of each barcode indicates the position of the polynucleotide within the array. In some embodiments, each polynucleotide is linked to the capture probe via a bead. In some embodiments, each polynucleotide comprises a capture probe. In some embodiments, the capture probe is 3' of the primer binding site. In some embodiments, the capture probe is 5' of the barcode. In some embodiments, a capture probe comprises a sequence that differs from another capture probe. In some embodiments, a capture probe differs from another capture probe by fewer than five different nucleotides. In some embodiments, each capture probe comprises a different sequence. In some embodiments, the polynucleotides are randomly distributed on the surface of the substrate. In some embodiments, a barcode comprises a sequence that is different from another barcode. In some embodiments, each barcode comprises a different sequence. In some embodiments, each primer binding site comprises the same sequence. In some embodiments, the polynucleotides are attached to beads. In some embodiments, the beads are dispensed into wells. In some embodiments, each polynucleotide comprises a cleavable linker. In some embodiments, the cleavable linker is adapted to remove the capture probe from the primer binding site and the barcode. In some embodiments, the substrate comprises a well. In some embodiments, each polynucleotide comprises a spacer. In some embodiments, the spacer is attached to the substrate. In some embodiments, the spacer is attached to a bead.Some embodiments also include hybridizing the target nucleic acid to a capture probe. In some embodiments, hybridizing the target nucleic acid to the capture probe is performed after determining the sequence of the barcode. In some embodiments, hybridizing the target nucleic acid to the capture probe is performed before determining the sequence of the barcode. Some embodiments also include extending the hybridized target nucleic acid, or polynucleotide. In some embodiments, extending includes ligation. Some embodiments also include amplifying the target nucleic acid.

[0183] Some embodiments include methods for sequencing a target nucleic acid. Some such methods include: (a) decoding the position of a polynucleotide in an array according to any one of the methods described above; (b) hybridizing the target nucleic acid to a capture probe; (c) extending the capture probe hybridized to the target nucleic acid; and (d) detecting the position of the extended capture probe. In some embodiments, (d) detecting the position of the extended capture probe is performed before (a) decoding the position of the polynucleotide in the array. In some embodiments, the capture probe is extended by a ligase. In some embodiments, the capture probe is extended by a polymerase. In some embodiments, the capture probe is extended by the addition of a single nucleotide. Some embodiments also include cleaving the primer binding site and barcode from the capture probe before hybridizing the target nucleic acid to the capture probe. Some embodiments also include cleaving the primer binding site and barcode from the capture probe after hybridizing the target nucleic acid to the capture probe.

[0184] Some embodiments involve sequencing target polynucleotides from different biological sources on an array. In some such embodiments, polynucleotides containing target nucleic acids can be prepared from nucleic acid samples. Examples of polynucleotide samples include genomic DNA samples, cDNA samples, RNA samples, and amplicons from an individual. The polynucleotide can include a target nucleic acid, an index, and an index primer binding site adjacent to the index. The index can be used to indicate the source of the target nucleic acid as a particular nucleic acid sample. For example, polynucleotides prepared from different nucleic acid samples can include different indexes. Each index can have a position designed to bind to a specific amplification primer. The index primer binding site can be used to sequence the index by extending a primer hybridized to the index primer binding site. In some embodiments, the index is a nucleic acid or region within the polynucleotide ranging from about 3 to 30 contiguous nucleotides. The index can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or longer. Some aspects useful in the methods and compositions provided herein are disclosed in U.S. Patent Application Publication Nos. 20180334711(A1) and 20190085384(A1), each of which is incorporated by reference in its entirety. In some embodiments, the barcode or index can include a unique molecular identifier (UMI).

[0185] Polynucleotides can be prepared by various methods. In some embodiments, a nucleic acid sample containing a target nucleic acid can be tagged with a transposome to obtain nucleic acid fragments with ends containing sequences from the transposome. In some embodiments, the transposome can include an index and an index primer binding site, whereby tagging adds the index and index primer binding site to the nucleic acid fragments. For example, an input nucleic acid containing a target nucleic acid can be contacted with multiple transposomes. The transposome can fragment the input nucleic acid and attach adapters to the ends of the nucleic acid fragments. An example of a tagging reaction is disclosed in U.S. Patent No. 9,040,256, which is incorporated by reference in its entirety.

[0186] In some embodiments, a polynucleotide comprising an index can be prepared by adding an adapter to the end of a nucleic acid fragment comprising a target nucleic acid, where the adapter can comprise an index and an index primer binding site. In some embodiments, a polynucleotide comprising an index can be prepared by amplifying a nucleic acid fragment comprising a target nucleic acid with a primer comprising an index and an index primer binding site, whereby the amplification product comprises an index and an index primer binding site.

[0187] In some embodiments, the target nucleic acid of the polynucleotide is hybridized to a capture probe. In some embodiments, the capture probe is attached to a bead. In some embodiments, the oligonucleotide comprises the capture probe. The oligonucleotide can comprise a capture probe, a barcode, and a barcode primer binding site. In some embodiments, the barcode can be sequenced by extending a primer hybridized to the primer binding site. In some embodiments, the barcode can comprise a nucleic acid sequence that can be used to identify a polynucleotide, such as a capture probe, within an array. The barcode can comprise a unique nucleotide sequence that can be distinguished from other barcodes. The barcode can also be distinguished from other nucleotide sequences within the polynucleotide and target nucleic acid by its sequence and its location within the polynucleotide, e.g., its location adjacent to the barcode primer binding site. For example, in some embodiments, the barcode sequence can occur more than once in a plurality of nucleic acids, but the barcode adjacent to the barcode primer binding site can be detected. The barcode can be of any desired sequence length sufficient to be a unique nucleotide sequence within the plurality of barcodes in the population and / or within the plurality of polynucleotides and target nucleic acids being analyzed or examined. In some embodiments, the barcode is a nucleic acid or region within a polynucleotide ranging from about 6 to 30 contiguous nucleotides. The barcode can be, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or longer. Suitable barcodes for some embodiments are disclosed in U.S. Patent No. 8,053,192, which is incorporated by reference in its entirety. In some embodiments, the barcode can distinguish one polynucleotide from another polynucleotide within an array, such that each barcode is different from another barcode. In some embodiments, the barcode can be used to identify the location of a bead within an array.In some embodiments, the bars can be used to identify the capture probes.

[0188] In some embodiments, the polynucleotide target nucleic acid is hybridized to a capture probe to obtain hybridized beads. In some embodiments, the hybridized beads can include an oligonucleotide containing a barcode, a barcode primer binding site, and a capture probe, and the capture probe can hybridize to the polynucleotide target nucleic acid, and the polynucleotide can include an index and an index primer binding site. The hybridized beads can be randomly distributed on the array.

[0189] In some embodiments, sequence information of the target nucleic acid can be obtained by extending a capture probe. In some embodiments, the capture probe can be extended to include a sequence complementary to the target nucleic acid. In some such embodiments, the extension can include polymerase extension. In some embodiments, the extension is single base extension (SBE). In some embodiments, SBE can be used to detect alleles, mutations, or other features in the target nucleic acid.

[0190] In some embodiments, the capture probe can be extended by ligation. For example, a locus-specific oligonucleotide can hybridize to the target nucleic acid at a position adjacent to the site at which the capture probe hybridizes to the target nucleic acid, and the locus-specific oligonucleotide can then be ligated to the capture probe. In some embodiments, the capture probe can be extended such that the extended capture probe incorporates a sequence complementary to the index and index primer binding site of the polynucleotide.

[0191] In some embodiments, the index is sequenced to determine the source of the target nucleic acid attached to the bead on the array. In some embodiments, the barcode is sequenced to decode the position of the bead on the array. In some embodiments, the barcode is sequenced to identify the capture probe attached to the bead on the array.

[0192] In some embodiments, the array is located on the surface of a flow cell. In some embodiments, beads are adapted to be attached to the array. For example, the beads can include an agent such as biotin, streptavidin, or a derivative thereof, and the array includes biotin, streptavidin, or a derivative thereof. In some embodiments, the beads and the array are magnetic.

[0193] Some embodiments include preparing multiple polynucleotides from different nucleic acid samples, hybridizing each of the multiple polynucleotides to a population of beads comprising a capture probe to obtain hybridized beads, and randomly distributing the hybridized beads on an array. An exemplary embodiment includes obtaining first and second populations of beads, wherein the first population of beads comprises a first capture probe, a first barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the first barcode, and the second population of beads comprises a second capture probe, a second barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the second barcode. The first and second multiples of polynucleotides are obtained, wherein the first multiple polynucleotides comprise a first target nucleic acid, the first multiple polynucleotides are in solution, the second multiple polynucleotides comprise a second target nucleic acid, and the second multiple polynucleotides are in solution. A first target nucleic acid is hybridized to a first capture probe to obtain a hybridized first bead, and a second target nucleic acid is hybridized to a second capture probe to obtain a hybridized second bead. The hybridized first beads and hybridized second beads on the array are randomly distributed on the array. The positions of the first and second beads on the array are decoded by sequencing the first and second barcodes to determine which beads are bound by each set of target nucleic acids. Nucleic acid sequence data of the first and second target nucleic acids is obtained by extending the first and second capture probes.

[0194] In some embodiments, polynucleotides are indexed as described herein. For example, first and second populations of beads are obtained, where the first population of beads comprises an oligonucleotide comprising a first capture probe, a first barcode, and a barcode primer binding site adjacent to the first barcode, and the second population of beads comprises an oligonucleotide comprising a second capture probe, a second barcode, and a barcode primer binding site adjacent to the second barcode. First and second pluralities of polynucleotides are obtained, where the first pluralities of polynucleotides comprise a first target nucleic acid, a first index, and an index primer binding site adjacent to the first index, the first pluralities of polynucleotides being in solution, the second pluralities of polynucleotides comprise a second target nucleic acid, a second index, and a second primer binding site adjacent to the second index, and the second pluralities of polynucleotides being in solution. A first target nucleic acid is hybridized to a first capture probe to obtain a hybridized first bead, and a second target nucleic acid is hybridized to a second capture probe to obtain a hybridized second bead. The hybridized first beads and hybridized second beads are randomly distributed on an array. The positions of the first and second beads on the array are decoded by sequencing the first and second barcodes. The first and second capture probes are extended to obtain nucleic acid sequence data of the first and second target nucleic acids. The source of the nucleic acid sequence data of the first and second target nucleic acids is determined by sequencing the first and second indexes.

