RU2816708C2 - Methods and compositions for determining ligands on matrices using indices and barcodes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A method for detecting a plurality of target ligands in a matrix is described, including: (a) obtaining a substrate having a matrix of granules distributed on the surface of the specified substrate, wherein the specified matrix of granules contains the first and second subpopulation of granules, including: (i) obtaining the first and second subpopulations of granules, each granule containing: a capture probe, which capable of specifically binding to the target ligand, the first polynucleotide, which contains a barcode and a primer binding site at the 3' end relative to the specified barcode, while the specified barcode indicates a capture probe, and the second polynucleotide, containing an index and an index primer binding site at the 3' end relative to the index, each the index of the first subpopulation of granules differs from each index of the second subpopulation of granules; (ii) ensuring specific binding of the first target ligands to the specified capture probes of the specified first subpopulation of granules in the first reaction volume, and specific binding of the second target ligands to capture probes of the specified second subpopulation of granules in the second reaction volume, and (iii) distribution of the specified the first and second subpopulations of granules containing specifically bound first and second target ligands on the substrate, thereby obtaining a substrate having a matrix of granules; (b) detection in the specified matrix of specifically bound first and second target ligands of the specified first and second subpopulation of granules; (c) sequencing the barcodes of the specified first and second subpopulations of granules by hybridizing a plurality of primers with the primer binding sites of the barcodes of the specified first and second subpopulations of granules; and elongation of hybridized primers, thereby determining the position of the capture probes of the specified first and second subpopulations of granules in the specified matrix, (d) sequencing the indices of the specified first and second subpopulations of granules, thereby determining the positions of the specified first and second subpopulations of granules in the matrix; and (e) decoding the positions of granules in the specified matrix of granules, including the detected the target ligand in the matrix, thereby ensuring the identification of the target ligands of the specified first target ligands or the specified second target ligands in the matrix. A method for identifying target ligands in the matrix is also described, including: (a) obtaining the first and second subpopulations of granules, wherein each granule contains: (i) a capture probe that is capable of specifically binding to the target ligand, (ii) the first polynucleotide containing a barcode, wherein the specified barcode indicates a capture probe, and (iii) a second polynucleotide containing an index, wherein each index of the first subpopulation of granules differs from each index of the second subpopulation of granules; (b) ensuring specific binding of the first target ligands to the specified capture probes of the specified first subpopulation of granules in the first reaction volume, and specific binding of the second target ligands to capture probes of the specified second subpopulation of granules in the second reaction volume, and (c) distribution of the first and second subpopulations of granules containing specifically bound first and second target ligands on the substrate surface, thereby obtaining a matrix of granules; (d) detection of the specifically bound first and second target ligands of the specified first and second subpopulations in the specified matrix granules; (e) sequencing the barcodes of the pellet matrix; (f) sequencing the indices of the pellet matrix; and (g) decoding the positions of the granules in the specified pellet matrix, including the detected target ligand in the matrix, thereby ensuring the identification of target ligands in the pellet matrix.

EFFECT: invention expands the arsenal of methods for detecting specific nucleotide sequences present in a biological sample.

24 cl, 8 dwg, 4 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Patent Application 62/903,108, filed September 20, 2019, entitled METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT GENOTYPING ON ARRAYS USING INDEXES AND BARCODES; and U.S. Provisional Patent Application No. 62/750,370, filed October 25, 2018, entitled POSITIONAL IDENTIFICATION OF MICROFEATURES IN ARRAYS USING BARCODES, each of which is incorporated herein by reference in its entirety.

FIELD OF APPLICATION OF THE INVENTION [0002]

Some embodiments provided herein include methods and compositions for detecting target ligands on a matrix. In some embodiments, the capture probe specifically binds to a target ligand from the sample, the position of the capture probe-containing beads in the matrix is determined, and the bead is decoded to determine the capture probe and the sample. In some embodiments, the barcode indicates the presence of a capture probe attached to the bead; and the index indicates a subpopulation of granules. In some embodiments, the barcode and index are determined through sequencing.

PREREQUISITES FOR CREATION OF THE INVENTION

[0003] Detection of specific nucleotide sequences present in a biological sample 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, studying genetic susceptibility to disease, and assessing response to different types of treatment. A common technique for detecting specific nucleotide sequences in a biological sample is nucleic acid sequencing.

[0004] Nucleic acid sequencing techniques have largely evolved from the chemical digestion techniques used by Maxam and Gilbert and the chain extension techniques used by Sanger. Currently, several sequencing techniques are used that allow parallel processing of thousands of nucleic acids, all on a single chip. Some platforms include bead- and microarray-based configurations in which silica beads are functionalized with probes depending on the use of such configurations in applications including sequencing, genotyping, and gene expression profiling.

SUMMARY OF THE INVENTION

[0005] Some embodiments include a method for detecting multiple target ligands, comprising: (a) producing first and second subpopulations of beads, each bead comprising: a capture probe that specifically binds to the target ligand, a first polynucleotide comprising a barcode indicating a capture probe, and a barcode primer binding site at the 3' end relative to the barcode, and a second polynucleotide containing the index and an index primer binding site at the 3' end relative to the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation; (b) contacting first target ligands with capture probes of a first subpopulation of beads and contacting second target ligands with capture probes of a second subpopulation of beads; (c) distributing first and second subpopulations of granules containing specifically bound first and second target ligands on the substrate; (d) detecting capture probes specifically bound to the first and second target ligands; and (f) decoding the positions of the beads containing the detected capture probes on the substrate.

[0006] In some embodiments, the capture probe contains a nucleic acid, the first and second target ligands contain nucleic acids, and step (d) includes extending the capture probes specifically bound to the first and second target ligands.

[0007] In some embodiments, the first polynucleotide comprises a capture probe.

[0008] In some embodiments, the first and second bead subpopulation capture probes each contain different nucleotide sequences.

[0009] In some embodiments, the different capture probes for the first and second bead subpopulations contain the same nucleotide sequences.

[0010] In some embodiments, the capture probes contain a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0011] In some embodiments, step (d) includes polymerase extension of the capture probes.

[0012] In some embodiments, step (d) includes ligase extension of the capture probes.

[0013] In some embodiments, step (d) includes single nucleotide extension of the capture probes.

[0014] In some embodiments, step (d) includes extending the capture probes to multiple nucleotides.

[0015] In some embodiments, the elongated capture probes contain a detectable label.

[0016] In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof.

[0017] In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands.

[0018] In some embodiments, different capture probes of the first and second bead subpopulations specifically bind to the same target ligands.

[0019] In some embodiments, detection involves bringing first and second target ligands specifically bound to the capture probes into contact with a secondary antibody or antigen binding fragment thereof, wherein the secondary antibody or antigen binding fragment thereof comprises a detectable label.

[0020] In some embodiments, the barcode primer binding sites (barcode primer) contain the same nucleotide sequence.

[0021] In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence.

[0022] In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence.

[0023] In some embodiments, contacting the first target ligand with the capture probes of the first subpopulation of beads and bringing the second target ligands into contact with the capture probes of the second subpopulation of beads are carried out in different reaction volumes.

[0024] Some embodiments also include combining the first and second bead subpopulations before distributing the first and second bead subpopulations onto the substrate.

[0025] In some embodiments, the first subpopulation of beads is distributed on the substrate before the second subpopulation of beads is distributed on the substrate.

[0026] In some embodiments, step (d) is performed before step (c).

[0027] In some embodiments, the detected capture probes contain a detectable label.

[0028] In some embodiments, step (d) further includes determining the position of the detected capture probes on the substrate.

[0029] In some embodiments, determining the position of detected capture probes involves at least one cycle of sequencing by synthesis.

[0030] In some embodiments, step (e) includes decoding the index position of beads containing the detected capture probe by sequencing the indexes on the substrate.

[0031] In some embodiments, step (e) includes decoding the position of the barcodes of the beads containing the detected capture probe by sequencing the barcodes on the substrate.

[0032] In some embodiments, the flow cell contains a substrate.

[0033] In some embodiments, the distributed first and second subpopulations of beads comprise a matrix.

[0034] In some embodiments, the first and second target ligands are obtained from different entities.

[0035] In some embodiments, each of the first and second bead subpopulations contains at least 50 capture probes that are different from each other.

[0036] Some embodiments also include at least 10 different subpopulations of beads, each subpopulation containing an index that is different from the other subpopulation.

[0037] Some embodiments include a method for detecting a target nucleic acid, comprising: (a) providing a target nucleic acid comprising a first region capable of hybridizing with a first capture probe and a second region capable of hybridizing with a second capture probe; (b) producing a population of beads, each bead comprising: a first capture probe, a second capture probe, wherein the second capture probe is attached to the bead by a cleavable linker, wherein the second capture probe contains a detectable label, and a first polynucleotide containing a barcode indicating a first or second capture probe, and a barcode primer binding site at the 3' end of the barcode; (c) ligating the first capture probe to the second capture probe, comprising: hybridizing a target nucleic acid to the first and second capture probes of the bead population to produce a double-stranded nucleic acid containing a single-stranded gap between the first and second capture probes, and filling the gap between the first and second capture probes. second capture probes; (d) cleaving the cleavable linker to obtain a first capture probe containing a detectable label; (e) distribution of the population of granules on the substrate; and (f) decoding the position of the bead containing the first capture probe containing the detectable label on the substrate.

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

[0039] In some embodiments, step (e) is performed before step (d).

[0040] In some embodiments, each bead contains a second polynucleotide containing an index indicating the source of the target nucleic acid and an index primer binding site at the 3' end of the index.

[0041] In some embodiments, step (f) includes determining the position of a first capture probe containing a detectable label on the substrate.

[0042] In some embodiments, step (f) includes decoding the barcode position of a bead containing a first capture probe containing a detectable label on the substrate by sequencing the barcode on the substrate.

[0043] In some embodiments, the flow cell contains a substrate.

[0044] In some embodiments, the distributed population of beads comprises a matrix.

[0045] In some embodiments, the population of beads comprises a first and a second subpopulation of beads, each bead containing a second polynucleotide containing an index and an index primer binding site at the 3' end of the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation.

[0046] In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence.

[0047] In some embodiments, the ligation or cleavage step with the first subpopulation of beads is performed at a different reaction volume than the ligation or cleavage step with the second subpopulation of beads.

[0048] Some embodiments also include combining the first and second subpopulations of beads before distributing the subpopulation of beads on the substrate.

