Methods and compositions for bead-based combinatorial indexing of nucleic acids

Abstract

Some embodiments relate to methods and compositions for preparing combinatorially indexed beads. Some embodiments include sequential addition of different indexes to polynucleotides attached to the beads. In some embodiments, the indexes are added by chemical ligation, polymerase extension, ligation of partial double-stranded adapters, or short splint ligation.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent No. 63 / 214,693, filed June 24, 2021, entitled "METHODS AND COMPOSITIONS FOR COMBINATORIAL INDEXING OF BEAD-BASED NUCLEIC ACIDS," which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application has been filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled ILLINC608WOSEQLIST, created on June 17, 2022, which is approximately 3.15 kilobytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] Some embodiments relate to methods and compositions for preparing combinatorially indexed beads. Some embodiments include sequential addition of different indexes to polynucleotides attached to the beads. In some embodiments, the indexes are added by chemical ligation, polymerase extension, ligation of partial double-stranded adapters, or short splint ligation. [Background technology]

[0004] Detection of specific nucleic acid sequences present in biological samples has been used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, determining genetic susceptibility to disease, and measuring response to various types of treatments. A common technique for detecting specific nucleic acid sequences in biological samples is nucleic acid sequencing.

[0005] Nucleic acid sequencing methods have evolved significantly from the chemical degradation method used by Maxam and Gilbert and the strand extension method used by Sanger. Several sequencing methods are now in use that allow parallel processing of thousands of nucleic acids on a single chip. Several platforms include bead-based and microarray formats, where silica beads are functionalized with probes for the application of such formats in applications including sequencing, genotyping, and gene expression profiling.

[0006] Current methods for genotyping different samples on bead-based arrays require a gasket to physically subdivide different areas of the beadchip into multiple sectors. Individual samples are then loaded into each separate section created by the gasket. However, such methods, while used with relatively low sample number inputs, have proven difficult and unmanageable as the density of samples per beadchip increases from 24 to 96, 384, 1536, or more samples per beadchip. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Provisional Patent No. 63 / 214,693 Summary of the Invention [Means for solving the problem]

[0008] Some embodiments of the methods and compositions provided herein include a method of preparing a plurality of combinatorial indexed beads, the method comprising: (a) obtaining a population of primary indexed beads comprising a first polynucleotide comprising a first index, the population of primary indexed beads comprising a plurality of subpopulations of first indexed beads, the subpopulations of first indexed beads comprising first indices that are different from each other; (b) dividing the population of primary indexed beads into a plurality of subpopulations of second beads; and (c) obtaining a population of secondary indexed beads, the method comprising: (i) extending the first polynucleotide of the subpopulations of the plurality of second beads with a second polynucleotide comprising a second index to obtain a subpopulation of second indexed beads, the subpopulations of second indexed beads comprising second indices that are different from each other. and (ii) combining the second subpopulations of indexed beads to obtain a population of secondary indexed beads, optionally further comprising (d) dividing the population of secondary indexed beads into a plurality of third subpopulations of beads; and (e) obtaining a population of tertiary indexed beads, comprising (i) extending the second polynucleotide of the plurality of subpopulations of third beads with a third polynucleotide comprising a third index to obtain a subpopulation of third indexed beads, wherein the subpopulations of third indexed beads comprise third indices that are different from each other; and (ii) combining the subpopulations of third indexed beads to obtain a population of tertiary indexed beads.

[0009] In some embodiments, (c) comprises extending the first polynucleotide with the second polynucleotide by chemical ligation.

[0010] In some embodiments, a first polynucleotide comprises a terminal 3' modified deoxynucleotide (dNTP) that comprises a 3' functional moiety that can participate in a click chemistry reaction, and a second polynucleotide comprises a terminal 5' modified dNTP that comprises a compatible 5' functional moiety that can participate in a click chemistry reaction with the 3'-functional moiety, and the 3' functional moiety and the 5' functional moiety can react with each other to form a modified backbone linkage.

[0011] In some embodiments, a secondary indexed polynucleotide is obtained by extending the first polynucleotide with a second polynucleotide, and the method further comprises modifying the secondary indexed polynucleotide to obtain a modified polynucleotide comprising a terminal 3' modified deoxynucleotide (dNTP) that comprises a 3' functional moiety capable of participating in a click chemistry reaction.

[0012] In some embodiments, the modifying comprises contacting the secondary indexed polynucleotide with a template-independent polymerase. In some embodiments, the template-independent polymerase is selected from terminal deoxynucleotidyl transferase (TdT), polyA polymerase, or CCA-adding RNA polymerase. In some embodiments, the template-independent polymerase is TdT.

[0013] Some embodiments also include extending the modified polynucleotide with a third polynucleotide by a chemical ligation reaction, the third polynucleotide comprising a terminal 5' modified dNTP that comprises a compatible 5' functional moiety that can participate in a click chemistry reaction with the 3' functional moiety.

[0014] In some embodiments, the 3' functional moiety is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone.

[0015] In some embodiments, the 5' functional moiety is different from and compatible with the 3' functional moiety and is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone.

[0016] In some embodiments, the 3'-functional moiety and the 5'-functional moiety are selected from the following pairs: (i) 3'-azido / 5'-alkynyl, (ii) 3'-alkynyl / 5'-azide, (iii) 3'-thiol / 5'-alkynyl, (iv) 3'-thiol / 5'-alkenyl, (v) 3'-alkynyl / 5'-thiol, (vi) 3'-alkenyl / 5'-thiol, (vii) 3'-azido / 5'-cyclooctynyl, (viii) 3'-cyclooctynyl / 5'-azido, (ix) 3'-nitrone / 5'-cyclooctynyl, and (x) 3'-cyclooctynyl / 5'-nitrone. In some embodiments, the 3'-functional moiety is 3'-azido and the 5'-functional moiety is 5'-alkynyl.

[0017] In some embodiments, the click chemistry reaction comprises copper catalyzed azide-alkyne cycloaddition (CuAAC) to form a triazolyl-containing modified backbone linkage.

[0018] In some embodiments, (c) comprises extending the first polynucleotide by polymerase extension, wherein the first polynucleotide comprises a first linker.

[0019] Some embodiments also include (i) obtaining a first adaptor that includes a region capable of hybridizing to the first linker and a region that includes the second index or a complement of the second index, (ii) hybridizing the first adaptor to the first linker, and (iii) extending the first polynucleotide to obtain a secondary indexed polynucleotide.

[0020] In some embodiments, the first adaptor comprises a non-extendable 3' end. In some embodiments, the non-extendable 3' end comprises a 3'2' dideoxynucleotide, or a C3 linker.

[0021] Some embodiments also include removing the first adaptor from the secondary indexed polynucleotide, in some embodiments removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0022] In some embodiments, the first linker has a length of less than 10 contiguous nucleotides. In some embodiments, the first linker has a length of less than 5 contiguous nucleotides.

[0023] In some embodiments, (e) comprises extending the second polynucleotide by polymerase extension.