[0195] In some embodiments, the first plurality of polynucleotides comprises a first index and an index primer binding site adjacent to the first index, and the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index. In some embodiments, the first or second plurality of polynucleotides is obtained by tagging a nucleic acid sample with a plurality of transposomes. In some embodiments, the plurality of transposomes comprises a first or second index. Some embodiments also include adding an adapter to the tagged nucleic acid sample, the adapter comprising the first or second index. Some embodiments also include amplifying the tagged nucleic acid sample with a primer comprising the first or second index. In some embodiments, extending the first and second capture probes incorporates sequences complementary to the first and second indexes and the first and second index primer binding sites into the extended capture probes.

[0196] In some embodiments, the beads can be indexed. For example, the beads can comprise a nucleic acid comprising an index and an index primer binding site. In some embodiments, the oligonucleotides attached to the beads can comprise a barcode, a barcode primer binding site, an index, and an index primer binding site. For example, a first population of beads can comprise a first index and an index primer binding site adjacent to the first index, and a second population of beads can comprise a second index and an index primer binding site adjacent to the second index. In some embodiments, the oligonucleotides of the first population of beads comprise a first index, and the oligonucleotides of the second population of beads comprise a second index.

[0197] In some embodiments, first and second populations of beads are obtained, the first population of beads comprising an oligonucleotide including a first capture probe, a first barcode, a barcode primer binding site adjacent to the first barcode, a first index, and an index primer binding site adjacent to the first index, and the second population of beads comprising an oligonucleotide including a second capture probe, a second barcode, a barcode primer binding site adjacent to the second barcode, a second index, and an index primer binding site adjacent to the second index. First and second pluralities of polynucleotides are obtained, the first pluralities of polynucleotides comprising a first target nucleic acid, the first pluralities of polynucleotides being in solution, the second pluralities of polynucleotides comprising a second target nucleic acid, and the second pluralities of polynucleotides being in solution. The first target nucleic acid is hybridized to the first capture probe to obtain hybridized first beads, and the second target nucleic acid is hybridized to the second capture probe to obtain hybridized second beads. The hybridized first beads and the hybridized second beads are randomly distributed on the array. The positions of the first and second beads on the array are decoded by sequencing the first and second barcodes. The first and second capture probes are extended to obtain nucleic acid sequence data of the first and second target nucleic acids. In some embodiments, the oligonucleotides of the first population of beads include a first index, and the oligonucleotides of the second population of beads include a second index.

[0198] In some embodiments, the first index indicates the source of the first target nucleic acid and the second index indicates the source of the second target nucleic acid. In some embodiments, the first indexes are the same as each other and the second indexes are the same as each other. Some embodiments also include sequencing the first and second indexes. In some embodiments, the first and second indexes include extending primers hybridized to the index primer binding sites. In some embodiments, the index binding primer sites are the same. In some embodiments, the first and second target nucleic acids are obtained from different nucleic acid samples. In some embodiments, the first and second target nucleic acids are obtained from genomic DNA.

[0199] In some embodiments, the first and second barcodes represent the nucleic acid sequence of the first or second capture probe. In some embodiments, the first barcodes are different from each other and the second barcodes are different from each other. In some embodiments, sequencing the first and second barcodes comprises extending a primer hybridized to the barcode primer binding site. In some embodiments, the barcode primer binding sites are the same.

[0200] In some embodiments, extending the first and second capture probes comprises polymerase extension. In some embodiments, extending the first and second capture probes comprises adding a single nucleotide to the capture probe. Some embodiments also comprise ligating a locus-specific oligonucleotide to the extended capture probe. In some embodiments, extending the first and second capture probes comprises ligating a locus-specific oligonucleotide to the capture probe.

[0201] In some embodiments, hybridizing a first target nucleic acid to a first capture probe to obtain a hybridized first bead and hybridizing a second target nucleic acid to a second capture probe to obtain a second bead are performed in solution.

[0202] In some embodiments, the flow cell comprises an array. In some embodiments, the array comprises a plurality of wells, each well comprising one or more beads. In some embodiments, the first and second beads are adapted to be attached to the array. In some embodiments, the first and second beads comprise biotin, streptavidin, or derivatives thereof, and the array comprises biotin, streptavidin, or derivatives thereof. In some embodiments, the beads and the array are magnetic.

[0203] An exemplary embodiment of sequencing a target nucleic acid on an array, in which the polynucleotide containing the target nucleic acid also contains an index, is shown in Figure 9. A DNA sample containing a target nucleic acid containing a single nucleotide polymorphism (star shape) is fragmented and tagged by adding an amplification primer binding site to each end of the fragment. The fragments are amplified by PCR using primers that bind to the amplification primer binding sites and contain a sample index and an index read primer binding site. The sample index and index read primer sequences are incorporated into the amplification product. Beads are prepared by attaching oligonucleotides to the beads. The oligonucleotides contain a linker, a decoding sequence (also known as a barcode sequence), a decoding read primer binding site, and a capture probe. The amplification product and beads are combined. The target nucleic acid is hybridized to the capture probe. Hybridization can occur in solution or on the array. The hybridized beads are randomly distributed on the array. The capture probe is extended with fully functionalized nucleotides (FFNs), and the extension is detected. The extended capture probe is further extended to incorporate sequences complementary to the sample index and index read primer binding sites. The strand containing the target nucleic acid is dissociated from the extended capture probe. The sample index of the extended capture probe is sequenced by extending a primer hybridized to the index read primer binding site. The decoded sequence of the extended capture probe is sequenced by extending a primer hybridized to the decode read primer binding site. The source of the target nucleic acid is identified from the sequence of the sample index. The bead is decoded and the capture probe is identified from the decoded sequence.

[0204] Another exemplary embodiment for sequencing a target nucleic acid on an array in which beads contain indexes is shown in Figure 10. The beads are prepared by attaching a capture probe oligonucleotide and a sample index probe oligonucleotide. The capture probe oligonucleotide includes a linker, a decoding sequence, a decoding read primer binding site, and a capture probe. The sample index probe oligonucleotide includes a linker, a sample index, and an index read primer binding site. The beads are combined with fragmented nucleic acids containing a target nucleic acid, such as a nucleic acid containing a single nucleotide polymorphism (star) of interest. The target nucleic acid is hybridized to the capture probe either in solution or on the array. The capture probe is extended with FFN. The extension is detected. The target nucleic acid is dissociated from the extended capture probe. The sample index is sequenced by extending a primer hybridized to the index read primer binding site. The decoding sequence of the extended capture probe is sequenced by extending a primer hybridized to the decoding read primer binding site. The source of the target nucleic acid is identified from the sequence of the sample index. The beads are decoded and the capture probes are identified from the decoding sequence. Specific dual-probe methods

[0205] Some embodiments include detecting a target nucleic acid with first and second capture probes. In some embodiments, the target nucleic acid comprises a first portion capable of hybridizing to the first capture probe and a second portion capable of hybridizing to the second capture probe. In some embodiments, a population of beads is obtained, each bead comprising a first capture probe and a second capture probe. In some embodiments, one of the two capture probes is attached to the bead via a cleavable linker. In some embodiments, one of the capture probes comprises a detectable label, such as a fluorescent label. In some embodiments, the target nucleic acid hybridizes to the capture probe to generate a double-stranded nucleic acid comprising a single-stranded gap. The gap is filled and the cleavable linker is cleaved. The extended capture probe is detected. In some embodiments, one of the capture probes comprises a detectable label, such as a fluorescent label. In some embodiments, the detectable label is incorporated into the extended capture probe during gap filling.

[0206] In some embodiments, the second capture probe is attached to the bead via a cleavable linker, and the second capture probe comprises a detectable label, such as a fluorescent label. Examples of cleavable linkers include linkers that can be cleaved by chemical means, by enzymes such as endonucleases, and by light of a specific frequency. In some embodiments, each bead also comprises a first polynucleotide comprising a barcode indicative of the first or second capture probe and a barcode primer binding site 3' of the barcode. In some embodiments, the first capture probe comprises the first polynucleotide. In some embodiments, the first capture probe is separate from the first polynucleotide.

[0207] In some embodiments, the end of the first capture probe is ligated to the end of the second capture probe. In some embodiments, the first capture probe is ligated to the second capture probe by hybridizing the target nucleic acid to the first and second capture probes of the beads of the population of beads to generate a double-stranded nucleic acid containing a single-stranded gap between the first and second capture probes, and bridging the gap between the first and second capture probes. In some embodiments, filling the gap can be performed using a polymerase and / or a ligase. In some embodiments, the cleavable linker is cleaved to generate beads containing the first capture probes that include a detectable label. In some embodiments, the population of beads is distributed on a substrate. In some embodiments, the population of beads is distributed on a substrate after the cleavable linker is cleaved. In some embodiments, the population of beads is distributed on a substrate before the cleavable linker is cleaved or before the first capture probe is ligated to the second capture probe. In some embodiments, the position of a bead containing a first capture probe that includes a detectable label on a substrate is determined.

[0208] In some embodiments, decoding the position of the bead comprising a first capture probe comprising a detectable label on the substrate comprises decoding the position of the barcode of the bead comprising a first capture probe comprising a detectable label on the substrate. Some such embodiments include hybridizing a barcode primer to the barcode primer site and extending the hybridized barcode primer. In some embodiments, extending the hybridized barcode primer comprises at least one cycle of sequencing-by-synthesis. In some embodiments, decoding the position of the barcode of the bead comprises sequencing the barcode on the substrate.

[0209] In some embodiments, each bead comprises a second polynucleotide comprising an index indicative of the source of the target nucleic acid and an index primer binding site 3' of the index.