[0049] In some embodiments, the first subpopulation of beads is distributed on the substrate before the second subpopulation of beads is distributed on the substrate.

[0050] In some embodiments, step (f) includes decoding the index position of beads containing the detected capture probe by sequencing the indexes on the substrate.

[0051] Some embodiments include a set comprising: first and second bead subpopulations, each bead comprising: a capture probe that specifically binds to a target ligand, a first polynucleotide containing a barcode indicative of the capture probe, and a barcode primer binding site on 3'-end relative to the barcode, and a second polynucleotide containing the index and an index primer binding site at the 3'-end relative to the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation, wherein the first volume contains the first subpopulation of beads, and the second volume contains the second subpopulation of granules.

[0052] In some embodiments, the capture probe is selected from a nucleic acid, an antibody, or an antigen-binding fragment thereof.

[0053] In some embodiments, the first polynucleotide comprises a capture probe.

[0054] In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands.

[0055] In some embodiments, different capture probes of the first and second bead subpopulations specifically bind to the same target ligands.

[0056] Some embodiments include a method of decoding the positions of polynucleotides in a matrix, comprising: (a) providing a substrate having a matrix of polynucleotides distributed on the surface of the substrate, each polynucleotide containing a primer binding site 3' relative to the barcode, wherein each polynucleotide is associated with a probe capture; (b) hybridizing multiple primers to primer binding sites; and (c) determining the barcode sequences by extension of the hybridized primers, the sequence of each barcode indicating the position of the polynucleotide in the template.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0057] In FIG. 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.

[0058] In FIG. 2 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, with a cleavable linker provided between the capture probe and the barcode.

[0059] In FIG. 3 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.

[0060] In FIG. 4 shows an exemplary embodiment of a polynucleotide containing a spacer, a barcode, a primer binding site, and a capture probe attached to a bead via a 5' linker.

[0061] In FIG. 5A shows an exemplary embodiment of a bead with an attached first polynucleotide containing a barcode, a barcode primer binding site, and a capture probe, and with an attached second polynucleotide containing an index and an index primer binding site.

[0062] In FIG. 5B shows an example embodiment of a target nucleic acid containing a single nucleotide polymorphism (SNP) hybridized to a bead-attached capture probe.

[0063] In FIG. 5C illustrates an example embodiment of a capture probe extended with a detectable marker.

[0064] In FIG. 5D shows an exemplary embodiment of a barcode primer hybridized to a barcode primer binding site and extension of the barcode primer.

[0065] In FIG. 5E shows an exemplary embodiment of an index primer hybridized to an index primer binding site and extension of the index primer.

[0066] In FIG. 6A shows an exemplary embodiment of a bead containing a single polynucleotide containing a capture probe (left); a bead containing a nucleic acid capture probe, a polynucleotide containing a barcode indicating the capture probe, and a polynucleotide containing an index to distinguish one subpopulation of beads from another subpopulation of beads (middle); and a bead containing a capture probe containing an antibody or antigen-binding fragment of an antibody, a polynucleotide containing a barcode indicating the capture probe, and a polynucleotide containing an index allowing one subpopulation of beads to be distinguished from another subpopulation of beads (right).

[0067] In FIG. 6B shows exemplary embodiments of: a bead containing first and second capture probes hybridized to a target nucleic acid, the first capture probe containing a cleavable linker (left); and a bead containing a protein capture probe that transiently binds a substrate to generate a signal containing a detectable label (right).

[0068] In FIG. 6C shows an example implementation of a two-probe assay in which a target nucleic acid is hybridized to first and second capture probes on a bead, the first capture probe is ligated to a second capture probe, and the cleavable linker is cleaved to produce a bead containing an extended first capture probe containing the detectable label.

[0069] In FIG. 7A is a photograph of a pool of beads immobilized on a flow cell.

[0070] In FIG. 7B shows a copy number histogram for specific bead types in specific bins.

[0071] In FIG. 7C is a graph comparing the average intensity C and the average intensity T. The average intensity C is the fluorescence intensity coming from the labeled cytosine in the nucleotide pair, and the average intensity T is the fluorescence intensity from the labeled thymidine in the nucleotide pair.

[0072] In FIG. Figure 8 shows a histogram of the number of certain types of beads in certain cells for a representative sample.

DETAILED DESCRIPTION

[0073] Some embodiments provided herein include methods and compositions for detecting target ligands on a matrix. In some embodiments, the target ligand may comprise a nucleic acid, protein, or other antigen. In some embodiments, the capture probe specifically binds to a target ligand from the sample, the position of the capture probe-containing beads in the matrix is determined, and the bead is decoded to determine the capture probe and the sample. In some embodiments, the barcode indicates the presence of a capture probe attached to the bead; and the index indicates a subpopulation of granules. In some embodiments, the barcode and index are determined through sequencing. Some embodiments also include a two-probe assay in which the first and second capture probes attached to the bead are ligated to each other in the presence of a target nucleic acid, the end of the ligation product is cleaved from the bead, and the bead containing the extended capture probe is detected and decoded to matrix.

[0074] Some embodiments presented herein relate to high-throughput array genotyping. Some embodiments relate to decoding the positions of microelements in the matrix. In some embodiments, the trace elements comprise polynucleotides having barcodes and indexes. Some embodiments include sequencing barcodes and indexes to determine the positions of polynucleotides in the array.

[0075] Hybridization decoding involves determining the position of a capture probe in a randomly distributed capture probe matrix. The method typically involves several successive cycles of hybridizing labeled hybridization probes to one or more capture probe sites, visualizing the hybridization events, and removing the hybridization probes. Decoding by hybridization requires special reagents, special liquid devices and special detectors. In some embodiments, hybridization decoding can take up to 8 hours with 7-8 consecutive cycles.

[0076] Embodiments of the invention include randomly distributed arrays of polynucleotides containing a primer binding site and a barcode. In some embodiments, the barcode can be quickly sequenced to decode the template using a high-throughput sequencing system. Some embodiments can significantly reduce the time required to decode an array without additional reagents, hybridization probes, or specialized decoding equipment.

[0077] Some embodiments include the use of next generation sequencing (NGS) techniques and bead-based microarrays. Some such embodiments provide high-throughput, low-cost, high-throughput genotyping assays that can be performed on a standard NGS sequencing platform with minor modifications to substrates and reagents.

[0078] In some multiplexing methods, multiple nucleic acid samples from different sources can be processed in parallel while maintaining physical separation of each sample. Some embodiments presented herein include a nucleic acid indexing method that, by indexing each sample by index on a corresponding bead, eliminates the need for physical barriers separating individual samples at all stages. In some embodiments, index demultiplexing is performed by sequencing, and this can be achieved at the customer's site using standard synthetic sequencing (SBS) chemistry. Additionally, by adopting a decoding-by-sequencing (DBS) approach, which can be provided at the customer's site using a standard platform and chemical SBS, the cost, space and time constraints associated with in-house decoding of the bead matrix are reduced.

[0079] Some embodiments comprise a pool of indexed enrichment beads containing the bead shown in FIG. 5A. In some such embodiments, the bead comprises a first polynucleotide comprising a locus-specific capture probe, a barcode to identify the position of the corresponding probe on the array, and a barcode primer binding site for reading the barcode by SBS; and a second polynucleotide containing an index for sample multiplexing and an index primer binding site for reading the index by SBS.

[0080] In some embodiments, the complexity of the bead pool is determined by the number of capture probes or the number of composites (N) and the number of samples supported (S). Thus, a pool of granules that supports a number of composites N and samples S will consist of S × N unique granule types. In some embodiments, the capture probes and/or index primers include additional 3' orthogonal blockers to prevent interference during SBS on one of the two oligonucleotides.

[0081] Some embodiments include conducting a genotyping assay in which a multiwell plate containing S wells is loaded with S pools of beads, each pool of beads having a unique sample index, with each well containing N unique types of beads. After generating a nucleic acid library from the samples, processing the nucleic acid sample in steps involving random primer amplification followed by enzymatic fragmentation and purification, each sample library is added to an index well and allowed to hybridize with the capture probes. Once hybridization is complete by adding an incorporation mixture that includes fluorescent nucleotides and the appropriate polymerase, a single base extension assay is performed to probe the SNP of interest. At the end of the embedding, all bead capture samples on the plate are combined and loaded into the flow cell. The flow cell can be smooth or structured, and the surface can be suitably modified to maintain bead immobilization at the desired density. In some embodiments, after immobilization of the beads, an SNP readout is performed, which involves one scan cycle to read the signal obtained from the fluorescent incorporation at the SNP site. This cycle may include an SBS cycle on the device. To determine the capture probe and the position of a particular bead within the flow cell, a barcode reading is also performed, which involves 12-20 cycles of SBS, depending on the number of constituent bead pools. In some embodiments, this step can be replaced with additional rounds of sequencing after a specific SNP. In addition, a sample index reading is performed, which includes 6-12 SBS cycles to read the sample index. In some embodiments, the entire flow cell analysis may involve less than about 30 SBS cycles and may be completed in less than 4 hours.

[0082] As used herein, the term “matrix” may refer to a population of various trace elements, such as trace elements containing polynucleotides, that are associated or attached to a surface such that the different trace elements can be distinguished from each other according to relative position. A single matrix element may include a single copy of a trace element, or multiple copies of a trace element may be present as a population of trace elements within a single matrix element. The population of microelements in each element is usually homogeneous and has one type of microelements. Thus, multiple copies of a single nucleotide sequence may be present on an element, for example, multiple nucleic acid molecules having the same sequence.

[0083] In some embodiments, a heterogeneous population of trace elements may be present on the element. Thus, an element may not necessarily include only one type of trace element and may instead contain many different kinds of trace elements, such as a mixture of nucleic acids having different sequences. Adjacent matrix elements can be separated from each other without overlapping. Accordingly, elements can be located next to each other or separated by a gap. In embodiments in which the elements are spaced apart, adjacent sites may be spaced, for example, 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 the range of any two of the above distances. The arrangement of elements on a matrix can also be understood in terms of center-to-center distances between adjacent elements. The matrix used in the invention may have adjacent elements with a center-to-center distance 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 the range of any two of the above distances.