[0024] In some embodiments, the first adaptor comprises the complement of the second linker, such that the secondary indexed polynucleotide comprises the second linker.

[0025] Some embodiments also include (i) obtaining a second adaptor comprising a region capable of hybridizing to a second linker and a region comprising a third index or a complement of the third index, (ii) hybridizing the second adaptor to the second linker, and (iii) extending the secondary indexed polynucleotide to obtain a tertiary indexed polynucleotide.

[0026] In some embodiments, the second adaptor comprises a non-extendable 3' end.

[0027] In some embodiments, the non-extendable 3' end comprises a 3'2' dideoxynucleotide, or a C3 linker.

[0028] Some embodiments also include removing the second adaptor from the tertiary indexed polynucleotide, in some embodiments, removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0029] In some embodiments, the second linker has a length of less than 10 contiguous nucleotides. In some embodiments, the second linker has a length of less than 5 contiguous nucleotides.

[0030] In some embodiments, (c) comprises extending the first polynucleotide with the second polynucleotide by ligation.

[0031] Some embodiments also include (i) obtaining a double-stranded first adaptor comprising a second polynucleotide and a 3' single-stranded overhang capable of hybridizing to the first linker of the first polynucleotide, (ii) hybridizing the first adaptor to the first linker, and (iii) ligating the first polynucleotide to the second polynucleotide to obtain a secondary indexed polynucleotide.

[0032] Some embodiments also include extending the second polynucleotide with a third polynucleotide by ligation.

[0033] Some embodiments also include (i) obtaining a double-stranded second adaptor comprising a third polynucleotide and a 3' single-stranded overhang capable of hybridizing to a second linker of the second polynucleotide; (ii) hybridizing the second adaptor to the second linker and, optionally, hybridizing an additional oligonucleotide to the first index; and (iii) ligating the second polynucleotide to the third polynucleotide to obtain a tertiary indexed polynucleotide.

[0034] In some embodiments, ligation involves the use of a ligase.

[0035] In some embodiments, the ligation comprises a chemical ligation reaction.

[0036] In some embodiments, (c) comprises extending the first polynucleotide by ligation, wherein the first polynucleotide comprises a first linker and the second polynucleotide comprises a second linker.

[0037] Some embodiments also include (i) obtaining a first adaptor comprising a region capable of hybridizing to a first linker and a region capable of hybridizing to a second linker; (ii) hybridizing the first adaptor to the first linker; (iii) hybridizing a second oligonucleotide to the region capable of hybridizing to the second linker; and (iv) ligating the first polynucleotide to the second polynucleotide to obtain a secondary indexed polynucleotide.

[0038] In some embodiments, the first linker and / or the second linker has a length of less than 10 contiguous nucleotides. In some embodiments, the first linker and / or the second linker has a length of less than 5 contiguous nucleotides.

[0039] In some embodiments, the first linker and / or the second linker are modified to have an increased Tm compared to an oligonucleotide having the same length as the first adaptor.

[0040] In some embodiments, the first linker and / or the second linker comprises an increased G / C content or comprises modified nucleotides compared to an oligonucleotide having the same length.

[0041] Some embodiments also include removing the first adaptor from the secondary indexed polynucleotide, in some embodiments removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0042] In some embodiments, (e) comprises extending the second polynucleotide by ligation, wherein the second oligonucleotide comprises a third linker, such that the secondary indexed polynucleotide comprises a third linker, and the third polynucleotide comprises a fourth linker.

[0043] Some embodiments also include (i) obtaining a second adaptor comprising a region capable of hybridizing to a third linker and a region capable of hybridizing to a fourth linker; (ii) hybridizing the second adaptor to the third linker; (iii) hybridizing a third polynucleotide to the second adaptor via the region capable of hybridizing to the fourth index; and (iv) ligating the secondary indexed polynucleotide to the third polynucleotide to obtain a tertiary indexed polynucleotide.

[0044] In some embodiments, the third linker and / or the fourth linker has a length of less than 9 contiguous nucleotides. In some embodiments, the third linker and / or the fourth linker has a length of less than 5 contiguous nucleotides.

[0045] In some embodiments, the third linker and / or the fourth linker are modified to have an increased Tm compared to an oligonucleotide having the same length as the second adaptor.

[0046] In some embodiments, the third linker and / or the fourth linker comprises an increased G / C content or comprises modified nucleotides compared to an oligonucleotide having the same length.

[0047] Some embodiments also include removing the second adaptor from the tertiary indexed polynucleotide.

[0048] In some embodiments, removing comprises denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0049] In some embodiments, (a) comprises: (i) attaching a first polynucleotide to a plurality of first bead subpopulations to obtain first indexed bead subpopulations, where the first polynucleotide comprises a first index that differs for each first bead subpopulation; and (ii) combining the first indexed bead subpopulations to obtain a primary indexed bead population.

[0050] In some embodiments, the first polynucleotide is attached to the bead via a first binding partner and a second binding partner, in some embodiments, the first binding partner or the second binding partner is selected from the group consisting of biotin, streptavidin, a biotin derivative, a streptavidin derivative, an antibody, and an antigen-binding fragment of an antibody.

[0051] Some embodiments also include repeating (d) and (e) and adding additional indexes to the subpopulation of indexed beads.

[0052] In some embodiments, the first polynucleotide comprises a primer binding site selected from the group consisting of a P5 sequence, a P5' sequence, a P7 sequence, and a P7' sequence.

[0053] In some embodiments, the first index, the second index, and / or the third index have a length of less than 20 contiguous nucleotides. In some embodiments, the first index, the second index, and / or the third index have a length of less than 10 contiguous nucleotides.

[0054] In some embodiments, (b) comprises randomly distributing the population of primary indexed beads among the multiple compartments.

[0055] In some embodiments, (d) comprises randomly distributing the population of secondary indexed beads among the multiple compartments.

[0056] In some embodiments, the plurality of compartments comprises a compartment selected from a well, a channel, or a droplet.

[0057] In some embodiments, the plurality of combinatorial indexed beads comprises magnetic beads.

[0058] Some embodiments also include distributing a plurality of combinatorially indexed beads on an array. Some embodiments also include sequencing the combinatorially indexed beads on the array.

[0059] Some embodiments also decode the position of a bead among a plurality of combinatorially indexed beads on the array based on the combinatorial index.