[0210] In some embodiments, the population of beads comprises a first and a second subpopulation of beads, each bead comprising a second polynucleotide comprising an index and an index primer binding site 3' of the index, wherein the index of the first subpopulation is different from the index of the second subpopulation. In some embodiments, the nucleotide sequences of the indexes of the first subpopulation of beads comprise the same nucleotide sequence and the nucleotide sequences of the indexes of the second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence.

[0211] In some embodiments, ligating or cleaving a first subpopulation of beads is performed at a different location than ligating or cleaving a second subpopulation of beads. In some embodiments, the different locations comprise different reaction volumes. In some embodiments, the different locations comprise different wells.

[0212] Some embodiments also include combining the first and second subpopulations of beads prior to distributing the population of beads onto the substrate, while in other embodiments, the first subpopulation of beads is distributed onto the substrate before the second subpopulation of beads is distributed onto the substrate.

[0213] In some embodiments, decoding the position of the bead containing the first capture probe containing the detectable label on the substrate comprises determining the position of the index of the bead containing the detected capture probe. Some such embodiments include hybridizing a plurality of index primers to the index primer sites and extending the hybridized index primers. In some embodiments, extending the hybridized index primers comprises at least one cycle of sequencing by synthesis. In some embodiments, decoding the position of the bead index comprises sequencing the index on the substrate.

[0214] In some embodiments, the substrate comprises a plurality of discrete sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, a flow cell comprises the substrate. In some embodiments, the distributed population of beads comprises an array.

[0215] An embodiment in which beads 200 include a first capture probe 300 attached to the bead via a cleavable linker 310 is shown in the left panel of Figure 6B. A second capture probe 320 is attached to the bead. Target nucleic acid 220 from a particular sample is hybridized to the first and second capture probes, and the first capture probe is extended to fill the gap between the first and second capture probes with a nucleotide containing a detectable label 230. After the gap is filled, the target nucleic acid is removed, and the cleavable linker is cleaved to generate an extended second capture probe containing a detectable label attached to the bead. The beads can be distributed in an array on a substrate and decoded. Polynucleotides containing indexes indicating the sample attached to the bead and polynucleotides containing barcodes indicating the first or second capture probe are not shown. Decoding can include determining the location of the detectable label on the array, determining the barcodes attached to the beads on the array, and / or determining the indexes attached to the beads on the array.

[0216] An embodiment in which a first capture probe is attached to a bead 200 via its 5' end is shown in Figure 6C. A second capture probe 360 ​​is attached to a bead via its 3' end and a cleavable linker 310. The second capture probe comprises a detectable label 230. A target nucleic acid 220 is hybridized to the first and second capture probes, and the first capture probe is extended and ligated to the second capture probe. In some embodiments, the target nucleic acid can comprise a structural variant (SV). The cleavable linker is cleaved to generate an extended first capture probe that comprises a detectable label and is attached to a bead. The beads can be distributed in an array on a substrate and decoded. Polynucleotides comprising an index indicating the sample attached to the bead and a polynucleotide comprising a barcode indicating the first or second capture probe are not shown. Decoding can include determining the location of the detectable label on the array, determining the barcode attached to the bead on the array, and / or determining the index attached to the bead on the array. In the absence of target nucleic acid, the second capture probe and the detectable label are cleaved from the bead. Specific Methods for the Preparation and Use of Indexed Beads

[0217] Some embodiments include preparing indexed beads. Some such embodiments can include providing a population of index polynucleotides, each index polynucleotide comprising an index, an index primer binding site, and an anchor / adapter. In some embodiments, the anchor / adapter can bind or hybridize to an adapter binding site attached to the bead.

[0218] Some embodiments include a method of preparing a population of indexed beads, the method comprising: (a) obtaining a population of beads, each bead comprising an adapter, a capture probe, and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining a plurality of index polynucleotides, each index polynucleotide comprising an index and an index primer binding site; and (c) attaching the plurality of index polynucleotides to the population of beads via adapters, thereby obtaining the indexed population of beads. In some embodiments, (c) comprises extending the adapters by polymerase extension. In some embodiments, each index polynucleotide comprises an adapter binding site, and attaching comprises hybridizing the adapter binding site to the adapter. In some embodiments, (c) comprises ligating the index polynucleotides to the adapters. In some embodiments, attaching comprises hybridizing a splint polynucleotide to the adapter and the index polynucleotides. In some embodiments, (c) comprises attaching the plurality of index polynucleotides to the adapters of the population of beads via chemically reactive moieties. In some embodiments, the first polynucleotides of the population of beads comprise different capture probes. In some embodiments, the index of each index polynucleotide is the same. Aspects useful for methods and compositions useful for joining polynucleotides to each other by chemical ligation are disclosed in U.S. Patent Application Publication No. 20180127816, the entire contents of which are incorporated by reference.

[0219] Some embodiments also include contacting the population of indexed beads with a plurality of nucleic acids that includes the target nucleic acid. Some embodiments also include mixing the population of indexed beads that has been contacted with the plurality of nucleic acids that includes the target nucleic acid with an additional population of indexed beads, the additional population of indexed beads comprising index polynucleotides that have an index that differs from the index of the population of indexed beads that has been contacted with the plurality of nucleic acids.

[0220] In some embodiments, the first polynucleotide comprises a capture probe. Some embodiments also include contacting the population of indexed beads with a plurality of nucleic acids, including the target nucleic acid. Some embodiments also include mixing the population of indexed beads contacted with the plurality of nucleic acids, including the target nucleic acid, with an additional population of indexed beads, the additional population of indexed beads comprising an index polynucleotide having an index that is different from the index of the population of indexed beads contacted with the plurality of nucleic acids.

[0221] In some embodiments, the capture probe comprises a protein.

[0222] In some embodiments, the method is carried out on a flow cell.

[0223] An exemplary embodiment for preparing indexed beads is shown in Figure 11. The index polynucleotide comprises an anchor or adaptor, an index, and an index primer binding site. Multiple populations of index polynucleotides are prepared and distributed into wells of a multiwell plate. Each well can contain a population of index polynucleotides comprising the same index. For example, a first well can contain a population of index polynucleotides comprising a first index, and a second well can contain a population of index polynucleotides comprising a second index. Multiple beads can be distributed into the wells. Each bead can comprise a first polynucleotide comprising a capture probe and a second polynucleotide comprising an indexing anchor, such as a binding site capable of binding to the anchor or adaptor of the index polynucleotide. A single pool of beads is added to each well. The single pool of beads can comprise a population of beads whose capture probes differ from each other. A single pool of beads containing the same index can be used with a single sample, thereby allowing the source of subsequent products of the sample's engineered nucleic acids to be identified as originating from a single sample through identifying the associated index. As shown in Figure 11, a single indexed bead pool can be added to a single well in a multi-well plate, with each well containing a single sample.

[0224] The index polynucleotide index can be incorporated into the bead by several different methods. In some embodiments, the index polynucleotide hybridizes to the bead via an anchor and a second polynucleotide, and the second polynucleotide is extended to incorporate the complement of the index polynucleotide index into the extended second polynucleotide attached to the bead. In some embodiments, the index polynucleotide and the second polynucleotide are ligated together. In some embodiments, the index polynucleotide is attached to the polynucleotide attached to the bead via chemical or enzymatic methods. In some embodiments, the index polynucleotide hybridizes to the polynucleotide attached to the bead, and the index of the hybridized index polynucleotide is determined on the array.

[0225] Some embodiments include adding an index to a bead by chemical or enzymatic methods. An exemplary embodiment for adding an index to a bead by chemical or enzymatic methods is shown in FIG. 12A, which shows a bead containing first and second polynucleotides. The first polynucleotide includes a probe, such as a capture probe, a code, such as a barcode, and a primer A, such as a barcode primer binding site, that can be used to determine the sequence of the barcode. The second polynucleotide includes an index and a primer B, such as an index primer binding site, that can be used to determine the sequence of the index. The second polynucleotide can be attached to the bead via a spacer and a moiety XY, which links the second polynucleotide to the spacer. In some embodiments, the capture probe can include a sequence primer binding site. In some embodiments, a blocking group can be added to the index polynucleotide. In some embodiments, the index primer binding site can also be a hairpin with a reversibly blocked 3' end.

[0226] Some embodiments include adding an index to a bead by extending an adapter attached to the bead. In some embodiments, the adapter attached to the bead can be extended by ligation or by polymerase extension. An exemplary embodiment of extension by ligation is shown in FIG. 12B, which shows a bead containing first and second polynucleotides. The first polynucleotide contains a probe, such as a capture probe, a code, such as a barcode, and a primer A, such as a barcode primer binding site, that can be used to determine the sequence of the barcode. The second polynucleotide contains an adapter that can be extended by ligation to a third polynucleotide containing an index and primer B using a fourth polynucleotide containing a splint that can hybridize to both the second and third polynucleotides. In some embodiments, the capture probe can contain a sequence primer binding site. In some embodiments, a blocking group can be added to the index polynucleotide. In some embodiments, the index primer binding site can also be a hairpin with a reversibly blocked 3' end.

[0227] An exemplary embodiment of extension by polymerase extension is shown in Figure 12C, which shows a bead containing first and second polynucleotides. The first polynucleotide includes a probe, primer A, and a code. The second polynucleotide includes an adapter. A third polynucleotide, such as an index polynucleotide, includes an adapter binding site capable of binding to the adapter, an index, and primer B. The third polynucleotide hybridizes to the second polynucleotide such that the second polynucleotide is extended by polymerase extension to incorporate the index into the extension adapter. In some embodiments, the capture probe can include a sequence primer binding site. In some embodiments, a blocking group can be added to the index polynucleotide. In some embodiments, the index primer binding site can also be a hairpin with a reversibly blocked 3' end.