[0084] In some embodiments, the distance values described above or elsewhere herein may represent the average distance between adjacent matrix elements. Thus, not all adjacent elements need be within the specified range unless otherwise specifically stated, for example by specifically stating that the distance is a threshold distance between all adjacent matrix elements. Embodiments may be used with matrices having elements with different densities. Examples of density ranges for some embodiments include from about 10,000,000 elements/cm ² to about 200,000,000 elements/cm ² ; from about 100,000,000 elements/cm ² to about 1000,000,000 elements/cm ² ; from about 100,000 elements/cm ² to about 10,000,000 elements/cm ² ; from about 1,000,000 elements/cm ² to about 5,000,000 elements/cm ² ; from about 10,000 elements/cm ² to about 100,000 elements/cm ² ; from about 20,000 elements/cm ² to about 50,000 elements/cm ² ; from about 1000 elements/cm ² to about 5000 elements/cm ² or any density within the range of any two of the above densities.

[0085] As used herein, the term “surface” may refer to the portion of a substrate or support structure that is available for contact with reagents, beads, or analytes. The surface may be substantially level or flat. Alternatively, the surface may have a rounded or contoured shape. Examples of contours that can be used on a surface include dimples, recesses, pillars, ribs, channels, or the like. Examples of materials that can be used as a substrate or support structure include glass, such as modified or functionalized glass; plastic such as acrylic, polystyrene or styrene copolymer, polypropylene, polyethylene, polybutylene, polyurethane or TEFLON; polysaccharides or cross-linked polysaccharides such as agarose or sepharose; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; bundle of optical fibers or various other polymers. A surface suitable for use in the context of the invention may be made from a single material or a mixture of several different materials. In some embodiments, the surface contains dimples. In some embodiments, the support structure may comprise one or more layers. Examples of support structures may include a chip, film, multiwell plate, and flow cell.

[0086] As used herein, the term "pellet" may refer to a small body made of a rigid or semi-rigid material. The body may have a shape described, for example, as a sphere, oval, microsphere, or other conventional particle shape with symmetrical or asymmetrical dimensions. Examples of materials that can be used in the granules include glass, such as modified or functionalized glass; plastic such as acrylic, polystyrene or styrene copolymer, polypropylene, polyethylene, polybutylene, polyurethane or TEFLON; polysaccharides or cross-linked polysaccharides such as agarose or sepharose; nylon; nitrocellulose; resin; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metal; inorganic glass; or various other polymers. Examples of beads include controlled pore glass beads, paramagnetic beads, thorium dioxide sol, Sepharose beads, nanocrystals and other beads known in the art. Granules can be made from biological or non-biological materials. Magnetic beads are particularly suitable for use due to the ease with which magnetic beads can be manipulated using magnets at various stages of the methods described herein. The beads used in certain embodiments may have a diameter, width, or length from about 0.1 μm to about 100 μm, from about 0.1 nm to about 500 nm. In some embodiments, the beads used in certain embodiments of the invention may have a diameter, width, or length 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 diameter, width or length within the range of any two of the above diameters, widths or lengths. The size of the bead can be chosen so that it has a reduced size and therefore can provide more elements per unit area while maintaining sufficient signal (template copies per element) to analyze those elements.

[0087] In some embodiments, polynucleotides may be attached to beads. In some embodiments, the beads may be distributed into wells on the surface of the substrate. An example of bead matrices that can be used in certain embodiments include BEADARRAY random order technology (Illumina Inc., San Diego, Calif.). Such bead matrices are described in 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 herein by reference in its entirety.

[0088] As used herein, the terms “polynucleotide” and “nucleic acid” may be used interchangeably and may refer to the 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-stranded and single-stranded molecules. Examples of polynucleotides include a gene or gene fragment, genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, non-coding RNA (ncRNA) such as PIWI-interacting RNA (piRNA), small interfering RNA RNA (siRNA) and long non-coding RNA (lncRNA), small hairpin RNA (shRNA), small nuclear RNA (snRNA), microRNA (scrRNA), small nucleolar RNA (snoRNA) and viral RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, polynucleotide probe, primer or an amplified copy of any of the above options. The polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, including nucleotides with non-natural bases, nucleotides with modified natural bases, such as aza- or deazapurines. A polynucleotide may consist of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T). Uracil (U) may also be present, for example as a natural replacement for thymine if the polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term "sequence" refers to the literal representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

[0089] As used herein, the term “target nucleic acid” or its grammatical equivalent may refer to molecules or nucleic acid sequences that need to be sequenced, analyzed and/or further processed. In some embodiments, the target nucleic acid may be attached to a template. In some embodiments, a capture probe can be attached to an array, and the array can subsequently be used to detect a target nucleic acid in a sample that interacts with the probe. Accordingly, it should be understood that with respect to methods for detecting nucleic acids, in some embodiments, the terms “target” and “probe” may be used interchangeably.

[0090] As used herein, the term “capture probe” may refer to a polynucleotide having sufficient complementarity to specifically hybridize to a target nucleic acid. The capture probe may function as an affinity binding molecule to isolate the target nucleic acid from other nucleic acids and/or components in the mixture. In some embodiments, the target nucleic acid may be specifically bound by a capture probe via intermediate molecules. Examples of intermediate molecules include linkers, adapters, and other bridging nucleic acids having sufficient complementarity to specifically hybridize to both the target sequence and the capture probe.

[0091] As used herein, the term “hybridization,” “hybridize,” or their grammatical equivalent may refer to a reaction in which one or more polynucleotides react to form a complex that is at least partially formed through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur through Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner. The complex may have two chains forming a duplex structure, three or more chains forming a multistrand complex, one self-hybridizing chain, or any combination thereof. In addition to hydrogen bonding, chains can also be cross-linked or connected by other forces.

[0092] As used herein, the terms “extension,” “extension,” or any grammatical equivalent thereof, may refer to the addition of a dNTP to a primer, polynucleotide, or other nucleic acid molecule by an extension enzyme such as a polymerase. For example, in some of the methods described herein, the resulting extended primer contains RNA sequence information. Although some embodiments describe performing extension using a polymerase, such as DNA polymerase or reverse transcriptase, extension can be performed by any other method well known in the art. For example, extension can be accomplished by ligating short stretches of randomly selected oligonucleotides to each other, for example, oligonucleotides that have hybridized to the strand of interest.

[0093] As used herein, the terms “ligation,” “ligate,” or other grammatical equivalents thereof may refer to the joining of two nucleotide chains by a phosphodiester bond. This reaction can be catalyzed by a ligase. The term "ligase" refers to a class of enzymes that catalyze this reaction to hydrolyze ATP or a similar triphosphate.

Decoding by sequencing

[0094] Embodiments include decoding the position of microarray elements using high-throughput sequencing. In some embodiments, the matrix microelements comprise polynucleotides. In some embodiments, the polynucleotides may be randomly distributed on the surface of the substrate. In some embodiments, the polynucleotide may comprise a primer binding site and a barcode. In some embodiments, the polynucleotide may comprise a capture probe, a primer binding site, and a barcode.

[0095] Some embodiments for decoding the position of polynucleotides in a matrix may include (a) providing a substrate having a matrix of polynucleotides distributed on the surface of the substrate, each polynucleotide containing a barcode and a primer binding site; (b) hybridizing multiple primers to primer binding sites; and (c) determining barcode sequences by extending hybridized primers. In some such embodiments, the sequence of each barcode may indicate the position of the polynucleotide in the array. For example, in some embodiments, the array may be generated using polynucleotides, where barcodes are known to be associated with certain capture probes such that determining the position of the barcode on the array may indicate the position of the corresponding capture probe. In such embodiments, each polynucleotide may be linked to a capture probe through a common element. For example, each polynucleotide and capture probe may be associated with the same trace element, such as a bead. In further such embodiments, each polynucleotide may comprise a capture probe.

[0096] In some embodiments, the barcode may contain a nucleotide sequence that can be used to identify a polynucleotide in the array. A barcode may contain a unique nucleotide sequence that is distinguishable from other barcodes. It can also be distinguished from other nucleotide sequences in target polynucleotides and nucleic acids by the barcode sequence, as well as by the position of the barcode in the polynucleotide, for example, its position 5' from the primer binding site. For example, in some embodiments, a barcode sequence may be present more than once in multiple nucleic acids, but a barcode located 5' from the primer binding site may be detected. The barcode may be of any desired sequence length sufficient to provide a unique nucleotide sequence within a plurality of barcodes in a population and/or within a plurality of target polynucleotides and nucleic acids being analyzed or interrogated. In some embodiments, the barcode is a nucleic acid or region within a polynucleotide in the range of about 6-30 nucleotides. The barcode may contain, 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 it can be longer. For example, a barcode may be 35, 40, 45 or 50 nucleotides long, or it may be longer. Suitable barcodes for some embodiments are described in US Patent No. 8,053,192, which is incorporated herein by reference in its entirety. In some embodiments, the barcode may distinguish a polynucleotide from another polynucleotide in the array, such that each barcode is different from the other barcode. In some embodiments, the barcode may distinguish a population of polynucleotides from another population of polynucleotides in the array, such that a set of barcodes is different from another set of barcodes.

[0097] In some embodiments, the primer binding site may be 3' to the barcode such that a primer hybridized to the primer binding site can be extended to form a complement to the barcode. For example, the primer can be extended to obtain a barcode sequence. In some embodiments, the primer binding site may be immediately adjacent to a barcode in the polynucleotide. In some embodiments, each primer binding site in a population of polynucleotides may have the same sequence. In some embodiments, a subpopulation of polynucleotides may comprise primer binding sites for a first sequence, and another subpopulation of polynucleotides may contain primer binding sites for a second sequence. In some embodiments, hybridization of different primers to multiple different primer binding sites may be simultaneous, sequential, or iterative.

[0098] Some embodiments include polynucleotides containing a capture probe. In some embodiments, the capture probe may contain a sequence that can hybridize to a target nucleic acid. In some embodiments, the population of polynucleotides may contain capture probes that are different from each other. In some embodiments, each capture probe may be different from each other. In some embodiments, the capture probes may be similar to each other, for example, they may have similar sequences and/or similar sequences of similar length. In some embodiments, the capture probe may differ from one other capture probe by less than 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, nucleotides. 3 or 2, wherein the number of nucleotides can be represented by consecutive nucleotides, non-sequential nucleotides, inserted nucleotides or deleted nucleotides in the capture probe. In some embodiments, the capture probe may differ from one other capture probe by a single nucleotide. In some embodiments, the primer binding site and barcode may be 5' from the capture probe. In some embodiments, the primer binding site and barcode may be 3' from the capture probe.