[0060] In some embodiments, each bead of the plurality of combinatorial indexed beads comprises a capture probe. In some embodiments, the first polynucleotide, the second polynucleotide, or the third polynucleotide comprises a capture probe. Some embodiments also include hybridizing the plurality of target nucleic acids to the capture probe. Some embodiments also include extending the capture probe. [Brief description of the drawings]

[0061] [Figure 1] 1 illustrates an exemplary embodiment of combined indexing including pooling and splitting strategies. [Diagram 2] FIG. 1 shows an embodiment of a scheme for joining a first index (Index A), a second index (Index B), and a third index (Index C) attached to a substrate by sequential splint-ligation reactions using a linker-sprint. [Diagram 3] FIG. 1 shows one embodiment of a scheme for joining a second index (Index B) to a first index (Index A) attached to a substrate by click chemistry ligation. [Figure 4] 1 shows an embodiment of a scheme in which a second index (index B) and a third index (index C) are added to a first index (index A) attached to a substrate by sequential polymerase extension reactions. [Diagram 5] FIG. 1 shows an embodiment of a scheme in which an adaptor containing a double-stranded region and a single-stranded overhang is used to add a second index (Index B) and a third index (Index C) to a first index (Index A) attached to a substrate. [Figure 6] FIG. 1 shows an embodiment of a scheme in which a second index (Index B) and a third index (Index C) are added to a first index (Index A) attached to a substrate by sequential splinted ligation reactions using a short linker-sprint. [Figure 7A] A schematic diagram is shown including beads with capture oligonucleotides (P5-IndexA-Link1a) hybridized with extension templates (Link1a'-IndexB'-Hyb') for polymerase extension and the location of primers useful for measuring the amount of capture oligonucleotides, as well as beads with extension products and the location of primers useful for measuring the amount of full-length extension products. [Figure 7B]Graphs are shown for the amount of capture oligonucleotide and the amount of full length product from ligation extension of the capture oligonucleotide or polymerase extension of the capture oligonucleotide under various conditions. [Figure 8A] 1 is a schematic outlining the steps and conditions tested in a method for three-level indexing involving extension of a capture oligonucleotide by either polymerase extension or ligation. Numbering corresponds to the conditions tested. [Figure 8B] FIG. 8B is a table summarizing various experimental conditions for comparing splint ligation (SL) and polymerase extension (PE), including the various conditions (conditions, cond) numbered in FIG. 8A for level 1 indexing (L1), level 2 indexing (L2), and level 3 indexing (L3). [Figure 9] 1 is a graph showing the amount of capture oligonucleotide (CO), second level extension product, and third level extension product under various conditions. [Figure 10] FIG. 13 is a graph of the concentration of capture oligonucleotides with first extension products attached to beads via biotin or dual desthiobiotin (ddbiotin) and treated under various denaturing conditions, as measured by quantitative PCR. [Figure 11A] FIG. 1 is a schematic diagram showing the sequencing read orientation of synthesis products on beads, including three-level indexing products, PCR products derived from synthesis products, and sequencing reads derived from PCR products. [Figure 11B]Graph showing the percentage of sequencing reads containing sequences that are either completely correct or both indexes are usable for extension products generated by polymerase extension or splint ligation. Usable indexes include those that can be decoded and corrected with index error correction implementation, contain no more than one "SNP" within the index designed at a 3 nucleotide Hamming distance, and do not contain any insertions or deletions throughout the "index" region, for example, in a 3-level index oligonucleotide, the index region contains three indexes. [Figure 11C] 1 is a graph showing the error rate per base, including whether the error is a deletion, insertion, or base change (SNP), for sequencing reads containing sequences that are either completely correct or both indexes are usable for extension products generated by polymerase extension or splint ligation. Usable indexes include those that can be decoded and corrected with index error correction implementation, contain no more than one "SNP" within the index designed at a 3-nucleotide Hamming distance, and do not contain insertions or deletions throughout the "index" region, for example, in a 3-level index oligonucleotide, the index region contains three indexes. [Figure 12A] Schematic diagram showing steps in standard splint ligation (left panel) and double stranded splint ligation (right panel). In some embodiments, the double stranded splint ligation may include an additional oligonucleotide (index 1'). [Figure 12B] FIG. 1 is a schematic showing the conditions tested for steps in the method for standard splint ligation and double-stranded splint ligation. [Figure 12C] FIG. 1 is a schematic showing the location of the forward or reverse primer for measuring extension products from either standard ligation or double stranded ligation. [Figure 13A]12C is a graph showing the concentration of extension products measured by quantitative PCR, where the capture oligonucleotide was measured using F2 and R1 primers, and the full-length product was measured using either F2 and R2 or F3 and R2 primers, as shown in FIG. [Figure 13B] 1 is a graph showing the relative concentrations of extension products normalized to full-length controls. [Figure 13C] 1 is a graph showing the relative concentration of extension products normalized to capture oligonucleotide concentration. [Figure 13D] 1 is a graph showing the concentration of extension products produced in the presence of various amounts of double-stranded splint oligos. [Figure 13E] FIG. 12B is a graph showing the concentration of extension products produced in the presence of an additional oligonucleotide complementary to index 1 shown in FIG. 12A. [Figure 13F] 1 is a graph showing the concentration of extension products produced in the presence of an oligonucleotide with a non-extendible 3′ ddC end. [Figure 13G] FIG. 13 is a graph showing the concentration of extension products generated in the pre-ligation step at room temperature or at 75° C. [Figure 14A] Graph showing the percentage of sequencing reads that contain sequences that are either completely correct or that both indexes are usable for the extension products generated by splint ligation extension or double-stranded splint ligation.Usable indexes include those that can be decoded and corrected with index error correction implementation, contain no more than one "SNP" within the index designed at a 3-nucleotide Hamming distance, and do not contain any insertions or deletions throughout the "index" region, for example, in a 3-level index oligonucleotide, the index region contains three indexes. [Figure 14B] 1 is a graph showing the error rate per base, including whether the error is a deletion, insertion, or base change (SNP), for sequencing reads of extension products generated by splint ligation extension or double-stranded splint ligation. [Figure 15A] FIG. 1 is a schematic showing the steps in three-level indexing for the standard splint ligation method (left panel) and the double-stranded splint ligation method (right panel). [Figure 15B] FIG. 1 is a schematic showing the conditions tested in the steps in the method for three-level indexing using standard splint ligation or double-stranded splint ligation. [Figure 16A] 1 is a graph showing the concentrations of capture oligonucleotide extension products, second level products, and third level products as measured by quantitative PCR. [Figure 16B] Graph showing the percentage of sequencing reads that contain sequences that are either completely correct or all three indexes are usable for the extension products generated by splint ligation extension or double-stranded splint ligation.Usable indexes include those that can be decoded and corrected with index error correction implementation, contain no more than one "SNP" within the index designed at a 3-nucleotide Hamming distance, and do not contain any insertions or deletions throughout the "index" region, for example, in a 3-level index oligonucleotide, the index region contains three indexes. [Figure 16C] 1 is a graph showing the error rate per base, including whether the error is a deletion, insertion, or base change (SNP), for sequencing reads of extension products generated by splint ligation extension or double-stranded splint ligation. [Figure 17] Schematic diagram showing extension of a capture oligonucleotide by standard splint ligation or by splint ligation using an abbreviated splint (top panel). The bottom panel shows the position of the primers for measuring the extension products. [Figure 18A] 18A is a schematic diagram showing various splint oligonucleotides. The sequences shown in Figure 18A include SEQ ID NOs: 07-16. [Figure 18B]1 is a table showing various experimental conditions including a capture oligonucleotide with a level 1 index (L1 oligo), a splint oligonucleotide (sprint), and a level 2 oligonucleotide (L2 oligo). [Figure 18C] 13 is a graph of full length extension products relative to control full length extension products for extension products generated by splint ligation extension with various splint oligonucleotides. [Figure 19A] FIG. 1 is a schematic diagram of a combinatorial indexing method involving a double-stranded splint ligation containing linker sequences "KS-3'" and "MS-3'". [Figure 19B] FIG. 1 is a schematic overview of an exemplary workflow for a combinatorial indexing method involving double-stranded splint ligation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Some embodiments relate to methods and compositions for preparing combinatorially indexed beads. Some embodiments include sequential addition of different indexes to polynucleotides attached to the beads. In some embodiments, the indexes are added by chemical ligation, polymerase extension, ligation of partial double-stranded adapters, or short splint ligation.