[0228] Some embodiments include the use of index polynucleotides hybridized to first polynucleotides attached to beads. Some embodiments include a method for detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a capture probe and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adapter capable of binding to the barcode primer binding site; (c) specifically binding the target ligand to the capture probe; (d) hybridizing the index polynucleotide to the first polynucleotide via the adapter; (e) detecting the target ligand on the array; and (f) determining the index and barcode of the first polynucleotide. In some embodiments, (e) comprises distributing the population of beads on the array. In some embodiments, (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index. In some embodiments, the method includes dehybridizing an index polynucleotide from the first polynucleotide, hybridizing a barcode primer to the barcode primer binding site, and extending the barcode primer to determine the sequence of the barcode. In some embodiments, the index polynucleotide further includes a cleavable linker positioned between the adapter and the index, and (f) includes (i) cleaving the cleavable linker and (ii) extending the adapter to determine the sequence of the barcode. In some embodiments, the capture probe includes a protein. In some embodiments, the target ligand includes a target nucleic acid. In some embodiments, the first polynucleotide includes a capture probe. In some embodiments, (e) includes extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, the extension includes adding a detectable dideoxynucleotide. In some embodiments, the method is performed on a flow cell.

[0229] An exemplary embodiment for using an index polynucleotide hybridized to a first polynucleotide attached to a bead is shown in Figures 13A and 13B. Figure 13A shows beads containing a first polynucleotide. The first polynucleotide includes a probe, such as a capture probe, a primer, such as a primer binding site, and a code, such as a barcode. The index polynucleotide includes an index, a primer 2, such as an index primer binding site, a spacer, such as a cleavable spacer, and an adapter capable of hybridizing to the primer binding site of the first polynucleotide. A bead pool can be prepared using index polynucleotides as shown in Figure 11. The first panel of Figure 13B shows beads containing a first polynucleotide in which the target nucleic acid is hybridized to the first polynucleotide via the capture probe, and the index polynucleotide is hybridized to the first polynucleotide via the primer binding site. The second panel of Figure 13B shows an extended first polynucleotide with a single fluorescent dideoxynucleotide (star) that can be detected on the bead array. The index and barcode can be determined. The third panel of Figure 13B shows the fluorescent dideoxynucleotides removed from the extended first polynucleotide. An index primer can be hybridized to the index primer binding site and extended, and the sequence of the index can be determined. The fourth panel of Figure 13B shows the target nucleic acid dehybridized from the first polynucleotide. The index polynucleotide is cleaved at the cleavable linker, and the adapter now corresponds to the barcode primer hybridized to the primer binding site, which can be extended, and the sequence of the barcode can be determined.

[0230] Some embodiments include the use of index polynucleotides containing multiple index sequences and index primer binding sites. In some such embodiments, multiple index sequences can result in increased signal at locations on the array when the indexes are sequenced. An exemplary embodiment is shown in FIG. 14. FIG. 14 shows beads containing a first polynucleotide and a second polynucleotide. The first polynucleotide includes a code, such as a barcode, a primer A, such as a barcode primer binding site, and a probe, such as a capture probe. The second polynucleotide includes repeats of the index and index primer binding site. In some embodiments, the repeats improve signal intensity on beads with a small number of immobilized indexes. In some embodiments, the repeats can limit the amount of surface area occupied by the primers without reducing the signal.

[0231] Some embodiments include enzymatic addition of index polynucleotides to beads. In some embodiments, the index polynucleotides can be attached to beads by extending polynucleotides attached to the beads via reactive groups. In some embodiments, the index polynucleotides can be attached to beads before mixing the bead pool with other bead pools, for example, before loading the mixture onto an array. Some embodiments include a method of detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a first polynucleotide comprising a capture probe, a barcode, and a barcode primer binding site, and a second polynucleotide; (b) obtaining index polynucleotides comprising an index, an index primer binding site, and an adapter; (c) specifically binding the target ligand to the capture probe; (d) attaching the index polynucleotide to the second polynucleotide via the adapter; (e) detecting the target ligand on the array; and (f) determining the index and barcode of the first polynucleotide. In some embodiments, the second polynucleotide comprises a barcode and a barcode primer binding site. In some embodiments, (d) comprises adding a reactive moiety to the second polynucleotide, wherein the adapter can be attached to the reactive moiety. Aspects useful for methods and compositions useful for adding reactive moieties to polynucleotides are disclosed in U.S. Patent Application Publication No. 20180127816, which is incorporated by reference in its entirety. In some embodiments, (e) comprises distributing the population of beads onto an array. In some embodiments, (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index. In some embodiments, (f) comprises hybridizing a barcode primer to the barcode primer binding site and determining the sequence of the index. In some embodiments, the capture probe comprises a protein. In some embodiments, the target ligand comprises a target nucleic acid.In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. In some embodiments, the extension comprises adding a detectable dideoxynucleotide. In some embodiments, the method is performed on a flow cell.

[0232] An exemplary embodiment of enzymatic addition of an index polynucleotide to a bead is shown in Figures 15A and 15B. Figures 15A and 15B illustrate hybridization of a target nucleic acid to a bead via a capture probe of a first polynucleotide, addition of an index polynucleotide to a bead via a second polynucleotide, and determination of a barcode and index attached to the bead. The first panel of Figure 15A shows a bead containing first and second polynucleotides. The first polynucleotide includes a code, such as a barcode, a primer, such as a barcode primer binding site, and a probe, such as a capture probe. The second panel of Figure 15A shows hybridization of a target nucleic acid to a capture probe. The third panel of Figure 15A shows single-base extension (SBE) of a first polynucleotide with a fluorescent dideoxynucleotide (star). The fourth panel of Figure 15A shows addition of a reactive group (triangle) to a second polynucleotide, such as a reactive dideoxynucleotide. Addition can include the use of terminal deoxynucleotidyl transferase (TdT). The first panel of Figure 15B shows the attachment of an index polynucleotide, including an index, an index primer binding site, and an adapter, to a second polynucleotide via a reactive group and an adapter. The beads can be loaded onto an array, the position of the bead can be determined from the fluorescent dideoxynucleotide, and the fluorescent dideoxynucleotide can be removed. The second panel of Figure 15B shows hybridization of an index primer to the index primer binding site to determine the sequence of the index. The index primer and target nucleic acid can be dehybridized. The third panel of Figure 15B shows hybridization of a barcode primer to the barcode primer binding site to determine the sequence of the barcode. Spacer

[0233] Some embodiments provided herein include the use of a spacer. The spacer can comprise a polynucleotide having a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, or 500 consecutive nucleotides, or a length within any two of the foregoing ranges. In some embodiments, a spacer can be positioned between two polynucleotide elements to increase the efficiency of a particular process by reducing steric hindrance. For example, a spacer can be useful for increasing the efficiency of enzymatic processes, such as the use of polymerases, ligases, and / or terminal transferases, for substrates such as polynucleotides attached to beads. In some embodiments, a spacer can be positioned between any two elements, including beads, such as the attached end of a polynucleotide to a bead, an index, an index primer binding site, a barcode, a barcode primer binding site, and a capture probe. Kits and Systems

[0234] Some embodiments include kits and systems for decoding microfeatures, such as polynucleotides, on an array. Some such kits and systems can include a substrate, such as a chip, or a fluidic cell having an array of polynucleotides randomly distributed on the surface of the substrate. The polynucleotides can include a primer binding site 3' of a barcode. In some such embodiments, each polynucleotide can include a capture probe. In more such embodiments, each polynucleotide can be associated with the capture probe via a common element. For example, the polynucleotide and the capture probe can each be attached to the same microfeature, such as a bead. Some embodiments include a detector adapted to detect a signal from a reagent hybridized to the polynucleotides in the array, where such a reagent can include a sequencing reagent, such as a nucleotide containing a detectable label. Some embodiments include a detector adapted to detect a signal that can result from the incorporation of a nucleotide into the polynucleotide, such as pyrophosphate, or a change in a hydrogen ion.

[0235] Some embodiments include kits or systems comprising at least first and second subpopulations of beads. In some embodiments, each bead of a subpopulation can comprise a first polynucleotide comprising a capture probe, a barcode indicative of the capture probe of the same bead, and a barcode primer binding site 3' of the barcode. In some embodiments, each bead of a subpopulation can also comprise a second polynucleotide comprising an index and an index primer binding site 3' of the index. In some embodiments, the index of a subpopulation of beads can indicate that particular subpopulation of beads. In some embodiments, the index of the first subpopulation is different from the index of the second subpopulation. In some embodiments of the kits and systems provided herein, a first volume comprises the first subpopulation of beads and a second volume comprises the second subpopulation of beads.

[0236] In some embodiments, the capture probes of the first and second subpopulations of beads each comprise a different nucleotide sequence. In some embodiments, the different capture probes of the first and second subpopulations of beads comprise the same nucleotide sequence. In some embodiments, the capture probe comprises a nucleotide sequence that can hybridize to a single nucleotide polymorphism (SNP) or its complement.

[0237] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple barcode primers that can hybridize to the barcode primer sites.

[0238] In some embodiments, the nucleotide sequences of the indexes of a first subpopulation of beads comprise the same nucleotide sequence, and the nucleotide sequences of the indexes of a second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple index primers that can hybridize to the index primer sites.

[0239] In some embodiments, the substrate comprises a plurality of distinct sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, a flow cell comprises the substrate. In some embodiments, the substrate is adapted such that combination of the first subpopulation and the second subpopulation of beads forms an array of beads on the surface of the substrate, and the array can be sequenced in multiple sequencing-by-synthesis cycles.

[0240] In some embodiments, the first and second subpopulations of beads each comprise at least 50 capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 500 capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 5,000 capture probes comprising different nucleotide sequences. In some embodiments, the first and second subpopulations of beads each comprise at least 50,000 capture probes comprising different nucleotide sequences.

[0241] Some embodiments include at least 10 different subpopulations of beads, each subpopulation comprising a different index than another subpopulation. Some embodiments include at least 100 different subpopulations of beads, each subpopulation comprising a different index than another subpopulation. Some embodiments include at least 1000 different subpopulations of beads, each subpopulation comprising a different index than another subpopulation. Some embodiments include at least 10,000 different subpopulations of beads, each subpopulation comprising a different index than another subpopulation.