[0099] Some embodiments include polynucleotides containing cleavable linkers. In some such embodiments, the cleavable linker may be positioned such that cleavage of the linker can separate the capture probe from the primer-barcode binding site. In some embodiments, the cleavable linker may be located in the polynucleotide between the capture probe and the primer and barcode binding site. In some embodiments, the cleavable linker may remove a polynucleotide containing a primer binding site and a barcode associated with a capture probe. For example, the polynucleotide and the capture probe may both be associated with a bead. Cleavage of the cleavable linker may remove the polynucleotide containing the primer binding site and barcode from the bead.

[0100] In some embodiments, the cleavable linker may have a length corresponding to at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 500 nucleotides, or a length within the range of any two of the above length. In some embodiments, the cleavable linker is cleavable by agents such as light, base, acid, and enzymes, such as sequence-specific restriction enzymes or proteases. The cleavable linker may include a specific nucleotide sequence, such as an enzyme recognition site, and/or may include certain modified nucleotides amenable to cleavage by the agent. In some embodiments, the cleavable linker may contain uracil, which can be cleaved by an exogenous base-cleaving agent such as DNA glycosylase (uracil-DNA glycosylase (UDG)). In some embodiments, the cleavable linker may contain 8-hydroxyguanine, which can be cleaved by 8-hydroxyguanine DNA glycosylase (formamidopyridine DNA glycosylase (FPG) protein). Other examples of cleavable linkers are described in US Patent Application Publication 2005/0181394, which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the polynucleotides are attached to a substrate. In some embodiments, the substrate may comprise a granule. By means of a single-point covalent bond to a substrate surface at or near the 5' end or 3' end of a polynucleotide, the polynucleotides can be immobilized on a substrate, such as a bead or other surface. In some embodiments, the polynucleotide may comprise a spacer that is attached to a substrate. In some embodiments, the spacer may have a length corresponding to at least 2, 3, 5, 10, 15, 20, 25, 30, 50, 100, 500 nucleotides, or a length within the range of any two of the above lengths. For this purpose, any suitable covalent coupling means known in the art can be used. The addition chemistry chosen will depend on the nature of the solid support and any derivatization or functionalization applied to it. The polynucleotide may contain a functional group, which may be a non-nucleotide chemical modification, to facilitate attachment. In some embodiments, the polynucleotide may contain a sulfur-containing nucleophilic substance, such as phosphorothioate or thiophosphate, for example, located at the 5' end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile will bind to the bromoacetamide group present in the hydrogel. An example of a means of attaching a polynucleotide to a solid support is the 5'-phosphorothioate coupling to a hydrogel consisting of polymerized acrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), which is described in US Pat. No. 8,168,388, which is incorporated herein in its entirety by links.

[0102] In some embodiments, the position of the polynucleotides in the array can be decoded by sequencing the barcodes of the polynucleotides. For example, a barcode sequence may be associated with a site on an array, and the site may be associated with a specific capture probe. Some embodiments include next generation sequencing (NGS), which may refer to sequencing methods that enable 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. SBS monitors nucleic acid primer extension along a nucleic acid template to determine the sequence of nucleic acids in the template. The underlying chemical process may be polymerization. In a specific embodiment of the extension polymerase-based SBS, fluorescently labeled nucleotides are added to the primer in a template-dependent manner such that detection of the order and type of nucleotides added to the primer can be used to determine the template sequence.

[0103] One or more amplified nucleic acids can be subjected to SBS or another detection technique that involves repeated delivery of reagents in cycles. For example, to initiate the first round of SBS, one or more labeled nucleotides, DNA polymerase, etc. may be allowed to flow. into (or through) a hydrogel bead containing one or more amplified nucleic acid molecules. Those sites where primer extension results in the incorporation of a labeled nucleotide can be detected. The nucleotides may additionally have a reversible termination property that terminates additional primer extension upon addition of a nucleotide to the primer. For example, a nucleotide analog having a reversible terminator fragment can be added to the primer so that subsequent extension cannot occur until an unblocking agent is delivered to remove the fragment. Thus, for embodiments that employ reversible termination, delivery of the unblocking reagent to the flow cell may occur before or after detection. Washings can be carried out between different delivery stages. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n.

[0104] Some embodiments of SBS include detection of a proton released upon insertion of a nucleotide into the extension product. For example, sequencing based on the detection of released protons may use an electrical detector and related techniques that are available on the market. Examples of such sequencing systems are pyrosequencing, such as the commercially available platform from 454 Life Sciences, a subsidiary of Roche; sequencing using γ-phosphate 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, which is described in US Patent Application Publication 2005/0130173 and 2006/0134633 and US Patent Nos. 4,971,903; 6,258,568 and 6,210,891, which are incorporated herein by reference in their entirety. Some embodiments include ligation sequencing, which is described in US Patent No. 5,599,675 and US Patent No. 5,750,341, each of which is incorporated herein by reference in its entirety.

[0105] In some embodiments, methods may be used that include real-time monitoring of DNA polymerase activity. For example, nucleotide insertions can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-carrying polymerase and γ-phosphate-labeled nucleotides or through zero-mode waveguides (ZMWs). Another useful sequencing technique is nanopore sequencing. In some embodiments of the nanopore, the target nucleic acid or individual nucleotides removed from the target nucleic acid pass through the nanopore. As a nucleic acid or nucleotide passes through a nanopore, each type of nucleotide can be identified by measuring fluctuations in the electrical conductivity of the pore.

[0106] As shown in FIG. 1, in some embodiments, the array microelement may comprise a polynucleotide attached to bead 10 via a 5' linker 20. The polynucleotide may comprise a barcode 30, a primer binding site 40, and a capture probe 50. Primer 60 can hybridize to the primer binding site and can be extended to produce a barcode sequence to decode the position of the microelement in the matrix. In some embodiments, the capture probe can hybridize to target nucleic acids, and the capture probe can be extended, for example, by a polymerase or by a ligase. In some embodiments, the capture probe may participate in bridging amplification. Bridging amplification methods are described in US Patent Nos. 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety.

[0107] As shown in FIG. 2, in some embodiments, the array microelement may comprise a polynucleotide comprising a capture probe 50, a barcode 30, and a primer binding site 40, wherein the polynucleotide is attached to the bead 10 via a 5' linker 20. In some embodiments, the polynucleotide may include a cleavable linker 70 between the capture probe and the barcode. In some embodiments, the primer 60 can be hybridized to the primer binding site and the barcode sequence can be determined, thereby decoding the position of the trace element in the matrix. In some embodiments, the polynucleotide may be cleaved and the barcode and primer binding site removed from the microcell containing the bead and capture probe. Some such embodiments provide decoded templates before hybridizing target nucleic acids to capture probes. In some embodiments, the target nucleic acid may be hybridized to a capture probe at a decoded position in the array. In some embodiments, the hybridized capture probe may be extended, for example, by a polymerase or by a ligase. In some embodiments, the capture probe may participate in bridging amplification.

[0108] As shown in FIG. 3, in some embodiments, the array microelement may comprise a polynucleotide comprising a barcode 30 and a primer binding site 40, as well as a capture probe 50 attached to the bead 10 via a 3' linker 25. In some embodiments, the primer binding site may be adjacent to the linker, attached to the granule. Some embodiments may include the use of such trace elements in assays for screening and developing certain polymerases. For example, polymerase activity can be screened against primer sites attached to beads without spacers versus primer sites attached to beads with spacers. In some embodiments, the target nucleic acid may be hybridized to a capture probe. In some embodiments, hybridized target nucleic acids can be extended, for example, by a polymerase or by a ligase.

[0109] As shown in FIG. 4, some microarray embodiments may include a polynucleotide comprising a spacer 80, a barcode 30, a primer binding site 40, and a capture probe 50 attached to the bead 10 via a 5' linker 20.

Sequencing of multiple target nucleic acids

[0110] Some embodiments include sequencing multiple target nucleic acids. In some embodiments, target nucleic acids may be obtained from different sources, such as different subjects, such as genomic DNA from different subjects. In some embodiments, different target nucleic acids may be associated with different indexes such that the index may identify a particular population of target nucleic acids, such as a population derived from a single source.

[0111] Some embodiments include producing at least a first and a second bead subpopulation, each bead comprising a first polynucleotide containing a capture probe, a barcode indicating a capture probe of the same bead, and a barcode primer binding site at the 3' end relative to the barcode, and a second polynucleotide containing the index and an index primer binding site at the 3' end of the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation.

[0112] In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence.

[0113] In some embodiments, each of the first and/or second bead subpopulation capture probes contains different nucleotide sequences from each other. For example, the first subpopulation capture probes may be different from each other; and/or the first subpopulation capture probes may be different from each other. In some embodiments, each of the first bead subpopulation capture probes comprises capture probes having the same nucleotide sequences as the second bead subpopulation capture probes. In some embodiments, the capture probe may comprise a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement. In some embodiments, the barcode primer binding sites contain the same nucleotide sequence.

[0114] Some embodiments also include hybridizing the first target nucleic acids with the first bead subpopulation capture probes and hybridizing the second target nucleic acids with the second bead subpopulation capture probes. In some such embodiments, hybridization of the first target nucleic acids to the first bead subpopulation capture probes and hybridization of the second target nucleic acids to the second bead subpopulation capture probes are performed at different positions. For example, different positions provide different reaction volumes, such as different wells, such as different wells in a multiwell plate. For example, in a 96-well plate, 96 different bead subpopulations can be hybridized to 96 different target nucleic acids, in which each different bead subpopulation hybridizes to a different target nucleic acid in a different well.

[0115] In some embodiments, different subpopulations of beads containing hybridized capture probes and nucleic acids are distributed on a substrate, such as a planar substrate. In some embodiments, the distributed subpopulations of granules comprise a matrix. In some embodiments, the substrate contains multiple discrete sites. In some embodiments, the substrate contains a plurality of wells. In some embodiments, the substrate contains multiple channels. In some embodiments, the flow cell contains a substrate.

[0116] In some embodiments, different subpopulations of beads containing hybridized capture probes and target nucleic acids are combined prior to distribution on the substrate. In some embodiments, different subpopulations of beads containing hybridized capture probes and target nucleic acids are sequentially distributed on the substrate. For example, a first subpopulation of beads containing hybridized capture probes and target nucleic acids is distributed on a substrate before a second subpopulation of beads containing hybridized capture probes and target nucleic acids is distributed on the substrate.