[0063] Pools of bead-linked oligonucleotides, where each bead is uniformly coated with a single oligonucleotide sequence, are components of sequencing methods such as synthetic long-read sequencing. Combinatorial assembly of such bead pools can be used to achieve sufficient index diversity, however, some methods rely on splint ligation strategies to join successive levels of indexes. Splint ligation has the disadvantage of introducing invariant bases into the bead code, increasing the number of sequencing by synthesis (SBS) cycles required to read the complete bead code. Certain embodiments provided herein include several alternatives to the standard splint ligation bead code synthesis strategy that reduce the number of invariant bases in the bead code, increase the bead code information density, and enable more efficient bead code sequencing.

[0064] Combinatorial assembly of bead-linked oligonucleotides allows for the generation of bead pools containing a large number of unique indexes, where each bead is uniformly coated with a single unique index oligonucleotide. Starting with a common pool of magnetic beads derivatized for oligonucleotide capture, such as surface-attached streptavidin, the beads are aliquoted into "M" wells of a multiwell plate, each containing a single oligonucleotide sequence synthesized with a bead capture moiety, such as biotin. After binding, the beads are again pooled, mixed, and split into "N" wells of a multiwell plate to attach a second level of index. Since each well in the second indexing reaction contains a uniform mixture of the first level of oligonucleotides, the total number of unique indexes will be "M" x "N" after the second indexes are attached. By repeating this process multiple times, bead pools with millions of unique indexes can be obtained from a small set of individual index oligonucleotides that can easily fit into a standard multiwell plate, e.g., a 96-well plate, a 192-well plate, or a 384-well plate. An exemplary embodiment of combinatorial indexing, including pooling and splitting strategies, is shown in FIG. 1.

[0065] For indexes beyond the directly captured first level, a method is needed to covalently attach the single-stranded index oligonucleotide to the bead-bound index. This can be done using a long splint ligation approach (Figure 2). The directly captured first level oligonucleotide is designed to have an eight nucleotide capture sequence (L1A) at its 3' end, and the incoming index oligonucleotide has a second eight nucleotide capture sequence (L1B) at its 5' end, as well as a phosphate at the 5' end. In some embodiments, the LIA and / or LIB can have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides. These two pieces are then assembled in a ligation reaction with a 16 nucleotide splint oligonucleotide that is complementary to L1A at the 3' end and L1B at the 5' end; the combined splint hybridizes to both the capture oligonucleotide bound to the bead and the index oligonucleotide in solution, bringing the 3' and 5' ends of the two pieces directly adjacent to each other. The ligase enzyme in the reaction then covalently attaches the phosphorylated 5' end of the index oligonucleotide to the 3' end of the capture oligo. Table 1 lists the number of unique bead codes that can be obtained in a particular method using 12, 96, 192, or 384 and 768 multiwell plates. Some such methods include: (a) distributing beads into wells of a first multiwell plate, each well containing a different index A, attaching index A to the beads, and pooling the beads; (b) redistributing the beads into wells of a second multiwell plate, each well containing a different index B, attaching index B to the beads; and (c) redistributing the beads into wells of a third multiwell plate, each well containing a different index C, and attaching index C to the beads.

[0066] [Table 1]

[0067] The splinted ligation approach requires an invariant splint sequence of 16 nucleotides between the variable index regions for each level of index in the bead code. Since the bead code is typically sequenced in a single read, these invariant regions must be read in chemistry-only cycles, such as "dark" cycles during SBS. For example, each base in the invariant region requires an additional SBS cycle to maintain correct phasing. These are typically "chemistry-only" or "dark" SBS cycles, where a polymerization chemistry step is used without an imaging step. For a three-level index with individual sub-indexes of 8 nucleotides, this means that at least 32 total dark cycles are required just to sequence the 24 nucleotides of the informative index sequence. This large number of cycles has been observed to result in a decrease in read quality, both for the index sequence and any subsequent insert sequencing reads.

[0068] Certain embodiments provided herein include bead code synthesis strategies in addition to long splint ligation strategies to reduce the number of invariant bases, resulting in SBS cycles of chemistry only required to read the bead code. Some such embodiments include bead code synthesis by chemical ligation using azide-alkyne click chemistry, bead code synthesis by templated polymerase extension, bead code synthesis by enzymatic ligation of double-stranded fragments, and bead code synthesis by splinted ligation with short splints containing non-standard nucleotides.

[0069] Some embodiments provided herein relate to high-throughput genotyping on an array. Some embodiments relate to decoding the location of microfeatures in an array. In some embodiments, the microfeatures include polynucleotides having barcodes and indexes. Some embodiments include sequencing the barcodes and indexes to identify the location of the polynucleotides in the array. Certain aspects that may be useful for the methods and compositions disclosed herein are disclosed in International Publication No. WO2020 / 086746, which is incorporated by reference in its entirety.

[0070] Decoding by hybridization involves identifying the location of a capture probe within a randomly distributed array of capture probes. The method typically involves several successive cycles of hybridizing a labeled hybridization probe to one or more portions of the capture probe, imaging the hybridization event, and removing the hybridization probe. Decoding by hybridization requires specialized reagents, specialized fluidic devices, and specialized detectors. In some embodiments, decoding by hybridization can take up to 8 hours with 7-8 successive cycles.

[0071] The embodiments provided herein include randomly distributed arrays of polynucleotides that include primer binding sites and barcodes. In some embodiments, the barcodes can be easily sequenced to decode the array using a high-throughput sequencing system. Some embodiments can significantly reduce the time it takes to decode the array without additional reagents, hybridization probes, or specialized decoding equipment.

[0072] Some embodiments include the use of next generation sequencing (NGS) technology and bead-based microarrays. Some such embodiments result in high performance, low cost, and high throughput genotyping assays that can be run on common NGS sequencing platforms with minor modifications to substrates and reagents.