[0242] Some embodiments include kits comprising an array of polynucleotides randomly distributed on the surface of a substrate, each polynucleotide comprising a primer binding site 3' of a barcode and linked to a capture probe, each polynucleotide comprising a different barcode and linked to a different capture probe. In some embodiments, each polynucleotide is linked to the capture probe via a bead. In some embodiments, each polynucleotide comprises a capture probe, and in some embodiments, the substrate is planar. In some embodiments, the substrate comprises a well. In some embodiments, the polynucleotides are attached to beads. In some embodiments, the beads are distributed within the wells. In some embodiments, the flow cell comprises an array.

[0243] Some embodiments include kits and systems comprising first and second subpopulations of beads. In some embodiments, each bead comprises a capture probe that specifically binds to a target ligand. Examples of target ligands include nucleic acids, proteins, or other antigens. Examples of capture probes include nucleic acids, antibodies, and antigen-binding fragments of antibodies. In some embodiments, each bead comprises a first polynucleotide comprising a barcode indicative of the capture probe of the same bead and a barcode primer binding site 3' of the barcode. In some embodiments, each bead comprises a second polynucleotide comprising an index and an index primer binding site 3' of the index. In some such embodiments, the index of the first subpopulation is different from the index of the second subpopulation. For example, the index of the first subpopulation of beads can be used to distinguish the first subpopulation of beads from the second subpopulation of beads. In some embodiments, the first subpopulation of beads is distinct from the second subpopulation of beads. For example, a first volume comprises the first subpopulation of beads, and a second volume comprises the second subpopulation of beads.

[0244] In some embodiments, the capture probe comprises a nucleic acid and the target ligand comprises a target nucleic acid. In some such embodiments, the first polynucleotide comprises a capture probe. In some embodiments, the capture probe comprises a nucleotide sequence that can hybridize to a single nucleotide polymorphism (SNP) or its complement.

[0245] In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment of an antibody.

[0246] In some embodiments, the capture probes of the first and second subpopulations of beads each specifically bind to a different target ligand. For example, the capture probes of the first subpopulation of beads each specifically bind to a different target ligand, and the capture probes of the second subpopulation of beads each specifically bind to a different target ligand. In some embodiments, the different capture probes of the first and second subpopulations of beads specifically bind to the same target ligand. For example, a set of different capture probes of the first subpopulation of beads specifically binds to the same target ligand as a set of different capture probes of the first subpopulation of beads.

[0247] In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple barcode primers that can hybridize to the barcode primer sites.

[0248] In some embodiments, the nucleotide sequences of the indexes of a first subpopulation of beads comprise the same nucleotide sequence, and / or the nucleotide sequences of the indexes of a second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple index primers that can hybridize to the index primer sites.

[0249] In some embodiments, the substrate comprises a plurality of distinct sites. In some embodiments, the substrate comprises a plurality of wells. In some embodiments, the substrate comprises a plurality of channels. In some embodiments, a flow cell comprises the substrate. In some embodiments, the substrate is adapted such that combination of the first subpopulation and the second subpopulation of beads forms an array of beads on the surface of the substrate, and the array can be sequenced in multiple sequencing-by-synthesis cycles.

[0250] In some embodiments, the first and second subpopulations of beads each comprise at least 50, 100, 500, 1000, or 5000 capture probes that are different from one another, or any number between any two of the foregoing numbers. Some embodiments also comprise at least 5, 10, 20, 50, 100, 200, 500, 1000 different subpopulations of beads, each subpopulation comprising a different index from another subpopulation, or any number between any two of the foregoing numbers.

[0251] Some embodiments include a kit comprising first and second subpopulations of beads, each bead comprising a capture probe that specifically binds to a target ligand, a first polynucleotide comprising a barcode indicative of the capture probe and a barcode primer binding site 3' of the barcode, and a second polynucleotide comprising an index and an index primer binding site 3' of the index, wherein the index of the first subpopulation is different from the index of the second subpopulation, a first volume comprising the first subpopulation of beads, and a second volume comprising the second subpopulation of beads. In some embodiments, the capture probe comprises a nucleic acid and the target ligand comprises a target nucleic acid. In some embodiments, the first polynucleotide comprises a capture probe. In some embodiments, the capture probes of the first and second subpopulations of beads each comprise a different nucleotide sequence from each other. In some embodiments, the different capture probes of the first and second subpopulations of beads comprise the same nucleotide sequence. In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the capture probes of the first and second subpopulations of beads each specifically bind to a different target ligand. In some embodiments, the different capture probes of the first and second subpopulations of beads specifically bind to the same target ligand. In some embodiments, the barcode primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple barcode primers that can hybridize to the barcode primer sites. In some embodiments, the nucleotide sequences of the indexes of the first subpopulation of beads comprise the same nucleotide sequence, and the nucleotide sequences of the indexes of the second subpopulation of beads comprise the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites comprise the same nucleotide sequence. Some embodiments also include multiple index primers that can hybridize to the index primer sites. In some embodiments, the flow cell includes a substrate.In some embodiments, the substrate is adapted so that the combination of the first and second subpopulations of beads forms an array of beads on the surface of the substrate, and the array can be sequenced in multiple sequencing-by-synthesis cycles. In some embodiments, the first and second subpopulations of beads each comprise at least 50 capture probes that are different from each other. In some embodiments, the first and second subpopulations of beads each comprise at least 500 capture probes that are different from each other. Some embodiments also comprise at least 10 different subpopulations of beads, each subpopulation comprising an index that is different from another subpopulation.

[0252] Many embodiments of the kits and systems provided herein can include multiple populations of beads comprising oligonucleotides attached to the beads, where the oligonucleotides comprise an index, an index primer binding site adjacent to the index, a capture probe, a barcode, and a barcode primer binding site adjacent to the barcode, and the index differs between the populations of beads. In some embodiments, the index primer binding site is the same in the multiple populations. In some embodiments, the barcode indicates the nucleic acid sequence of the capture probe. In some embodiments, the barcode differs in the populations of beads. In some embodiments, the barcode primer binding site is the same in the multiple populations. In some embodiments, the beads comprise biotin, streptavidin, or derivatives thereof. In some embodiments, the beads are magnetic.

[0253] Some embodiments also include a reagent selected from a locus-specific oligonucleotide, a transposome for tagging a nucleic acid sample, a transposome comprising an index and an index primer binding site, an adapter comprising an index and an index primer binding site, a primer capable of hybridizing to an index primer binding site or its complement, and / or a primer capable of hybridizing to a barcode primer binding site or its complement. Some embodiments also include an array, such as an array on the surface of a flow cell. Some embodiments include a detector adapted to detect a signal from a reagent hybridized to a polynucleotide in the array, where such a reagent can include a sequencing reagent, such as a nucleotide comprising a detectable label. Some embodiments include a detector adapted to detect a signal that can result from incorporation of a nucleotide into a polynucleotide, such as pyrophosphate, or a change in a hydrogen ion. [Example]

[0254] Example 1 - Decoding arrays by sequencing Multiple polynucleotides are synthesized, each containing, from 5' to 3', a spacer, a unique barcode, a primer binding site, and a unique capture probe. The sequences of the barcode and capture probe are known. The sequence of the primer binding site is the same for each polynucleotide. Each polynucleotide is attached to a bead. The beads are randomly distributed into the wells of the chip. The bead array is decoded by hybridizing a primer to the primer binding site, extending the primer, and detecting the sequence of the barcode. The location of the barcode identifies the location of the associated capture probe. Example 2 - Decoding barcodes on arrays by sequencing

[0255] A nucleic acid library prepared from human genomic DNA was prepared. A subpopulation of beads was prepared. A first and a second polynucleotide were attached to each bead. The first polynucleotide contained a capture probe, a barcode primer binding site, and a barcode representing the capture probe. The second polynucleotide contained an index and an index primer binding site.

[0256] A bead pool containing 18,816 different code / probe types was loaded onto a HiSeq flow cell (Figure 7A). After immobilization, the 20-nucleotide-long code was sequenced using SBS chemistry, and the identity of each bead was determined by aligning the code sequence to a list of bead types. Figure 7B shows a histogram of the number of replicates of a particular bead type within a particular bin, demonstrating that 97.5% of the expected contents were identified using the sequencing-based decoding process and that the majority of bead types were present in sufficient concentrations for genotyping testing. Example 3 - Genotyping performance on HiSeq using FFN detection

[0257] To demonstrate genotyping performance on the HiSeq using FFN detection, oligonucleotide target DNA was hybridized to a suspension of beads conjugated to probe oligos. The beads were loaded onto a HiSeq flow cell. The probes bound to the target DNA were extended by a single base using fluorescent nucleotides. The bead-loaded flow cell was imaged to obtain genotyping bead intensities. The fluorescent nucleotides were cleaved, and the beads were decoded using SBS chemistry. The decoded and genotyping reads were aligned to measure assay performance. Individual points were colored according to the expected genotype. Figure 7C shows a graph of C intensity versus T intensity, where each point is the average of all replicates of a given bead type and is colored according to the expected genotype, demonstrating that single-base probe extension with fluorescent nucleotides enables accurate genotyping. Example 4 - Multiplexing 12 Samples with a 10,368-plex Bead Pool

[0258] This example demonstrates the multiplexing of multiple samples on a single flow cell, specifically the ability to multiplex 12 samples with a 10,368-plex bead pool. A single bead pool was hybridized separately to 12 different index sequences. After hybridization, the samples were pooled and loaded onto a HiSeq flow cell. Two separate reads were then performed: one to identify the sample based on the hybridized index, and another to identify the bead type based on the decoding read. Figure 8 shows a histogram of the number of specific bead types within a specific binding for a representative sample, demonstrating that the majority of bead types were present at sufficient concentrations for genotyping experiments for a given sample. The table in Figure 7 summarizes the consistency of bead multiplexing and decoding across the 12 indexed samples pooled and loaded simultaneously, demonstrating uniform sample representation and that the majority of probes were present across all samples. Example 5 - Massively parallel SNP genotyping

[0259] This is an exemplary workflow for genotyping 384 samples in a single sequencing run. Polynucleotides containing target nucleic acids containing single nucleotide polymorphisms (SNPs) of interest are prepared in a 384-well plate. Each well contains DNA from a different subject. The DNA is tagged with transposomes, which fragment the DNA and add amplification primer binding sites to each end of the fragments. The fragments are amplified with primers containing an index and an index primer binding site to obtain amplified fragments in which at least one end of each amplified fragment contains an index and an index primer binding site. The indexes are different for each well so that polynucleotides from a particular well can be identified by their specific index. The prepared polynucleotides contain an index, an index primer binding site, and the target nucleic acid.