[0117] In some embodiments, the hybridized capture probes are elongated. In some embodiments, the hybridized capture probes are extended before distributing various subpopulations of beads containing the hybridized capture probes and target nucleic acids onto the substrate. In some embodiments, the hybridized capture probes are extended after distributing beads containing the hybridized capture probes and target nucleic acids onto a substrate. In some embodiments, extension of the hybridized capture probes may involve polymerase extension. In some embodiments, extension of hybridized capture probes may involve ligase-based extension, for example, ligation of the extension probe to the capture probe in the presence of a ligase. In some embodiments, a detectable marker may be added to the elongated capture probe during the extension step. In some embodiments, the detectable marker may comprise a fluorescent marker.

[0118] Some embodiments include decoding beads on a substrate. Some embodiments include decoding the beads by determining the positions of elongated capture probes, barcodes, and indexes on the substrate. In some embodiments, the presence of a specific barcode at a specific position on the substrate indicates a specific capture probe at that position. In some embodiments, the presence of a particular index at a position on a substrate indicates that the target nucleic acid of a particular subpopulation of target nucleic acids is associated with a position on the substrate. In some embodiments, the presence of an extended capture probe at a position on the substrate indicates the presence of a particular target nucleic acid in subpopulations of target nucleic acids. In some embodiments, the barcode identity, the index identity, and the presence of an extended capture probe at one position on the surface indicate the presence of a specific target nucleic acid in a specific subpopulation of target nucleic acids.

[0119] In some embodiments, decoding beads on a substrate includes detecting the positions of hybridized capture probes or extended capture probes. In some embodiments, detecting the position of the hybridized capture probes or extended capture probes includes extending the hybridized capture probes with a detectable marker. In some embodiments, determining the position of hybridized capture probes or extended capture probes involves at least one round of sequencing by synthesis.

[0120] In some embodiments, decoding the beads on the substrate includes decoding the position of the indices of the beads containing the elongated capture probe. Some embodiments include hybridizing multiple index primers to index primer sites and extending the hybridized index primers. In some embodiments, extension of the hybridized index primers involves at least one round of sequencing by synthesis. In some embodiments, decoding the position of bead indices involves sequencing the indices on the substrate.

[0121] In some embodiments, decoding beads on a substrate includes decoding the position of bead barcodes containing an elongated capture probe. Some embodiments include hybridizing multiple barcode primers to barcode primer sites and extending the hybridized barcode primers. In some embodiments, decoding the beads on the substrate includes extending the hybridized barcode primers, involves at least one round of sequencing by synthesis. In some embodiments, decoding the beads on the substrate includes decoding the position of the bead barcodes, includes sequencing the barcodes on the substrate.

[0122] In some embodiments, each of the first and second bead subpopulations contains at least 50 capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 100, 200, 300, 400, or 500 or more capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 5000 capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 50,000 capture probes containing different nucleotide sequences.

[0123] Some embodiments include at least 10 different subpopulations of beads, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 100 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 1000 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 10,000 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation.

[0124] Aspects of some embodiments are shown in FIG. 5A-FIG. 5E. As shown in FIG. 5A, a subpopulation of beads comprises a bead with an attached first polynucleotide containing a barcode, a barcode primer binding site, and a capture probe, and with an attached second polynucleotide containing an index and an index primer binding site. Different subpopulations of granules may include different indices. Different subpopulations of beads, each subpopulation having a specific index, can be distributed among the wells of a 96-well plate such that each well contains one subpopulation of beads.

[0125] As shown in FIG. 5B, a target nucleic acid from a population of target nucleic acids is hybridized to a capture probe, where 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 may be obtained from a different source, such as a different subject. For example, a subpopulation of target nucleic acids can be obtained by preparing a nucleic acid library from a single nucleic acid source, such as a single nucleic acid sample from a subject. In some embodiments, target nucleic acids do not require adapter sequencing.

[0126] As shown in FIG. 5C, a capture probe that is hybridized to a SNP-containing target nucleic acid can be extended. In some embodiments, extension can be accomplished by adding an incorporation mixture that contains fluorescent nucleotides and an appropriate polymerase. In some embodiments, the extension is an extension along one base.

[0127] In some embodiments, all of the beads are combined and distributed over the surface of the flow cell. The flow cell may have a structured surface, and the surface may be modified to maintain immobilization of the beads at the desired density.

[0128] In some embodiments, an SNP read is performed. In some embodiments, a scan cycle is performed to read the signal from the incorporation of a capture probe at the SNP site. In some embodiments, this cycle may include at least one cycle of sequencing by synthesis. In some embodiments, the 3' ends of the extended capture probes are cleaved off and blocked.

[0129] As shown in FIG. 5D, the barcode primer is hybridized to the barcode primer binding site, and the barcode primer is extended. In some embodiments, extending the barcode primer involves reading the barcode. In some embodiments, extension may include at least one round of sequencing by synthesis. In some embodiments, the number of synthetic sequencing cycles may depend on the number of different barcodes in a subpopulation of beads. In some embodiments, the extension may involve 12-20 cycles of sequencing by synthesis. In some embodiments, the extension allows identification of the capture probe and the position of a particular bead within the flow cell.

[0130] As shown in FIG. 5E, the index primer is hybridized to the index primer binding site, and the index primer is extended. In some embodiments, extension of the index primer includes reading the index. In some embodiments, extension may include at least one round of sequencing by synthesis. In some embodiments, the number of synthetic sequencing cycles may depend on the number of different indices in multiple bead subpopulations. In some embodiments, extension may involve 6-12 cycles of sequencing by synthesis.

Specific methods for detecting target ligands

[0131] Some embodiments include methods for detecting target ligands. In some embodiments, target ligands may comprise nucleic acids, proteins, or other antigens. In some embodiments, target ligands are obtained from different sources, such as from different samples, different individual subjects, or different populations of subjects.

[0132] In some embodiments, methods for detecting target ligands may include producing a population of beads, each bead containing a capture probe that specifically binds to the target ligand. For example, the capture probe may contain a nucleic acid, an antibody, or an antigen-binding fragment of an antibody. In some embodiments, the bead contains a first polynucleotide containing a barcode indicating a capture probe of the same bead, and a barcode primer binding site at the 3' end of the barcode. In some embodiments, each bead also contains a second polynucleotide containing an index and an index primer binding site at the ≈/-terminal to the index. In some embodiments, the granule population comprises first and second granule subpopulations. In some embodiments, the indices of the first subpopulation of beads are different from the indices of the second subpopulation of beads. For example, the indices of the first subpopulation of beads can be used to determine the first subpopulation of beads from the indices of the second subpopulation of beads.

[0133] Some embodiments include contacting first target ligands with capture probes of a first bead subpopulation and contacting second target ligands with capture probes of a second bead subpopulation. For example, first target ligands can be obtained from a first sample of ligands, and second target ligands can be obtained from a second sample of ligands. Some embodiments also provide for the distribution on the substrate of first and second subpopulations of beads containing specifically bound first and second target ligands. Some embodiments also include detecting capture probes specifically bound to first and second target ligands distributed on the substrate. Some embodiments also include decoding the positions of the beads containing the detected capture probes on the substrate.

[0134] In some embodiments, the capture probe contains a nucleic acid and the target ligands contain nucleic acids. In some embodiments, the first polynucleotide comprises a capture probe. In other embodiments, the capture probe is different from the first polynucleotide. In some embodiments, the bead subpopulation capture probes contain different nucleotide sequences from each other. In some embodiments, different subpopulations of beads may contain the same or different capture probes. In some embodiments, the capture probes contain a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0135] In some such embodiments, detecting capture probes specifically bound to target ligands involves extending capture probes specifically bound to target ligands. In some embodiments, extension may include polymerase extension and/or ligase extension. In some embodiments, the extension includes a detectable nucleotide, such as a fluorescently labeled nucleotide. In some embodiments, the extension includes single nucleotide extension of the capture probes. In some embodiments, the extension includes extending the capture probes to multiple nucleotides.

[0136] In some such embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof. In some embodiments, bead subpopulation capture probes specifically bind to distinct target ligands. In some embodiments, different subpopulations of beads may contain the same or different capture probes. In some such embodiments, different bead subpopulation capture probes specifically bind to the same target ligands.

[0137] In some embodiments, detection of capture probes specifically bound to target ligands involves an immunoassay. For example, target ligands specifically bound to capture probes are contacted with a secondary antibody or antigen-binding fragment thereof, wherein the secondary antibody or antigen-binding fragment thereof contains a detectable label, such as a fluorescent label.

[0138] In some embodiments, the barcode primer binding sites contain the same nucleotide sequence.

[0139] In some embodiments, the bead subpopulation index nucleotide sequences contain the same nucleotide sequence and allow one bead subpopulation to be distinguished from another bead subpopulation. For example, the index nucleotide sequences of the first granule subpopulation and the index nucleotide sequences of the second granule subpopulation contain the same nucleotide sequence.

[0140] In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence.

[0141] In some embodiments, bringing the first target ligand into contact with the capture probes of the first subpopulation of beads and bringing the second target ligands into contact with the capture probes of the second subpopulation of beads are at different positions. For example, different positions may provide different reaction volumes, such as different volumes in different wells in a microtiter plate.

[0142] Some embodiments also include combining the first and second bead subpopulations before distributing the first and second bead subpopulations onto the substrate. In other embodiments, the first subpopulation of beads is distributed on the substrate before the second subpopulation of beads is distributed on the substrate. In some embodiments, subpopulations of beads are distributed on the substrate prior to detection of a capture probe specifically bound to a ligand.

[0143] In some such embodiments, detecting capture probes specifically bound to target ligands may also include determining the position of capture probes specifically bound to target ligands on the substrate.

[0144] In some embodiments, decoding the position of the detected capture probes includes decoding the position of the indices of the beads containing the detected capture probe. Some embodiments also include hybridizing multiple index primers to index primer sites and extending the hybridized index primers. In some embodiments, the extension of the hybridized index primers comprises at least one round of sequencing by synthesis. In some embodiments, decoding the position of bead indices involves sequencing the indices on the substrate.

[0145] In some embodiments, decoding the position of the detected capture probes includes decoding the position of barcodes of the beads containing the detected capture probe. Some such embodiments include hybridizing multiple barcode primers to barcode primer sites and extending the hybridized barcode primers. In some embodiments, extension of the hybridized barcode primers involves at least one round of sequencing by synthesis. In some embodiments, decoding the position of the bead barcodes involves sequencing the barcodes on the substrate.