[0073] Some embodiments include performing a genotyping assay in which S bead pools are loaded into a multi-well plate containing "S" wells, each bead pool having a unique sample index and each well containing "N" unique bead types. After nucleic acid library generation from the samples, such as processing the nucleic acid samples with steps including random primer amplification, followed by enzymatic fragmentation, and cleanup, each sample library is added to an indexed well and hybridized to a capture probe. After hybridization is complete, a single base extension assay is performed to probe the SNPs of interest by adding an incorporation mix containing fluorescent nucleotides and a suitable polymerase. At the end of this incorporation, all bead capture samples in the plate are pooled and loaded into a flow cell. The flow cell can be left as is or can be patterned, and the surface can be appropriately modified to support bead immobilization at a desired density. In some embodiments, upon bead immobilization, an SNP readout is performed, which includes one scan cycle to read the signal from fluorescent incorporation at the SNP sites. This cycle may include an SBS cycle on the instrument. A barcode readout is also performed, which includes 12-20 SBS cycles, depending on the complexity of the bead pool, to identify the capture probe and location of the specific bead in the flow cell. In some embodiments, this step can be replaced by an additional cycle of sequencing past the identified SNPs. A sample index readout is also performed, which includes 6-12 SBS cycles to read the sample index. In some embodiments, the entire assay on the flow cell can include less than about 30 SBS cycles and can be performed in less than 4 hours.

[0074] definition As used herein, "nucleic acid" is intended to be consistent with its use in the art and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize to nucleic acids in a sequence-specific manner or can be used as templates to replicate specific nucleotide sequences. Naturally occurring nucleic acids generally have backbones containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art. Naturally occurring nucleic acids generally have deoxyribose sugars (e.g., as found in deoxyribonucleic acid (DNA)) or ribose sugars (e.g., as found in ribonucleic acid (RNA)). Nucleic acids can contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can include natural or unnatural bases. In this regard, natural deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Useful unnatural bases that can be included in nucleic acids are known in the art. Examples of unnatural bases include locked nucleic acids (LNA) and bridged nucleic acids (BNA). LNA and BNA bases can be incorporated into DNA oligonucleotides to increase the hybridization strength and specificity of the oligonucleotides. LNA and BNA bases and the use of such bases are known and routine to those skilled in the art.

[0075] As used herein, the term "nucleotide analog" refers to a synthetic analog having modified nucleotide base moieties, modified pentose moieties, and / or modified phosphate moieties, and in the case of polynucleotides, modified internucleotide linkages. Modified internucleotide linkages include phosphate analogs, analogs having achiral and uncharged intersubunit linkages, and uncharged morpholino-based polymers having achiral intersubunit linkages. Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles. Examples of phosphate analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and boranophosphates, including, but not limited to, associated counterions, such as H+, NH4+, Na+, when such counterions are present. Examples of modified nucleotide base moieties include, but are not limited to, 5-methylcytosine (5mC); C-5-propynyl analogs, including but not limited to C-5 propynyl-C and C-5 propynyl-U; 2,6-diaminopurine (also known as 2-aminoadenine or 2-amino-dA); hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methylisoC, and isoguanine (isoG). Examples of modified pentose moieties include, but are not limited to, locked nucleic acid (LNA) analogs including Bz-A-LNA, 5-Me-Bz-C-LNA, dmf-G-LNA, and T-LNA, as well as 2' or 3' modifications in which the 2' or 3' position is hydrogen, hydroxy, alkoxy (e.g., methoxy, methoxy-ethyl, -O-methyl, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, and phenoxy), azido, amino, alkylamino, fluoro, chloro, or bromo. Other examples include 2-aminopurine; 5-bromodu, deoxyuridine, deoxyinosine, hydroxymethyl dC, 5-methyl dC, 5-nitroindole, 5-hydroxybutyne-2'-deoxyuridine, and 8-aza-7-deazaguanosine.

[0076] As used herein, "target", when used in reference to a nucleic acid, is intended as a semantic identifier of the nucleic acid in the context of the methods or compositions described herein and does not necessarily limit the structure or function of the nucleic acid other than as otherwise expressly indicated. A target nucleic acid may essentially be any nucleic acid of known or unknown sequence. It may be, for example, a fragment of genomic DNA or cDNA. Sequencing may result in the determination of the sequence of the entire or part of the target molecule. Targets may be derived from primary nucleic acid samples, such as nuclear or cell-free samples. In one embodiment, targets can be processed into templates suitable for amplification by placing universal sequences at the ends of each target fragment. Targets can also be obtained from primary RNA samples by reverse transcription into cDNA.

[0077] As used herein, "universal" when used to describe a nucleotide sequence refers to a region of sequence common to two or more nucleic acid molecules, the molecules also having regions of sequence that differ from each other. A universal sequence present in different members of a collection of molecules can capture multiple different nucleic acids using a population of universal capture nucleic acids, such as a capture oligonucleotide that is complementary to a portion of the universal sequence, e.g., a universal capture sequence. Non-limiting examples of universal capture sequences include sequences identical to or complementary to P5 and P7 primers. Similarly, a universal sequence present in different members of a collection of molecules can amplify or replicate (e.g., sequence) multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, such as a universal anchor sequence. Thus, the capture oligonucleotide or universal primer comprises a sequence that can specifically hybridize to the universal sequence. Two hybridizing universal sequences are referred to as a universal binding pair. For example, a hybridizing capture oligonucleotide and a universal capture sequence are a universal binding pair.

[0078] As used herein, "P5" and "P7" may be used when referring to a primer sequence or primer binding site. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complements of P5 and P7, respectively. It will be understood that any suitable amplification primers may be used in the methods presented herein, and the use of P5 and P7 is only an exemplary embodiment. The use of amplification primers such as P5 and P7 on flow cells is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, each of which is incorporated herein by reference in its entirety. For example, any suitable forward amplification primer can be useful in the methods presented herein for amplifying complementary sequences and sequences, whether immobilized or in solution. Similarly, any suitable reverse amplification primer can be useful in the methods presented herein for amplifying complementary sequences and sequences, whether immobilized or in solution. Those skilled in the art will understand how to design and use suitable primer sequences for capturing and / or amplifying nucleic acids as presented herein.

[0079] As used herein, "compartment" is intended to mean an area or volume that separates or isolates something from another. Exemplary compartments include vials, tubes, wells, droplets, boluses, beads, containers, surface features, or areas or volumes separated by physical forces such as fluid flow, magnetism, electric current, etc. In one embodiment, the compartments are wells of a multi-well plate, such as a 96 or 384 well plate.

[0080] As used herein, the term "primer" and its derivatives generally refer to any nucleic acid that can hybridize to a target sequence of interest. Typically, a primer serves as a substrate onto which nucleotides can be polymerized by a polymerase. In some embodiments, a primer can be incorporated into a synthesized nucleic acid strand to provide a site to which another primer can hybridize to prime synthesis of a new strand complementary to the synthesized nucleic acid molecule. A primer can include any combination of nucleotides or analogs thereof. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length and can include ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. It should be understood that these terms include, as equivalents, analogs of either DNA or RNA made from nucleotide analogs and are applicable to single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, which is a complementary or copy DNA generated from an RNA template, for example, by the action of reverse transcriptase. The term refers only to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA").