[0260] A population of beads is added to each well. The population of beads includes oligonucleotides attached to the beads. The oligonucleotides include a barcode, a barcode primer binding site, and a capture probe. The capture probe is 50 nucleotides long and specific to a particular target nucleic acid. The barcode can be used to identify the capture probe. The population of beads includes different capture probes. The target nucleic acid hybridizes with the capture probe in solution in each well to obtain hybridized beads. The hybridized beads from each well are pooled together and loaded into a bead array on a flow cell. The hybridized beads are randomly distributed across the array.

[0261] On the array, the capture probe is extended by a single nucleotide to identify the SNP. The index is sequenced by extending a primer hybridized to the index primer binding site to identify the source of the target nucleic acid. The barcode is sequenced by extending a primer hybridized to the index primer binding site to decode the position of the bead on the array. Specific SNPs are identified and associated with specific DNA samples from specific subjects.

[0262] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude further, unrecited elements or method steps.

[0263] The above description discloses some methods and materials of the present invention. The invention is susceptible to modifications of the methods and materials, and to changes in the manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Accordingly, the present invention is not intended to be limited to the particular embodiments disclosed herein, but rather is intended to cover all modifications and alternatives falling within the true scope and spirit of the invention.

[0264] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated by reference in their entirety and made a part of this specification. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede such conflicting material. The present invention provides, for example, the following items. (Item 1) 1. A method for sequencing a target nucleic acid on an array, comprising: (a) obtaining a first population and a second population of beads, the first population of beads comprises a first capture probe, a first barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the first barcode; the second population of beads comprises a second capture probe, a second barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the second barcode; (b) obtaining a first plurality of polynucleotides and a second plurality of polynucleotides, the first plurality of polynucleotides comprises a first target nucleic acid, the plurality of first polynucleotides is in solution; the second plurality of polynucleotides comprises a second target nucleic acid, and the second plurality of polynucleotides is in solution; (c) hybridizing the first target nucleic acid to the first capture probe to obtain a hybridized first bead, and hybridizing the second target nucleic acid to the second capture probe to obtain a hybridized second bead; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; (e) decoding the positions of the first beads and the second beads on the array by sequencing the first barcode and the second barcode; (f) extending the first capture probe and the second capture probe to obtain nucleic acid sequence data of the first target nucleic acid and the second target nucleic acid; A method comprising: (Item 2) the first plurality of polynucleotides comprises a first index and an index primer binding site adjacent to the first index; 2. The method of claim 1, wherein the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index. (Item 3) 3. The method of claim 2, wherein the first plurality of polynucleotides or the second plurality of polynucleotides are obtained by tagging a nucleic acid sample with a plurality of transposomes. (Item 4) 4. The method of claim 3, wherein the plurality of transposomes comprises the first index or the second index. (Item 5) 5. The method of claim 4, further comprising adding an adapter to the tagged nucleic acid sample, wherein the adapter comprises the first index or the second index. (Item 6) 6. The method of claim 5, further comprising amplifying the tagged nucleic acid sample with a primer comprising the first index or the second index. (Item 7) 7. The method of any one of items 2 to 6, wherein extending the first capture probe and the second capture probe incorporates sequences complementary to the first index and the second index, and the first index primer binding site and the second index primer binding site, into the extended capture probe. (Item 8) the first population of beads comprises a first index and an index primer binding site adjacent to the first index; 2. The method of claim 1, wherein the second population of beads comprises a second index and an index primer binding site adjacent to the second index. (Item 9) 9. The method of claim 8, wherein the oligonucleotides of the first population of beads comprise the first index and the oligonucleotides of the second population of beads comprise the second index. (Item 10) 10. The method according to any one of items 2 to 9, wherein the first index indicates a source of the first target nucleic acid and the second index indicates a source of the second target nucleic acid. (Item 11) 11. The method according to any one of items 2 to 10, wherein the first indexes are the same as each other and the second indexes are the same as each other. (Item 12) 12. The method of any one of items 2 to 11, further comprising sequencing the first index and the second index. (Item 13) Item 13. The method of item 12, wherein sequencing the first index and the second index comprises extending a primer hybridized to the index primer binding site. (Item 14) 14. The method according to any one of Items 2 to 13, wherein the index primer binding sites are the same. (Item 15) 15. The method according to any one of items 1 to 14, wherein the first target nucleic acid and the second target nucleic acid are obtained from different nucleic acid samples. (Item 16) 16. The method according to any one of items 1 to 15, wherein the first target nucleic acid and the second target nucleic acid are obtained from genomic DNA. (Item 17) 17. The method according to any one of items 1 to 16, wherein the first barcode and the second barcode indicate the nucleic acid sequence of the first capture probe or the second capture probe. (Item 18) 18. The method according to any one of items 1 to 17, wherein the first barcodes are different from each other and the second barcodes are different from each other. (Item 19) 19. The method of any one of items 1 to 18, wherein sequencing the first barcode and the second barcode comprises extending a primer hybridized to the barcode primer binding site. (Item 20) 20. The method according to any one of items 1 to 19, wherein the barcode primer binding sites are the same. (Item 21) 21. The method of any one of items 1 to 20, wherein extending the first capture probe and the second capture probe comprises polymerase extension. (Item 22) Extending the first capture probe and the second capture probe comprises extending the capture probes. 22. The method of claim 21, comprising the addition of a single nucleotide to (Item 23) 23. The method of claim 21 or 22, further comprising ligating a locus-specific oligonucleotide to the extended capture probe. (Item 24) 24. The method of any one of items 21 to 23, wherein extending the first capture probe and the second capture probe comprises ligating a locus-specific oligonucleotide to the capture probe. (Item 25) 25. The method according to any one of items 1 to 24, wherein step (c) is carried out in solution. (Item 26) 26. The method of any one of items 1 to 25, wherein the array is located on the surface of a flow cell. (Item 27) 27. The method of any one of items 1 to 26, wherein the first bead and the second bead are adapted to be attached to the array. (Item 28) 28. The method of claim 27, wherein the first bead and the second bead comprise biotin, streptavidin, or a derivative thereof, or the array comprises biotin, streptavidin, or a derivative thereof. (Item 29) 28. The method of claim 27, wherein the first barcode and the second barcode are magnetic. (Item 30) 1. A method for sequencing a target nucleic acid on an array, comprising: (a) obtaining a first population and a second population of beads, the first population of beads comprises a first capture probe, a first barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the first barcode; the second population of beads comprises a second capture probe, a second barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the second barcode; (b) obtaining a first plurality of polynucleotides and a second plurality of polynucleotides, the first plurality of polynucleotides comprises a first target nucleic acid, a first index, and an index primer binding site adjacent to the first index, the plurality of first polynucleotides being in solution; the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and a second primer binding site adjacent to the second index, and the plurality of second polynucleotides is in solution; (c) hybridizing the first target nucleic acid to the first capture probe to obtain a hybridized first bead, and hybridizing the second target nucleic acid to the second capture probe to obtain a hybridized second bead; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; and (e) decoding the positions of the first beads and the second beads on the array by sequencing the first barcode and the second barcode; (f) extending the first capture probe and the second capture probe to obtain nucleic acid sequence data of the first target nucleic acid and the second target nucleic acid; (g) determining the source of the nucleic acid sequence data for the first target nucleic acid and the second target nucleic acid by sequencing the first index and the second index. And, A method comprising: (Item 31) the first plurality of polynucleotides comprises a first index and an index primer binding site adjacent to the first index; 31. The method of claim 30, wherein the second plurality of polynucleotides comprises a second target nucleic acid, a second index, and an index primer binding site adjacent to the second index. (Item 32) 32. The method of claim 31, wherein the first plurality of polynucleotides or the second plurality of polynucleotides are obtained by tagging a nucleic acid sample with a plurality of transposomes. (Item 33) 33. The method of claim 32, wherein the plurality of transposomes comprises the first index or the second index. (Item 34) 34. The method of claim 33, further comprising adding an adapter to the tagged nucleic acid sample, wherein the adapter comprises the first index or the second index. (Item 35) 35. The method of claim 34, further comprising amplifying the tagged nucleic acid sample with a primer comprising the first index or the second index. (Item 36) 36. The method of claim 35, wherein extending the first capture probe and the second capture probe incorporates sequences complementary to the first index and the second index, and the first index primer binding site and the second index primer binding site, into the extended capture probe. (Item 37) 1. A method for sequencing a target nucleic acid on an array, comprising: (a) obtaining a first population and a second population of beads, the first population of beads comprises an oligonucleotide comprising a first capture probe, a first barcode, a barcode primer binding site adjacent to the first barcode, a first index, and an index primer binding site adjacent to the first index; the second population of beads comprises an oligonucleotide comprising a second capture probe, a second barcode, and a barcode primer binding site adjacent to the second barcode, and a second index and an index primer binding site adjacent to the second index; (b) obtaining a first plurality of polynucleotides and a second plurality of polynucleotides, the first plurality of polynucleotides comprises a first target nucleic acid, the plurality of first polynucleotides is in solution; the second plurality of polynucleotides comprises a second target nucleic acid, and the second plurality of polynucleotides is in solution; (c) hybridizing the first target nucleic acid to the first capture probe to obtain a hybridized first bead, and hybridizing the second target nucleic acid to the second capture probe to obtain a hybridized second bead; (d) randomly distributing the hybridized first beads and the hybridized second beads on an array; and (e) sequencing the first barcode and the second barcode; decoding the positions of the first beads and the second beads on the array; (f) extending the first capture probe and the second capture probe to obtain nucleic acid sequence data of the first target nucleic acid and the second target nucleic acid; A method comprising: (Item 38) 38. The method of any one of items 30 to 37, wherein the oligonucleotides of the first population of beads comprise the first index and the oligonucleotides of the second population of beads comprise the second index. (Item 39) 39. The method according to any one of items 30 to 38, wherein the first index indicates a source of the first target nucleic acid and the second index indicates a source of the second target nucleic acid. (Item 40) 40. The method according to any one of items 30 to 39, wherein the first indexes are the same as each other and the second indexes are the same as each other. (Item 41) 41. The method of any one of items 30 to 40, further comprising sequencing the first index and the second index. (Item 42) 42. The method of claim 41, wherein sequencing the first index and the second index comprises extending a primer hybridized to the index