[0146] In some embodiments, the substrate contains multiple discrete sites. In some embodiments, the substrate contains a plurality of wells. In some embodiments, the substrate contains multiple channels. In some embodiments, the flow cell contains a substrate. In some embodiments, the distributed first and second subpopulations of beads comprise a matrix.

[0147] In some embodiments, each of the first and second bead subpopulations contains at least 50, 100, 500, 1000, or 5000 distinct capture probes, or any number of capture probes ranging between any two of the above amounts. Some embodiments also contain at least 5, 10, 20, 50, 100, 200, 500, 1000 different bead subpopulations, each subpopulation containing an index different from the other subpopulation, or any number of different bead subpopulations between any two of the above amounts.

[0148] In FIG. 6A on the left depicts an embodiment that includes a bead 200 having a polynucleotide 210 attached to the bead. The polynucleotide contains a capture probe that is hybridized to a target nucleic acid 220. The capture probe is extended with a nucleotide containing a detectable tag 230. In some embodiments, the polynucleotide may contain a barcode indicating the capture probe and a barcode primer binding site suitable for sequencing and identifying the barcode . In some embodiments, the polynucleotide may also include an index indicating a subpopulation of beads from another subpopulation of beads, and an index primer binding site suitable for sequencing and index determination. In some embodiments, the bead can be distributed into a matrix on a substrate and decoded. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix.

[0149] In FIG. 6A in the middle depicts an embodiment that includes a bead 200 with a capture probe 240 attached to the bead. The capture probe is hybridized to the target nucleic acid 220. Also attached to the bead is a first polynucleotide 260 containing a barcode indicating the capture probe and a barcode primer binding site suitable for sequencing and barcode detection. Also attached to the bead is a second polynucleotide 250 containing an index indicating a subpopulation of beads from another subpopulation of beads. The capture probe is extended with a nucleotide containing detectable tag 230. In some embodiments, the bead can be distributed in a matrix on a substrate and decoded. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix.

[0150] In FIG. 6A to the right depicts an embodiment that includes a bead 200 with a capture probe 270 attached to the bead, wherein 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, which contains a detectable tag 230. Also attached to the bead is a first polynucleotide 260 containing a barcode indicating the capture probe and a barcode primer binding site suitable for sequencing and barcode detection . Also attached to the bead is a second polynucleotide 250 containing an index indicating a subpopulation of beads from another subpopulation of beads. In some embodiments, the bead can be distributed into a matrix on a substrate and decoded. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix.

[0151] In FIG. 6B on the right depicts an embodiment that includes a bead 200 with a capture probe 330 containing a protein attached to the bead. In some embodiments, the granule may be distributed in a matrix on a substrate. Protein substrate 340 is used to contact the protein to generate a signal containing a detectable label 230. The position of the signal on the array can be determined. In some embodiments, the bead may comprise a first polynucleotide containing a barcode indicating a capture probe and a barcode primer binding site suitable for sequencing and barcode detection also attached to the bead. In some embodiments, the bead may comprise a second polynucleotide containing an index indicating a subpopulation of beads from another subpopulation of beads also attached to the bead. In some embodiments, the granule is decoded into a matrix. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix.

Sequencing and analysis of target nucleic acids

[0152] Some embodiments include sequencing and/or analysis of target nucleic acids. Some embodiments include decoding the positions of polynucleotides in a template in accordance with the methods provided herein; hybridization of target nucleic acid with capture probes; extension of capture probes; and detecting extension of capture probes hybridized to the target nucleic acid at a position on the array. In some embodiments, the positions of the polynucleotides on the array can be decoded before the target nucleic acid is hybridized to the polynucleotides. In some embodiments, the positions of the polynucleotides on the array can be decoded after detection of extension of capture probes hybridized to the target nucleic acid. In some embodiments, each polynucleotide may be linked to a capture probe through a common element. For example, each polynucleotide and capture probe may be associated with the same trace element, such as a bead. In further such embodiments, each polynucleotide may comprise a capture probe.

[0153] Some embodiments include single base extension (SBE) capture probes. In some embodiments, SBE can be used to detect alleles, mutations, or other elements of target nucleic acids. Briefly, SBE uses a capture probe that hybridizes to a target genomic fragment at a position that is proximal or adjacent to the detection position, with the detection position indicating a specific locus. The polymerase can be used to extend the 3' end of a capture probe with a detector-labeled nucleotide analogue. Based on the precision of the enzyme, a nucleotide is inserted into the capture probe only if it is complementary to the detection position of the target nucleic acid. If necessary, the nucleotide can be derivatized in such a way that the use of a blocking group, including reversible blocking groups, cannot lead to additional extensions and thus only one nucleotide is added. The presence of a labeled nucleotide in an extended capture probe can be detected, for example, at a specific location in the array, and the added nucleotide determined to establish the identity of a locus or allele. SBE can be carried out under known conditions, such as those described in US Pat. Nos. 9,441,267 and US Pat. Nos. 9,045,796, each of which is incorporated herein by reference in its entirety.

[0154] Some embodiments include allele-specific primer extension (ASPE). In some embodiments, ASPE may include extension of capture probes that differ in the nucleotide composition at their 3' end. The ASPE method can be carried out using a nucleoside or nucleotide containing a cleavable linker so that once the probe is detected the label can be removed. This makes it possible to use additional probes or confirm that the detected signal is due to a tag that has now been removed. Briefly, ASPE can be performed by hybridizing a target nucleic acid with a capture probe having a 3' sequence region that is complementary to the detection position and a 5' sequence region that is complementary to the sequence that is adjacent to the detection position. Template-directed modification of the 3' portion of the capture probe, for example by adding a labeled nucleotide using a polymerase, produces a labeled extension product, but only if the template includes the target sequence. The presence of such a tagged primer extension product can then be detected, for example based on its position in the array, indicating the presence of a specific allele. In some embodiments, ASPE can be performed with multiple capture probes that have similar 5' ends so that they are annealed adjacent to the same detection position in the target nucleic acid, but different 3' ends so that they are modified by the polymerase only capture probes having a 3' end that complements the detection position. A capture probe that has a 3'-terminal base that is complementary to a particular detection position is called a position-perfect match (PM) probe, whereas capture probes that have a 3'-terminal mismatched base and are not designed to be extended during the ASPE reaction are are mismatch probes (MM) for position. The presence of a labeled nucleotide in the PM probe can be detected and the sequence of the 3' capture probe determined to identify the specific allele at the detection position.

[0155] Some embodiments include methods for decoding the positions of polynucleotides in a matrix. Some such methods include (a) providing a substrate having a matrix of polynucleotides distributed on the surface of the substrate, each polynucleotide containing a primer binding site 3' from the barcode, each polynucleotide associated with a capture probe; (b) hybridizing multiple primers to primer binding sites; and (c) determining the barcode sequences by extension of the hybridized primers, the sequence of each barcode indicating the position of the polynucleotide in the template. In some embodiments, each polynucleotide is associated with a capture probe via a bead. In some embodiments, each polynucleotide contains a capture probe. In some embodiments, the capture probe is located 3' relative to the primer binding site. In some embodiments, the capture probe is located 5' relative to the barcode. In some embodiments, the capture probe contains a different sequence from the other capture probe. In some embodiments, the capture probe differs from another capture probe by less than 5 different nucleotides. In some embodiments, each capture probe contains a different sequence. In some embodiments, the polynucleotides are randomly distributed on the surface of the substrate. In some embodiments, the barcode contains a different sequence from another barcode. In some embodiments, each barcode contains a different sequence. In some embodiments, each primer binding site contains the same sequence. In some embodiments, the polynucleotides are attached to the beads. In some embodiments, the beads are distributed in the wells. In some embodiments, each polynucleotide contains a cleavable linker. In some embodiments, the cleavable linker is configured to remove the capture probe from the primer-barcode binding site. In some embodiments, the substrate contains wells. In some embodiments, each polynucleotide contains a spacer. In some embodiments, the spacer is attached to the substrate. In some embodiments, the spacer is attached to the bead. Some embodiments also include hybridizing the target nucleic acid with capture probes. In some embodiments, hybridization of the target nucleic acid with the capture probes is performed after determination of the barcode sequences. In some embodiments, hybridization of the target nucleic acid with the capture probes is performed before determining the barcode sequences. Some embodiments also include extension of the hybridized target nucleic acid or polynucleotide. In some embodiments, extension involves ligation. Some embodiments also include amplifying a target nucleic acid.

[0156] Some embodiments include methods for sequencing a target nucleic acid. Some such methods are (a) decoding the positions of polynucleotides in a matrix in accordance with any of the above methods; (b) hybridizing the target nucleic acid with the capture probes; (c) extension of capture probes hybridized to the target nucleic acid; and (d) detecting the position of the elongated capture probes. In some embodiments, (d) detecting the position of the extended capture probes is performed before (a) decoding the positions of the polynucleotides in the array. In some embodiments, the capture probes are extended using a ligase. In some embodiments, the capture probes are extended using a polymerase. In some embodiments, the capture probes are extended by the addition of a single nucleotide. Some embodiments also include cleaving the primer and barcode binding sites from the capture probes before hybridizing the target nucleic acid to the capture probes. Some embodiments also include cleaving the primer and barcode binding sites from the capture probes after hybridization of the target nucleic acid to the capture probes.

Some two-probe methods

[0157] Some embodiments include detecting a target nucleic acid using first and second capture probes. In some embodiments, the target nucleic acid comprises a first region capable of hybridizing to a first capture probe and a second region capable of hybridizing to a second capture probe. In some embodiments, a population of beads is produced, in which each bead contains 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 contains a detectable label, such as a fluorescent label. In some embodiments, the target nucleic acid hybridizes to the capture probes to form a double-stranded nucleic acid containing a single-stranded gap. The gap is filled and the cleavable linker is cleaved off. An elongated capture probe is detected. In some embodiments, one of the capture probes contains a detectable label, such as a fluorescent label. In some embodiments, the detectable label is incorporated into the elongated capture probe during gap filling.

[0158] In some embodiments, the second capture probe is attached to the bead via a cleavable linker, and the second capture probe contains a detectable label, such as a fluorescent label. Examples of cleavable linkers include linkers that can be cleaved chemically, enzymes such as endonucleases, and certain frequencies of light. In some embodiments, each bead also contains a first polynucleotide containing a barcode indicating a first or second capture probe, and a barcode primer binding site at the 3' end of the barcode. In some embodiments, the first capture probe comprises a first polynucleotide. In some embodiments, the first capture probe is different from the first polynucleotide.