[0081] As used herein, "adaptor" or "adapter" and its derivatives, e.g., universal adaptor, generally refers to any linear oligonucleotide that can be ligated to a nucleic acid molecule or form a splint in a ligation reaction. In some embodiments, the adaptor or a portion of the adaptor is substantially complementary or complementary to the 3' or 5' end of a target sequence, such as the linker region of the target nucleic acid. In general, an adaptor can include any combination of nucleotides and / or nucleic acids. In some aspects, an adaptor can include one or more cleavable groups at one or more positions. In another aspect, an adaptor can include a sequence that is substantially identical to or substantially complementary to at least a portion of a primer, e.g., a universal primer. In some embodiments, an adaptor can include a barcode or tag to assist in downstream error correction, identification, or sequencing. The terms "adaptor" and "adapter" are used interchangeably.

[0082] As used herein, an "array" refers to a collection of sites that can be distinguished from one another according to their relative positions. Different molecules at different sites of an array can be distinguished from one another according to the site's position within the array. An individual site of an array can contain one or more molecules of a particular type. For example, a site can contain a single target nucleic acid molecule with a particular sequence, or a site can contain several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of an array can be different features located on the same substrate. Exemplary features include, but are not limited to, wells in a substrate, beads (or other particles) in or on a substrate, protrusions from a substrate, ridges on a substrate, or channels within a substrate. The sites of an array can be separate substrates, each with a different molecule. The different molecules attached to the separate substrates can be identified according to the substrate's position on a surface to which the substrates are associated, or according to the substrate's position within a liquid or gel. Exemplary arrays with separate substrates located on a surface include those with beads in wells. In some embodiments, the arrays can be located on a flow cell.

[0083] Combination Indexing Some embodiments of the methods and compositions provided herein include preparation of indexed beads by combinatorial indexing, in which a first polynucleotide attached to a bead is extended by sequential addition of an index. In some embodiments, a first polynucleotide, such as a single-stranded DNA polynucleotide, is extended with a second polynucleotide, such as a single-stranded DNA polynucleotide, that includes an index by a variety of methods, including by chemical ligation, by polymerase extension, by ligation of a second polynucleotide and a double-stranded adapter that includes a single-stranded overhang, or by splint ligation.

[0084] Some embodiments include split and pool indexing. For example, a first population of beads is split into a first plurality of subpopulations, a different first index is added to each subpopulation by attaching a first polynucleotide comprising a first index to the beads, and the subpopulations are combined to obtain a second population of beads. The second population of beads is split into a second plurality of subpopulations, a different second index is added to each subpopulation by extending the attached first polynucleotide with a polynucleotide comprising a second index, and the subpopulations are combined to obtain a third population of beads. The third population of beads is split into a third plurality of subpopulations, a different third index is added to each subpopulation by extending the second polynucleotide with a polynucleotide comprising a third index, and the subpopulations are combined to obtain a fourth population of beads. Splitting the population into subpopulations and adding a different index to each subpopulation can be repeated to generate even more diverse combination indexes.

[0085] Some embodiments for preparing a plurality of combinatorial indexed beads include: (a) obtaining a population of primary indexed beads comprising a first polynucleotide comprising a first index, the population of primary indexed beads comprising a plurality of subpopulations of first indexed beads, the subpopulations of first indexed beads comprising first indices that are different from each other; (b) dividing the population of primary indexed beads into a plurality of subpopulations of second beads; and (c) obtaining a population of secondary indexed beads comprising: (i) extending the first polynucleotide of the subpopulations of second beads with a second polynucleotide comprising a second index to obtain a subpopulation of second indexed beads, the second subpopulations of indexed beads comprising second indices that are different from each other; and (ii) combining the subpopulations of second indexed beads to obtain a population of secondary indexed beads.

[0086] Some embodiments also include (d) dividing the population of secondary indexed beads into a plurality of subpopulations of third beads, and (e) obtaining a population of tertiary indexed beads, comprising (i) extending the second polynucleotide of the plurality of subpopulations of third beads with a third polynucleotide comprising a third index to obtain a subpopulation of third indexed beads, the subpopulations of third indexed beads comprising third indices that are different from each other, and (ii) combining the subpopulations of third indexed beads to obtain a population of tertiary indexed beads. Some embodiments also include repeating (d) and (e) and adding additional indexes to the subpopulations of indexed beads to generate even more diverse combined indexes.

[0087] In some embodiments, obtaining a population of primary indexed beads comprising a first polynucleotide comprising a first index comprises (i) attaching the first polynucleotide to a plurality of subpopulations of first beads to obtain a subpopulation of first indexed beads, where the first polynucleotide comprises a different first index for each subpopulation of first beads, and (ii) combining the subpopulations of first indexed beads to obtain a population of primary indexed beads. In some embodiments, the first polynucleotide is attached to one bead of the plurality of subpopulations of first beads via a first binding partner and a second binding partner. In some embodiments, the first binding partner or the second binding partner is selected from the group consisting of biotin, streptavidin, a biotin derivative, a streptavidin derivative, an antibody, and an antigen-binding fragment of an antibody. In some embodiments, the first polynucleotide is attached to the bead by covalent attachment, for example via a chemical reaction.

[0088] In some embodiments, the first polynucleotide comprises a primer binding site. Examples of primer binding sites can include P5, P5', P7, and P7' sequences. P5 primer: AAT GAT ACG GCG ACC ACC GA (SEQ ID NO: 01), and P7 primer: CAA GCA GAA GAC GGC ATA CGA GAT (SEQ ID NO: 02). In some embodiments, the first polynucleotide comprises a cleavable linker. In some embodiments, the first polynucleotide comprises a universal sequence.

[0089] In some embodiments, the first index, the second index, and / or the third index have a length greater than, less than, or equal to 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 consecutive nucleotides, or a length within any two of the foregoing numbers. In some embodiments, an index or a combination of indexes can be used to tag an element, such as an attached nucleic acid, or a bead. Certain methods and compositions useful with the embodiments provided herein are disclosed in U.S. Patent Application Publication Nos. 2018 / 0023119 and 2018 / 0355348, each of which is incorporated by reference in its entirety.

[0090] In some embodiments, splitting a population of indexed beads, such as a population of primary indexed beads or a population of secondary indexed beads, may include randomly distributing the population of indexed beads into a plurality of compartments. In some embodiments, the plurality of compartments comprises a compartment selected from a well, a channel, or a droplet. In some embodiments, the plurality of compartments comprises a 96-well plate, a 192-well plate, or a 384-well plate. In some embodiments, a flow cell comprises a plurality of compartments.

[0091] In some embodiments, the plurality of beads comprises magnetic beads.

[0092] Some embodiments also include distributing the plurality of combinatorially indexed beads on an array, hi some embodiments, the plurality of combinatorially indexed beads are randomly distributed on the array.

[0093] Some embodiments also include sequencing the combinatorially indexed beads. For example, the combinatorial index of the bead or its complement can be sequenced. In some embodiments, the combinatorial index or its complement can be sequenced on the array. In some embodiments, the sequencing can include sequencing by synthesis (SBS). Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with the methods and compositions provided herein are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, and U.S. Patent No. 7,405,281, each of which is incorporated herein by reference. Some embodiments also include decoding the position of a bead of a plurality of combinatorially indexed beads on the array based on the combinatorial index of the bead.