primer binding site. (Item 43) 43. The method according to any one of Items 30 to 42, wherein the index primer binding sites are the same. (Item 44) 44. The method according to any one of Items 30 to 43, wherein the first target nucleic acid and the second target nucleic acid are obtained from different nucleic acid samples. (Item 45) 45. The method according to any one of Items 30 to 44, wherein the first target nucleic acid and the second target nucleic acid are obtained from genomic DNA. (Item 46) 46. ​​The method according to any one of Items 30 to 45, wherein the first barcode and the second barcode indicate the nucleic acid sequence of the first capture probe or the second capture probe. (Item 47) 47. The method according to any one of items 30 to 46, wherein the first barcodes are different from each other and the second barcodes are different from each other. (Item 48) 48. The method of any one of items 30 to 47, wherein sequencing the first barcode and the second barcode comprises extending a primer hybridized to the barcode primer binding site. (Item 49) 49. The method according to any one of items 30 to 48, wherein the barcode primer binding sites are the same. (Item 50) 50. The method of any one of items 30 to 49, wherein extending the first capture probe and the second capture probe comprises polymerase extension. (Item 51) 51. The method of claim 50, wherein extending the first capture probe and the second capture probe comprises the addition of a single nucleotide to the capture probe. (Item 52) 52. The method of claim 50 or 51, further comprising ligating a locus-specific oligonucleotide to the extended capture probe. (Item 53) 53. The method of any one of items 50 to 52, wherein extending the first capture probe and the second capture probe comprises ligating a locus-specific oligonucleotide to the capture probe. (Item 54) 54. The method according to any one of items 30 to 53, wherein step (c) is carried out in solution. (Item 55) 55. The method of any one of items 30 to 54, wherein a flow cell comprises the array. (Item 56) 56. The method of any one of items 30 to 55, wherein the array comprises a plurality of wells. (Item 57) 57. The method of any one of items 30 to 56, wherein the first bead and the second bead are adapted to be attached to the array. (Item 58) 58. The method of claim 57, wherein the first bead and the second bead comprise biotin, streptavidin, or a derivative thereof, or the array comprises biotin, streptavidin, or a derivative thereof. (Item 59) Item 58. The method of item 57, wherein the first barcode and the second barcode are magnetic. (Item 60) 1. A kit comprising: a plurality of populations of beads comprising oligonucleotides attached to the beads, the oligonucleotides comprising an index, an index primer binding site adjacent to the index, a capture probe, a barcode, and a barcode primer binding site adjacent to the barcode, the index varying between the populations of beads. (Item 61) Item 61. The kit of item 60, wherein the index primer binding site is the same in the plurality of populations. (Item 62) 62. The kit of item 60 or 61, wherein the barcode indicates the nucleic acid sequence of the capture probe. (Item 63) 63. The kit of any one of items 60 to 62, wherein the barcodes differ in a population of beads. (Item 64) 64. The kit according to any one of Items 60 to 63, wherein the barcode primer binding site is the same in the plurality of populations. (Item 65) locus-specific oligonucleotides, transposomes for tagging nucleic acid samples; a transposome comprising an index and an index primer binding site; an adapter containing an index and an index primer binding site; a primer capable of hybridizing to the index primer binding site or its complement; and a primer capable of hybridizing to the barcode primer binding site or its complement; 65. The kit according to any one of items 60 to 64, further comprising a reagent selected from: (Item 66) 66. The kit of any one of items 60 to 65, further comprising a flow cell. (Item 67) 1. A method for preparing a population of indexed beads, comprising: (a) obtaining a population of beads, each bead comprising an adapter, a capture probe, and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining a plurality of index polynucleotides, each index polynucleotide comprising an index and an index primer binding site; (c) attaching said plurality of index polynucleotides to said population of beads via said adaptors to obtain a population of indexed beads. (Item 68) 68. The method of claim 67, wherein (c) comprises extending the adapter by polymerase extension. (Item 69) 69. The method of claim 68, wherein each index polynucleotide comprises an adapter binding site, and the attaching comprises hybridizing the adapter binding site to the adapter. (Item 70) 68. The method of claim 67, wherein (c) comprises ligating the index polynucleotide to the adaptor. (Item 71) 71. The method of claim 70, wherein the attaching comprises hybridizing a splint polynucleotide to the adapter and the index polynucleotide. (Item 72) 68. The method of claim 67, wherein (c) comprises attaching the plurality of index polynucleotides to the adapters of the population of beads via chemically reactive moieties. (Item 73) 73. The method of claim 72, wherein the attaching comprises a click chemistry reaction. (Item 74) 74. The method of any one of items 67 to 73, wherein the first polynucleotides of the population of beads comprise capture probes that differ from each other. (Item 75) 75. The method of any one of Items 67 to 74, wherein the index of each index polynucleotide is the same. (Item 76) 76. The method of any one of items 67 to 75, wherein the first polynucleotide comprises the capture probe. (Item 77) 77. The method of any one of items 67 to 76, further comprising contacting the population of indexed beads with a plurality of nucleic acids, including a target nucleic acid. (Item 78) 78. The method of claim 77, further comprising mixing the population of indexed beads contacted with a plurality of nucleic acids, including a target nucleic acid, with an additional population of indexed beads, wherein the additional population of indexed beads comprises index polynucleotides that comprise an index that is different from the index of the population of indexed beads contacted with a plurality of nucleic acids. (Item 79) 76. The method of any one of items 67 to 75, wherein the capture probe comprises a protein. (Item 80) 80. The method of any one of items 67 to 79, carried out in a flow cell. (Item 81) 1. A method for detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a capture probe and a first polynucleotide comprising a barcode and a barcode primer binding site; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adapter capable of binding to a barcode primer binding site; (c) specifically binding a target ligand to said capture probe; (d) hybridizing the index polynucleotide to the first polynucleotide via the adapter; (e) detecting said target ligand on the array; (f) determining the index and the barcode of the first polynucleotide. (Item 82) 82. The method of claim 81, wherein (e) comprises distributing the population of beads on an array. (Item 83) 83. The method of claim 81 or 82, wherein (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index. (Item 84) 84. The method of claim 83, further comprising dehybridizing the index polynucleotide from the first polynucleotide, hybridizing a barcode primer to the barcode primer binding site, and extending the barcode primer to determine the sequence of the barcode. (Item 85) the index polynucleotide further comprises a cleavable linker positioned between the adaptor and the index, and (f) (i) cleaving the cleavable linker; (ii) extending the adapter and determining the sequence of the barcode. (Item 86) 86. The method of any one of items 81 to 85, wherein the capture probe comprises a protein. (Item 87) 86. The method of any one of items 81 to 85, wherein the targeting ligand comprises a targeting nucleic acid. (Item 88) 88. The method of claim 87, wherein the first polynucleotide comprises the capture probe. (Item 89) 89. The method of claim 87 or 88, wherein (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. (Item 90) 90. The method of claim 89, wherein the extension comprises adding a detectable dideoxynucleotide. (Item 91) 91. The method of any one of items 81 to 90, carried out on a flow cell. (Item 92) 1. A method for detecting a target ligand, comprising: (a) obtaining a population of beads, each bead comprising a capture probe, a first polynucleotide comprising a barcode and a barcode primer binding site, and a second polynucleotide; (b) obtaining an index polynucleotide comprising an index, an index primer binding site, and an adaptor; (c) specifically binding the target ligand to the capture probe; (d) attaching the index polynucleotide to the second polynucleotide via the adaptor; (e) detecting said target ligand on the array; (f) determining the index and the barcode of the first polynucleotide. (Item 93) 93. The method of claim 92, wherein the second polynucleotide comprises a barcode and a barcode primer binding site. (Item 94) 94. The method of claim 92 or 93, wherein (d) comprises adding a reactive moiety to the second polynucleotide, and the adaptor is capable of attaching to the reactive moiety. (Item 95) 95. The method of claim 94, wherein the attaching of the reactive moiety comprises a click chemistry reaction. (Item 96) 96. The method of any one of items 92 to 95, wherein (e) comprises distributing the population of beads onto an array. (Item 97) 97. The method of any one of items 92 to 96, wherein (f) comprises hybridizing an index primer to the index primer binding site and determining the sequence of the index. (Item 98) 98. The method of any one of items 92 to 97, wherein (f) comprises hybridizing a barcode primer to the barcode primer binding site and determining the sequence of the barcode. (Item 99) 99. The method of any one of items 92 to 98, wherein the capture probe comprises a protein. (Item 100) 99. The method of any one of items 92 to 98, wherein the targeting ligand comprises a targeting nucleic acid. (Item 101) 101. The method of claim 100, wherein the first polynucleotide comprises the capture probe. (Item 102) 102. The method of claim 100 or 101, wherein (e) comprises extending the first polynucleotide hybridized to the target nucleic acid. (Item 103) 103. The method of claim 102, wherein the extension comprises adding a detectable dideoxynucleotide. (Item 104) 104. The method of any one of items 92 to 103, carried out on a flow cell. (Item 105) 1. A method for detecting a target ligand on an array, comprising: (a) obtaining a first population and a second population of beads, each bead comprising: a capture probe capable of specifically binding to a target ligand; a nucleic acid encoding a barcode and a barcode primer binding site, said barcode representing said capture probe; a nucleic acid encoding an index and an index primer binding site, wherein the index indicates the source of the bead from the first population or the second population; and (b) contacting the first population of beads with a first sample containing a first target ligand, wherein the first target ligand specifically binds to the capture probes of the first population of beads, thereby obtaining a first population of target-bound beads; (c) contacting the second population of beads with a second sample containing a second target ligand, wherein the second target ligand specifically binds to the capture probes of the second population of beads, thereby obtaining a second population of target-bound beads; (d) randomly distributing the first population of target-bound beads and the second population of target-bound beads onto an array; (e) detecting the location of the beads containing the first target ligand and the second target ligand on the array; (f) determining the sequence of the index and the barcode of the beads containing the first target ligand and the second target ligand on the array. (Item 106) 106. The method of claim 105, wherein the capture probe comprises a polynucleotide. (Item 107) 107. The method of claim 105 or 106, wherein the targeting ligand comprises a nucleic acid. (Item 108) 108. The method of any one of items 105 to 107, wherein detecting the positions of the beads comprising the first target ligand and the second target ligand on the array comprises extending the capture probe by polymerase extension or ligation. (Item 109) 106. The method of claim 105, wherein the capture probe comprises a protein. (Item 110) 100. The method according to any one of items 105 to 109, wherein step (e) is carried out after step (f). (Item 111) 111. The method of any one of items 105 to 110, wherein the barcodes of the first population of beads comprise barcodes that are different from each other and the barcodes of the second population of beads comprise barcodes that are different from each other. (Item 112) 112. The method according to any one of items 105 to 111, wherein the indices of the first population of beads are the same as each other and the indices of the second population of beads are the same as each other. (Item 113) 113. The method of any one of items 105 to 112, wherein the array is located on the surface of a flow cell. (Item 114) 114. The method of any one of items 105 to 113, wherein the first population and the second population of beads are adapted to be attached to the array. (Item 115) The first and second populations of beads are labeled with biotin, streptavidin, or 115. The method of claim 114, wherein the array comprises biotin, streptavidin, or derivatives thereof. (Item 116) 115. The method of claim 114, wherein the first population and the second population of beads are magnetic.