[0159] 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 a second capture probe by hybridizing a target nucleic acid to the first and second bead population capture probes to produce a double-stranded nucleic acid comprising a single-stranded gap between the first and second capture probes and filling the gap between the first and second capture probes. In some embodiments, gap filling can be accomplished using a polymerase and/or ligase. In some embodiments, the cleavable linker is cleaved to produce a bead containing a first capture probe containing a detectable label. In some embodiments, the population of beads is distributed on the substrate. In some embodiments, the population of beads is distributed on the substrate after cleavage of the cleavable linker. In some embodiments, the bead population is distributed on the substrate prior to cleavage of the cleavable linker or prior to ligation of the first capture probe to the second capture probe. In some embodiments, the position of the bead containing the first capture probe containing the detectable label on the substrate is determined.

[0160] In some embodiments, decoding the position of a bead containing a first capture probe containing a detectable label on a substrate includes decoding the position of a barcode of a bead containing a first capture probe containing a detectable label on the substrate. Some such embodiments may include hybridizing a barcode primer to a barcode primer site and extending the hybridized barcode primer. In some embodiments, extension of the hybridized barcode primer involves at least one round of sequencing by synthesis. In some embodiments, decoding the position of the bead barcode involves sequencing the barcode on the substrate.

[0161] In some embodiments, each bead contains a second polynucleotide containing an index indicating the source of the target nucleic acid and an index primer binding site at the 3' end of the index.

[0162] In some embodiments, the population of beads comprises a first and a second subpopulation of beads, each bead containing a second polynucleotide containing an index and an index primer binding site at the 3' end of the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation. In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence.

[0163] In some embodiments, the ligation or cleavage step with the first subpopulation of beads is performed at positions different from the ligation or cleavage step with the second subpopulation of beads. In some embodiments, different positions provide different reaction volumes. In some embodiments, different positions provide different wells.

[0164] Some embodiments also include combining the first and second subpopulations of beads before distributing the subpopulation of beads on the substrate. In other embodiments, the first subpopulation of beads is distributed on the substrate before the second subpopulation of beads is distributed on the substrate.

[0165] In some embodiments, decoding the position of a bead containing a first capture probe containing a detectable label on a substrate includes determining the position of indices of the beads containing the detected capture umbrella. Some such embodiments include hybridizing multiple index primers to index primer sites and extending the hybridized index primers. In some embodiments, extension of the hybridized index primers involves at least one round of sequencing by synthesis. In some embodiments, decoding the position of the bead indices involves sequencing the indices on the substrate.

[0166] In some embodiments, the substrate contains multiple discrete sites. In some embodiments, the substrate contains a plurality of wells. In some embodiments, the substrate contains multiple channels. In some embodiments, the flow cell contains a substrate. In some embodiments, the distributed population of beads comprises a matrix.

[0167] In FIG. 6B on the left depicts an embodiment in which bead 200 includes a first capture probe 300 attached to the bead via a cleavable linker 310. A second capture probe 320 is attached to the bead. A 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 nucleotides that contain detectable tags 230. Once the gap is filled, the target nucleic acid is removed and the cleavable linker cleaved to produce an elongated second capture probe containing detectable tags attached to the bead. The granule can be distributed in a matrix on a substrate and decoded. A polynucleotide containing an index indicating the sample attached to the bead and a polynucleotide containing a barcode indicating the first or second capture probes are not shown. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix.

[0168] In FIG. 6C depicts an embodiment in which the first capture probe is attached to the bead 200 via its 5' end. The second capture probe 360 is attached to the bead through its 3' end and a cleavable linker 310. The second capture probe contains detectable tags 230. The target nucleic acid 220 is hybridized to the first and second capture probes, the first capture probe is extended and ligated to the second capture probe. In some embodiments, the target nucleic acid may contain structural variants (SV). The cleavable linker is cleaved to produce an elongated first capture probe containing a detectable label and attached to the bead. The granule can be distributed in a matrix on a substrate and decoded. A polynucleotide containing an index indicating the sample attached to the bead and a polynucleotide containing a barcode indicating the first or second capture probes are not shown. Decoding may include determining the position of a detectable mark on the array; detecting a barcode attached to a bead on a matrix; and/or determining the index attached to the bead on the matrix. In the absence of a target nucleic acid, the second capture probe and detectable label are cleaved from the bead.

Kits and systems

[0169] Some embodiments include kits and systems for decoding microelements such as polynucleotides on an array. Some such kits and systems may include a substrate, such as a chip or fluid cell, with a matrix of polynucleotides randomly distributed on the surface of the substrate. The polynucleotides may contain a primer binding site 3' relative to the barcode. In some such embodiments, each polynucleotide may comprise a capture probe. In additional such embodiments, each polynucleotide may be linked to a capture probe through a common element. For example, each polynucleotide and capture probe may be associated with the same trace element, such as a bead. Some embodiments include a detector configured to detect signals from reagents hybridized to polynucleotides in the array; such reagents may include sequencing reagents, such as nucleotides containing detectable tags. Some embodiments include a detector configured to detect signals that may result from the incorporation of a nucleotide into a polynucleotide, such as pyrophosphate, or changes in hydrogen ions.

[0170] Some embodiments include kits or systems containing at least first and second subpopulations of beads. In some embodiments, each bead of the subpopulation may contain a first polynucleotide containing a capture probe, a barcode indicating a capture probe of the same bead, and a barcode primer binding site at the 3' end of the barcode. In some embodiments, each subpopulation bead may also contain a second polynucleotide containing the index and an index primer binding site 3' to the index. In some embodiments, the granule subpopulation index may indicate that particular granule subpopulation. In some embodiments, the indices of the first subpopulation are different from the indices of the second subpopulation. In some embodiments of the kits and systems provided herein, the first volume contains a first subpopulation of beads and the second volume contains a second subpopulation of beads.

[0171] In some embodiments, the first and second bead subpopulation capture probes each contain different nucleotide sequences. In some embodiments, the different capture probes for the first and second bead subpopulations contain the same nucleotide sequences. In some embodiments, the capture probes contain a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0172] In some embodiments, the barcode primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites.

[0173] In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites.

[0174] In some embodiments, the substrate contains multiple discrete sites. In some embodiments, the substrate contains a plurality of wells. In some embodiments, the substrate contains multiple channels. In some embodiments, the flow cell contains a substrate. In some embodiments, the substrate is configured to form a combination of first and second bead subpopulations of a bead matrix on the surface of the substrate and to sequence that template in multiple sequencing runs through synthesis runs.

[0175] In some embodiments, each of the first and second bead subpopulations contains at least 50 capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 500 capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 5000 capture probes containing different nucleotide sequences. In some embodiments, each of the first and second bead subpopulations contains at least 50,000 capture probes containing different nucleotide sequences.

[0176] Some embodiments include at least 10 different subpopulations of beads, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 100 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 1000 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation. Some embodiments include at least 10,000 different bead subpopulations, with each subpopulation containing an index that is different from the other subpopulation.

[0177] Some embodiments include a set comprising: an array of polynucleotides randomly distributed on the surface of a substrate, wherein each polynucleotide contains a primer binding site 3' to a barcode and is associated with a capture probe, wherein each polynucleotide contains a different barcode and is associated with a different probe capture. In some embodiments, each polynucleotide is associated with a capture probe via a bead. In some embodiments, each polynucleotide contains a capture probe. In some embodiments, the substrate is flat. In some embodiments, the substrate contains wells. In some embodiments, the polynucleotides are attached to the beads. In some embodiments, the beads are distributed in the wells. In some embodiments, the flow cell contains a matrix.

[0178] Some embodiments include kits or systems containing first and second bead subpopulations. In some embodiments, each bead contains 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 contains a first polynucleotide containing a barcode indicating a capture probe of the same bead, and a barcode primer binding site at the 3' end of the barcode. In some embodiments, each bead contains a second polynucleotide containing an index and an index primer binding site at the 3' end of the index. In some such embodiments, the indices of the first subpopulation are different from the indices of the second subpopulation. For example, the indices of the first subpopulation of granules can be used to distinguish the first subpopulation of granules from the second subpopulation of granules. In some embodiments, the first subpopulation of beads is separate from the second subpopulation of beads. For example, the first volume contains a first subpopulation of beads, and the second volume contains a second subpopulation of beads.

[0179] In some embodiments, the capture probe contains a nucleic acid and the target ligand contains the target nucleic acid. In some such embodiments, the first polynucleotide comprises a capture probe. In some embodiments, the capture probes contain a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement.

[0180] In some embodiments, the capture probe comprises an antibody or an antigen binding fragment of an antibody.

[0181] In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands. For example, each of the first bead subpopulation capture probes specifically binds to different target ligands; and each of the second bead subpopulation capture probes specifically binds to different target ligands. In some embodiments, different capture probes for the first and second bead subpopulations specifically bind to the same target ligands. For example, a set of different first bead subpopulation capture probes bind specifically to the same target ligands as a set of different first bead subpopulation capture probes.

[0182] In some embodiments, the barcode primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites.

[0183] In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and/or the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites.

[0184] In some embodiments, the substrate contains multiple discrete sites. In some embodiments, the substrate contains a plurality of wells. In some embodiments, the substrate contains multiple channels. In some embodiments, the flow cell contains a substrate. In some embodiments, the substrate is configured to form a combination of first and second bead subpopulations of a bead matrix on the surface of the substrate and to sequence that matrix in multiple sequencing runs through synthesis runs.

[0185] In some embodiments, each of the first and second bead subpopulations contains at least 50, 100, 500, 1000, or 5000 distinct capture probes, or any number of capture probes ranging between any two of the above amounts. Some embodiments also contain at least 5, 10, 20, 50, 100, 200, 500, 1000 different granule subpopulations, each subpopulation containing a different index from the other subpopulation, or any number of different granule subpopulations between any two of the above amounts.