[0094] In some embodiments, one bead of the plurality of combinatorial indexed beads comprises a capture probe. In some embodiments, the first polynucleotide, the second polynucleotide, or the third polynucleotide comprises a capture probe. Some embodiments also include hybridizing the target nucleic acid to the capture probe. Some embodiments also include extending the capture probe.

[0095] Combinatorial Indexing Including Chemical Ligation Some embodiments of the methods and compositions provided herein include the preparation of indexed beads by combinatorial indexing, in which polynucleotides attached to the beads are extended by sequential addition of indexes by chemical ligation. Certain methods and compositions useful with the embodiments provided herein are disclosed in U.S. Patent Application Publication No. 2018 / 0127816, which is incorporated by reference in its entirety.

[0096] In some embodiments, sequential addition of indexes by chemical ligation involves a copper-catalyzed azide-alkyne cycloaddition "click" reaction (CuAAC) to covalently attach successive levels of index oligonucleotides or polynucleotides (Figure 3). Click chemistry is compatible with DNA oligonucleotides, and the cyclic triazole product has dimensions similar to the phosphodiester bonds present in DNA. The similar dimensions of the cyclic triazole product to the phosphodiester bonds present in DNA make the triazole bond a viable template for many DNA polymerases, and triazole-containing DNA strands can be amplified by methods including polymerase chain reaction (PCR). Advantages of chemical ligation include how the ends of different single-stranded polynucleotides can be joined in the absence of splint oligonucleotides or splint adaptors by using an excess of index oligonucleotides or polynucleotides and eliminating all or only a handful of invariant base positions in the final bead code or combinatorial index. In addition, the absence of an enzymatic ligation step increases the flexibility of reaction conditions, since the CuAAC reaction can be performed in alternative solvents and at nonstandard temperatures.

[0097] In some embodiments, chemical ligation for bead code or combinatorial index synthesis involves a two-step synthesis strategy. For the first level of the bead code, an initial capture oligonucleotide, such as a first polynucleotide containing a first index, is generated with a CuAAC handle (azide or alkyne) at the 3' end, and this oligonucleotide is bound to a magnetic bead via a binding partner, such as 5'-biotin / streptavidin. For the second level of the bead code, an excess of index oligonucleotide, such as a second polynucleotide containing a second index, with a cognate CuAAC handle, such as 3'-azide / 5'alkyne or 3'-alkyne / 5'-azide, is added to the first polynucleotide bound to the bead with a copper catalyst. In some embodiments, the polynucleotide containing the second index will only have a click handle on the 5' end. Once the reaction is complete, the beads are pelleted and the remaining CuAAC reagent is washed away. This results in essentially "scarless" junctions between index levels, such as between a first polynucleotide and a second polynucleotide, with no invariant bases separating the levels.

[0098] In some embodiments, attaching a third polynucleotide containing a third index may involve the use of a second polynucleotide synthesized to have complementary click handles at both the 5' and 3' ends, however, this may result in the formation of long concatemers. To add additional index levels while avoiding the formation of concatemers, an enzyme such as terminal deoxynucleotidyl transferase (TdT) is used to insert a single base containing a click handle at the 3'OH position of the deoxyribose into the 3' end of the assembled two-level bead code. TdT attaches nucleotide triphosphates to the 3' end of single-stranded oligonucleotides with little specificity, but only a single base is attached to each oligo because the click handle blocks the 3'OH. ​​After washing away the TdT, an additional index level is added to the junction of the first two oligonucleotides using an identical procedure. Because the click reaction may not have 100% efficiency, further ligation can be blocked to avoid combinatorial indexes with "skipped" levels. This is accomplished by adding a small molecule containing a complementary click handle to block any unreacted 3' groups.

[0099] In some embodiments, chemical ligation can be used in a splinted ligation strategy to reduce the amount of oligonucleotide required for the reaction. Chemical ligation is compatible with the use of non-standard nucleotides. The use of certain non-standard nucleotides with higher hybridization specificity allows for shorter splint oligonucleotides. For example, modified nucleotides such as peptide nucleic acid (PNA), locked nucleic acid (LNA), or 2'-OMe bases can increase the melting temperature of an oligonucleotide without adding length.

[0100] FIG. 3 shows one embodiment of a scheme for joining a second index (index B) to a first index (index A) attached to a substrate by click chemistry ligation. As shown in FIG. 3, a first polynucleotide comprising a P5 sequence and index A is joined to a bead via a biotin / streptavidin binding pair. For example, the bead is coated with biotin and the first polynucleotide comprises a 5' end linked to streptavidin, or the bead is coated with streptavidin and the first polynucleotide comprises a 5' end linked to biotin. The first polynucleotide comprises a 3' end with a propargyl moiety. The second polynucleotide comprises index B and a 5' end with an azide moiety. A copper-catalyzed click reaction results in the joining of the first and second polynucleotides. An additional polynucleotide is added to the 3' end of the second polynucleotide by first treating the bead-linked polynucleotide with TdT to add a single nucleotide comprising a propargyl moiety. An additional polynucleotide containing a 5' end bearing an azide moiety can be used to further extend the bead-linked polynucleotide via an additional click reaction.

[0101] Some embodiments include extending a first polynucleotide, such as a bead-linked first polynucleotide, with a second polynucleotide by a chemical ligation reaction. In some embodiments, the first polynucleotide includes a terminal 3'-modified deoxynucleotide (dNTP) that includes a 3' functional moiety that can participate in a click chemistry reaction. In some embodiments, the second polynucleotide includes a terminal 5'-modified nucleotide that includes a compatible 5' functional moiety that can participate in a click chemistry reaction with the 3' functional moiety. In some embodiments, the 3' functional moiety and the 5' functional moiety can react with each other to form a modified backbone bond.

[0102] In some embodiments, a secondary indexed polynucleotide is obtained by extending the first polynucleotide with a second polynucleotide, and the method further comprises modifying the secondary indexed polynucleotide to obtain a modified polynucleotide comprising a terminal 3' modified nucleotide comprising a 3' functional moiety capable of participating in a click chemistry reaction. In some embodiments, the modifying comprises contacting the secondary indexed polynucleotide with a template independent polymerase. In some embodiments, the template independent polymerase is selected from terminal deoxynucleotidyl transferase (TdT), polyA polymerase, or CCA-adding RNA polymerase. In some embodiments, the template independent polymerase is TdT.

[0103] Some embodiments also include extending the modified polynucleotide with a third polynucleotide by a chemical ligation reaction, the third polynucleotide comprising a terminal 5' modified nucleotide that comprises a compatible 5' functional moiety that can participate in a click chemistry reaction with the 3' functional moiety.