Claims

1. 1. A method for determining the source of target nucleic acids from different sources on an array, comprising: (a) obtaining a first population and a second population of beads, the first population of beads comprises a first capture probe, a first barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the first barcode; the second population of beads comprises a second capture probe, a second barcode, and an oligonucleotide comprising a barcode primer binding site adjacent to the second barcode; (b) obtaining a first plurality of polynucleotides and a second plurality of polynucleotides, the first plurality of polynucleotides comprises a first target nucleic acid, the first plurality of polynucleotides is in solution; the second plurality of polynucleotides comprises a second target nucleic acid, the second plurality of polynucleotides being in solution; and (c) hybridizing the first target nucleic acid to the first capture probe in a first container to obtain a hybridized first bead comprising a first index, and hybridizing the second target nucleic acid to the second capture probe in a second container to obtain a hybridized second bead comprising a second index; the first indexes are the same as each other and indicate the source of the first target nucleic acid, the second indexes are the same as each other and indicate the source of the second target nucleic acid, and the first index is different from the second index; (d) randomly distributing the hybridized first beads and the hybridized second beads onto an array; (e) decoding the positions of the first and second beads on the array by sequencing the first and second barcodes on the array; (f) extending the first capture probe and the second capture probe to obtain nucleic acid sequence data of the first target nucleic acid and the second target nucleic acid; (g) determining the source of the nucleic acid sequences of the first target nucleic acid and the second target nucleic acid by sequencing the first index and the second index; A method comprising:

2. the first plurality of polynucleotides comprises the first index and an index primer binding site adjacent to the first index; 2. The method of claim 1, wherein the second plurality of polynucleotides comprises the second index and an index primer binding site adjacent to the second index.

3. 3. The method of claim 2, wherein the first plurality of polynucleotides or the second plurality of polynucleotides is obtained by tagging a nucleic acid sample with a plurality of transposomes.

4. The method of claim 3 , wherein the plurality of transposomes comprises the first index or the second index.

5. 5. The method of claim 4, further comprising adding an adaptor to the tagged nucleic acid sample, wherein the adaptor comprises the first index or the second index.

6. 6. The method of claim 5, further comprising amplifying the tagged nucleic acid sample with a primer comprising the first index or the second index.

7. 7. The method of any one of claims 2 to 6, wherein extending the first and second capture probes incorporates sequences complementary to the first and second indexes and the first and second index primer binding sites into the extended capture probes.

8. the first population of beads comprises a first index and an index primer binding site adjacent to the first index; 2. The method of claim 1, wherein the second population of beads comprises a second index and an index primer binding site adjacent to the second index.

9. 9. The method of claim 8, wherein the oligonucleotides of the first population of beads comprise the first index and the oligonucleotides of the second population of beads comprise the second index.

10. 10. The method of claim 2, wherein sequencing the first index and the second index comprises extending a primer hybridized to the index primer binding site.

11. The method of any one of claims 2 to 10, wherein the index primer binding sites are the same.

12. The method of any one of claims 1 to 11, wherein the first target nucleic acid and the second target nucleic acid are obtained from different nucleic acid samples.

13. The method of any one of claims 1 to 12, wherein the first target nucleic acid and the second target nucleic acid are obtained from genomic DNA.

14. The method of any one of claims 1 to 13, wherein the first barcode and the second barcode indicate the nucleic acid sequence of the first capture probe or the second capture probe.

15. The method of any one of claims 1 to 14, wherein the first barcodes are different from each other and the second barcodes are different from each other.

16. 16. The method of any one of claims 1 to 15, wherein sequencing the first barcode and the second barcode comprises extending a primer hybridized to the barcode primer binding site.

17. The method of any one of claims 1 to 16, wherein the barcode primer binding sites are the same.

18. The method of any one of claims 1 to 17, wherein extending the first capture probe and the second capture probe comprises polymerase extension.

19. 20. The method of claim 18, wherein extending the first capture probe and the second capture probe comprises the addition of a single nucleotide to the capture probe.

20. 20. The method of claim 18 or 19, further comprising ligating a locus-specific oligonucleotide to the extended capture probe.

21. 21. The method of any one of claims 18-20, wherein extending the first capture probe and the second capture probe comprises ligating a locus-specific oligonucleotide to the capture probe.

22. The method of any one of claims 1 to 21, wherein step (c) is carried out in solution.

23. The method of any one of claims 1 to 22, wherein the array is located on the surface of a flow cell.

24. The method of any one of claims 1 to 23, wherein the first bead and the second bead are adapted to be attached to the array.

25. 25. The method of claim 24, wherein the first bead and the second bead comprise biotin, streptavidin, or a derivative thereof, and the array comprises biotin, streptavidin, or a derivative thereof.

26. 25. The method of claim 24, wherein the first bead and the second bead are magnetic.

27. 27. A kit for carrying out the method of any one of claims 1 to 26, the kit comprising a plurality of populations of beads comprising oligonucleotides attached to the beads, the oligonucleotides comprising an index, an index primer binding site adjacent to the index, a capture probe, a barcode, and a barcode primer binding site adjacent to the barcode, the index varying between the populations of beads.

28. 28. The kit of claim 27, wherein the index primer binding site is the same in the plurality of populations.

29. 29. The kit of claim 27 or 28, wherein the barcode indicates the nucleic acid sequence of the capture probe.

30. 30. The kit of any one of claims 27 to 29, wherein the barcodes differ in a population of beads.

31. The kit of any one of claims 27 to 30, wherein the barcode primer binding site is the same in the plurality of populations.

32. locus-specific oligonucleotides, transposomes for tagging nucleic acid samples; a transposome comprising an index and an index primer binding site; an adapter containing an index and an index primer binding site; a primer capable of hybridizing to the index primer binding site or its complement; and a primer capable of hybridizing to the barcode primer binding site or its complement; The kit of any one of claims 27 to 31, further comprising a reagent selected from:

33. The kit of any one of claims 27 to 32, further comprising a flow cell.

34. Step (a) (i) obtaining a plurality of index polynucleotides, each of which comprises the first index or the second index and an index primer binding site adjacent to the first index or the second index, respectively; 2. The method of claim 1, further comprising: (ii) attaching the plurality of index polynucleotides to the first population or the second population of beads via adapters.

35. 35. The method of claim 34, wherein step (ii) comprises extending the adapter by polymerase extension.

36. 36. The method of claim 35, wherein each index polynucleotide comprises an adapter binding site, and said attaching comprises hybridizing said adapter binding site to said adapter.

37. 35. The method of claim 34, wherein step (ii) comprises ligating the index polynucleotide to the adaptor.

38. 38. The method of claim 37, wherein said attaching comprises hybridizing a splint polynucleotide to said adaptor and said index polynucleotide.

39. 35. The method of claim 34, wherein step (ii) comprises attaching the plurality of index polynucleotides to the adaptors of the population of beads via chemically reactive moieties.

40. 40. The method of claim 39, wherein said attaching comprises a click chemistry reaction.

41. 41. The method of any one of claims 34 to 40, wherein the first polynucleotides of the population of beads comprise capture probes that differ from each other.

42. The method of any one of claims 34 to 41, wherein the index of each index polynucleotide is the same.

43. The method of any one of claims 34 to 42, wherein the first polynucleotide comprises the capture probe.

44. A method described in any one of claims 34 to 43, further comprising contacting the first population or the second population of beads attached to the plurality of index polynucleotides via adapters with a plurality of nucleic acids including a target nucleic acid.

45. 45. The method of claim 44, further comprising mixing the first or second population of beads attached to the plurality of index polynucleotides via adaptors that have been contacted with a plurality of nucleic acids comprising a target nucleic acid with an additional population of indexed beads, wherein the additional population of indexed beads comprises index polynucleotides that comprise index polynucleotides that comprise indexes that differ from the indexes of the first or second population of beads attached to the plurality of index polynucleotides via adaptors that have been contacted with a plurality of nucleic acids.

46. The method of any one of claims 34 to 45, wherein the capture probe is attached to a protein.

47. The method of any one of claims 34 to 46, which is carried out in a flow cell.

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