[0186] Some embodiments include a set containing first and second bead subpopulations, each bead comprising: a capture probe that specifically binds to a target ligand, a first polynucleotide containing a barcode indicating the capture probe, and a barcode primer binding site at 3 '-end relative to the barcode, and a second polynucleotide containing the index and an index primer binding site at the 3'-end relative to the index, wherein the indices of the first subpopulation are different from the indices of the second subpopulation, wherein the first volume contains the first subpopulation of beads, and the second volume contains the second subpopulation granules 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 first and second bead subpopulation capture probes each contain different nucleotide sequences. In some embodiments, the different capture probes for the first and second bead subpopulations contain the same nucleotide sequences. In some embodiments, the capture probe comprises an antibody or an antigen-binding fragment thereof. In some embodiments, each of the first and second bead subpopulation capture probes specifically binds to different target ligands. In some embodiments, different capture probes for the first and second bead subpopulations specifically bind to the same target ligands. In some embodiments, the barcode primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of barcode primers capable of hybridizing to the barcode primer sites. In some embodiments, the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence, and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. In some embodiments, the nucleotide sequences of the index primer binding sites contain the same nucleotide sequence. Some embodiments also include a plurality of index primers capable of hybridizing to the index primer sites. In some embodiments, the flow cell contains a substrate. In some embodiments, the substrate is configured to form a combination of first and second bead subpopulations of a bead matrix on the surface of the substrate and to sequence that matrix in multiple sequencing runs through synthesis runs. In some embodiments, each of the first and second bead subpopulations contains at least 50 distinct capture probes. In some embodiments, each of the first and second bead subpopulations contains at least 500 distinct capture probes. Some embodiments also include at least 10 different subpopulations of beads, each subpopulation containing an index that is different from the other subpopulation.

EXAMPLES

Example 1: Decoding a matrix by sequencing

[0187] A plurality of polynucleotides are synthesized, with each polynucleotide containing from 5' to 3': a spacer, a unique barcode, a primer binding site, and a unique capture probe. The barcode and capture probe sequences 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 template is decoded by hybridizing the primer to the primer binding site, extending the primer, and detecting the barcode sequence. The position of the barcode determines the position of the corresponding capture probe.

Example 2: Decoding Barcodes on an Array by Sequencing

[0188] A nucleic acid library derived from human genomic DNA was prepared. A subpopulation of pellets was prepared. The first and second polynucleotides were attached to each bead. The first polynucleotide contained a capture probe, a barcode primer binding site, and a barcode indicating the capture probe. The second polynucleotide contained an index and a binding site for the index primer.

[0189] A bead pool containing 18,816 different code/probe types was loaded into a HiSeq flow cell (FIG. 7A). After immobilization, the 20-nt codes were sequenced using SBS chemistry, and the identity of each bead was determined by aligning the code sequences to a list of bead types. In FIG. 7B provides a copy number histogram for specific granule types in specific columns and demonstrates that 97.5% of the expected composition was identified through the sequencing-based decoding process and that the vast majority of granule types are present at levels sufficient for genotyping studies.

Example 3: HiSeq Genotyping Performance Using Feed Forward Neural Network (FFN) Detection

[0190] To demonstrate genotyping performance on HiSeq using FFN detection, a target DNA oligonucleotide was hybridized to a probe-conjugated oligonucleotide bead suspension. The beads were loaded into a HiSeq flow cell. Probes bound to the target DNA were extended one base at a time using fluorescent nucleotides. The bead-loaded flow cell was imaged to obtain bead genotyping intensity values. Fluorescent nucleotides were digested and the beads were decoded using SBS chemistry. Decoding and genotyping reads were aligned to measure assay performance. Individual dots were colored according to the expected genotype. In FIG. Figure 1C shows a plot of C intensity versus T intensity, where each point represents the average of all copies for a given bead type and is colored according to the expected genotype, and demonstrates that single-base extension of the probe with fluorescent nucleotides provides accurate genotyping.

Example 4: Multiplexing 12 Samples with a 10368-plex Bead Pool

[0191] This example demonstrates the demultiplexing of multiple samples on a single flow cell and, in particular, the ability to multiplex 12 samples with a 10,368-plex bead pool. One pool of beads was separately hybridized to 12 different index sequences. After hybridization, 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 a separate read to identify the bead type based on the read-decoding. In FIG. Figure 8 provides a histogram for a representative sample of a number of specific granule types in specific relationships, and demonstrates that most granule types are present at a level sufficient for genotyping experiments for a given sample. In the table in Fig. Figure 7 provides a summary of bead demultiplexing and decoding consistency for 12 pooled and simultaneously loaded indexed samples, indicating that sample representation is uniform and that most probes are present in all samples.

[0192] As used herein, the term “comprising” is synonymous with the terms “including,” “comprising,” or “characterized by,” and is inclusive, not limiting, and does not exclude additional unspecified elements or method steps.

[0193] The description above sets forth several methods and materials of the present invention. The present invention is subject to modifications in methods and materials, as well as changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art upon examination of the present disclosure or upon practice of the invention described herein. Therefore, the present invention is not intended to be limited to the specific embodiments described herein, but is intended to cover all modifications and alternatives within the true scope and spirit of the invention.

[0194] All references cited herein, including, without limitation, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are therefore a part of this specification. To the extent that publications, patents, or patent applications incorporated by reference conflict with the teachings set forth herein, it is intended that this specification supersedes and/or takes precedence over any such conflicting material.

Claims (49)

1. A method for identifying target ligands in a matrix, including: (a) providing a substrate having a matrix of beads distributed on the surface of said substrate, said matrix of beads comprising a first and a second subpopulation of beads, comprising: (i) providing first and second subpopulations of granules, each granule comprising: a capture probe that is capable of specifically binding to a target ligand, a first polynucleotide that contains a barcode and a primer binding site at the 3' end of said barcode, wherein said barcode indicates a capture probe, and a second polynucleotide comprising an index and an index primer binding site at the 3' end of the index, each index of the first subpopulation of beads being different from each index of the second subpopulation of beads; (ii) causing the first target ligands to specifically bind to said capture probes of said first bead subpopulation in the first reaction volume and the second target ligands to specifically bind to the capture probes of said second bead subpopulation in the second reaction volume, and (iii) distributing said first and second subpopulations of beads containing specifically bound first and second target ligands onto a substrate, thereby obtaining a substrate having a bead matrix; (b) detecting in said matrix specifically bound first and second target ligands of said first and second subpopulations of granules; (c) sequencing the barcodes of said first and second bead subpopulations by hybridizing a plurality of primers to the primer binding sites of the barcodes of said first and second bead subpopulations; and extending the hybridized primers, thereby determining the position of the capture probes of said first and second bead subpopulations in said matrix, (d) sequencing the indices of said first and second subpopulations of granules, thereby determining the positions of said first and second subpopulations of granules in the matrix; And (e) decoding positions of beads in said bead array including a detected target ligand in the array, thereby identifying target ligands of said first target ligands or said second target ligands in the array. 2. The method according to claim 1, wherein said first and second target ligands each contain a nucleic acid, and wherein step a) further comprises: (iv) extension of capture probes specifically bound to the first and second target ligands. 3. The method of claim 2, wherein step (iv) includes contacting the first capture probes with the polymerase. 4. The method of claim 2, wherein step (iv) includes contacting the first capture probes with the ligase. 5. Method according to any one of paragraphs. 2-4, wherein the elongated capture probe contains a detectable mark. 6. Method according to any one of paragraphs. 2-5, in which step (iv) is performed before step (iii). 7. Method according to any one of paragraphs. 2-6, in which step (iv) includes performing at least one round of sequencing by synthesis. 8. A method for identifying target ligands in a matrix, including: (a) providing first and second subpopulations of granules, each granule comprising: (i) a capture probe that is capable of specifically binding to a target ligand, (ii) a first polynucleotide containing a barcode, said barcode indicating a capture probe, and (iii) a second polynucleotide comprising an index, wherein each index of the first subpopulation of beads is different from each index of the second subpopulation of beads; (b) causing the first target ligands to specifically bind to said capture probes of said first bead subpopulation in the first reaction volume and the second target ligands to specifically bind to the capture probes of said second bead subpopulation in the second reaction volume, and (c) distributing first and second subpopulations of beads containing specifically bound first and second target ligands on the surface of the substrate, thereby obtaining a matrix of beads; (d) detecting in said matrix specifically bound first and second target ligands of said first and second subpopulations of granules; (e) sequencing bead array barcodes; (f) sequencing of bead matrix indices; And (g) decoding bead positions in said bead array including a detected target ligand in the array, thereby allowing identification of target ligands in the bead array. 9. The method according to claim 8, in which: the capture probe contains a nucleic acid, the first and second target ligands contain nucleic acids, and step (d) involves extending capture probes specifically bound to the first and second target ligands. 10. Method according to any one of paragraphs. 2-9, wherein the first polynucleotide contains a capture probe. 11. Method according to any one of paragraphs. 2-10, wherein the first and second bead subpopulation capture probes each contain different nucleotide sequences. 12. Method according to any one of paragraphs. 2-11, wherein the first bead subpopulation capture probes contain the same nucleotide sequences as the second bead subpopulation capture probes. 13. Method according to any one of paragraphs. 2-12, wherein the capture probes contain a nucleotide sequence capable of hybridizing to a single nucleotide polymorphism (SNP) or its complement. 14. The method of claim 1 or 8, wherein the capture probe contains an antibody or an antigen-binding fragment thereof. 15. The method of claim 14, wherein each of the first and second bead subpopulation capture probes specifically binds to different target ligands. 16. The method of claim 14 or 15, wherein the different capture probes for the first and second bead subpopulations specifically bind to the same target ligands. 17. Method according to any one of paragraphs. 14-16, wherein detection involves bringing first and second target ligands specifically bound to the capture probes into contact with a secondary antibody or antigen binding fragment thereof, wherein the secondary antibody or antigen binding fragment thereof contains a detectable label. 18. Method according to any one of paragraphs. 1-17, wherein each of the first and second bead subpopulations contains at least 50 distinct capture probes. 19. Method according to any one of paragraphs. 1-18, wherein the index nucleotide sequences of the first bead subpopulation contain the same nucleotide sequence and the index nucleotide sequences of the second bead subpopulation contain the same nucleotide sequence. 20. Method according to any one of paragraphs. 1-19, further comprising combining a first subpopulation of beads containing the specifically bound first target ligands and a second subpopulation of beads containing the specifically bound second target ligands prior to distribution on a substrate. 21. Method according to any one of paragraphs. 1-19, wherein a first subpopulation of beads containing specifically bound first target ligands is distributed on a substrate before a second subpopulation of beads containing specifically bound second target ligands is distributed on the substrate. 22. Method according to any one of paragraphs. 1-21, in which the flow cell contains a substrate. 23. Method according to any one of paragraphs. 1-22, in which the first and second target ligands are obtained from different subjects. 24. Method according to any one of paragraphs. 1-23, further including at least 10 different subpopulations of granules, each subpopulation containing an index different from the index of the other subpopulation.