[0104] In some embodiments, the 3' functional moiety is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone. In some embodiments, the 5' functional moiety is different from and compatible with the 3' functional moiety and is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone. In some embodiments, the 3'-functional moiety and the 5'-functional moiety are selected from the following pairs: (i) 3'-azido / 5'-alkynyl, (ii) 3'-alkynyl / 5'-azide, (iii) 3'-thiol / 5'-alkynyl, (iv) 3'-thiol / 5'-alkenyl, (v) 3'-alkynyl / 5'-thiol, (vi) 3'-alkenyl / 5'-thiol, (vii) 3'-azido / 5'-cyclooctynyl, (viii) 3'-cyclooctynyl / 5'-azido, (ix) 3'-nitrone / 5'-cyclooctynyl, and (x) 3'-cyclooctynyl / 5'-nitrone. In some embodiments, the 3'-functional moiety is 3'-azido and the 5'-functional moiety is 5'-alkynyl. In some embodiments, if an orthogonal click reaction is used between the L1 / L2 and L2 / L3 indexes, the TdT step can be avoided.

[0105] In some embodiments, the click chemistry reaction comprises a copper-catalyzed azide-alkyne cycloaddition to form a triazolyl-containing modified backbone linkage.

[0106] Combinatorial indexing including polymerase extension Some embodiments of the methods and compositions provided herein include the preparation of indexed beads by combinatorial indexing, in which polynucleotides attached to the beads are extended by sequential addition of indexes by polymerase extension.

[0107] In some embodiments, the adapter comprises an oligonucleotide that contains both a 3' region complementary to the 3' linker (L1A) region of the first polynucleotide bound to the bead containing the first index, and the reverse complement of the second index sequence (Figure 4). The adapter oligonucleotide is hybridized to the bead-bound first polynucleotide, and DNA polymerase and dNTPs extend the first polynucleotide, resulting in a covalently linked, bead-bound oligonucleotide that contains the complement of the adapter at its 3' end. Because the adapter hybridizes directly to the first polynucleotide, half the number of bases, such as 8 base pairs 16 bases, is sufficient to achieve similar binding specificity in the current Sprint Ligation workflow. Once polymerization is complete, for example if the adapter contains deoxyuracil residues instead of thymine, the adapter can be removed by denaturation using heat, base (NaOH), or treatment with USER enzyme. Additional index levels can be added by hybridizing a second adaptor containing a third index to the 3' end of the extended first polynucleotide and further extending the extended first polynucleotide with a polymerization reaction and hybridized second adaptor. In some embodiments, the 3' end of the adaptor can be blocked, for example, with a 3'2' dideoxynucleotide, C3 linker, or other standard blocking chemistry, to prevent the polymerase from using the 3' end of the adaptor as a primer and generating undesired products.

[0108] FIG. 4 shows an embodiment of a scheme for adding a second index (index B) and a third index (index C) to a first index (index A) attached to a substrate by sequential polymerase extension reactions. As shown in FIG. 4, a first polynucleotide comprising a P5 sequence, index A, and a first linker (link 1a) is conjugated to a bead via a biotin / streptavidin binding pair. For example, the bead is coated with biotin and the first polynucleotide comprises a 5' end linked to streptavidin, or the bead is coated with streptavidin and the first polynucleotide comprises a 5' end linked to biotin. A first adaptor comprising a region capable of hybridizing to the first linker, index B', and linker 2a' is hybridized to the first linker, and the first polynucleotide is extended in the presence of a polymerase to obtain an extended polynucleotide comprising indexes A and B. The first adaptor is removed. A second adaptor comprising a region capable of hybridizing to linker 2a, index C', and capture probe' (Hyb') is hybridized to the linker 2a region of the extended polynucleotide comprising indexes A and B, and the extended polynucleotide comprising indexes A and B is further extended in the presence of a polymerase to obtain an extended polynucleotide comprising indexes A, B, and C, and capture probe (Hyb).

[0109] Some embodiments include extending the first polynucleotide by polymerase extension. In some embodiments, the first polynucleotide includes a first linker. Some embodiments also include (i) obtaining a first adaptor that includes a region capable of hybridizing to the first linker and a region that includes a second index or a complement of the second index, (ii) hybridizing the first adaptor to the first linker, and (iii) extending the first polynucleotide to obtain a secondary indexed polynucleotide. In some embodiments, the first adaptor includes a non-extendable 3' end. In some embodiments, the non-extendable 3' end includes a 3'2' dideoxynucleotide, or a C3 linker, such as a 3 carbon spacer arm. Some embodiments also include removing the first adaptor from the secondary indexed polynucleotide. In some embodiments, removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation. In some embodiments, the first linker has a length of less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 contiguous nucleotides. In some embodiments, the first linker has a length of less than 5 contiguous nucleotides.

[0110] In some embodiments, obtaining a population of tertiary indexed beads includes extending the second polynucleotide by polymerase extension. In some embodiments, the first adaptor includes a complement of the second linker, such that the secondary indexed polynucleotide includes the second linker. Some embodiments also include (i) obtaining a second adaptor including a region capable of hybridizing to the second linker and a region including the third index or the complement of the third index, (ii) hybridizing the second adaptor to the second linker, and (iii) extending the secondary indexed polynucleotide to obtain a tertiary indexed polynucleotide. In some embodiments, the second adaptor includes a non-extendable 3' end. In some embodiments, the non-extendable 3' end includes a 3'2' dideoxynucleotide, or a C3 linker. Some embodiments also include removing the second adaptor from the tertiary indexed polynucleotide. In some embodiments, removing comprises denaturing the first adaptor with heat or base, or degrading the first adaptor by enzymatic degradation. In some embodiments, the second linker has a length of less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 contiguous nucleotides. In some embodiments, the second linker has a length of less than 5 contiguous nucleotides.

[0111] Combinatorial indexing involving ligation of double-stranded fragments Some embodiments of the methods and compositions provided herein include the preparation of indexed beads by combinatorial indexing, where a polynucleotide attached to a bead is extended by sequential addition of an index by ligation of a second polynucleotide and an adapter that includes a double-stranded region. In some embodiments, the use of a partially double-stranded adapter means that additional indexes can be ligated to the bead-linked oligonucleotide using a single linker sequence, thereby reducing the length of the linker sequence in the combinatorial indexed oligonucleotide compared to some other methods. For example, FIG. 15A (left panel) shows an exemplary embodiment for standard splint ligation, where the splint contains two linker sequences, "KS-3" and "KS-5", that anneal to the "KS-3'" sequence in the bead-linked oligonucleotide and to the "KS-5'" sequence in the adapter sequence containing index 2, respectively. In contrast, the exemplary embodiment of double-stranded splint ligation shown in FIG. 15A (right panel) shows a double-stranded splint that contains a single linker sequence, "KS-3", sufficient to ligate the adapter sequence containing the index to the bead-linked oligonucleotide.

[0112] Some embodiments include the use of double-stranded adapters that include index oligonucleotides with complementary overhangs that extend from the 3'-end of both strands of the duplex (see, e.g., Figures 5, 15A, and 19A). Some such embodiments reduce the number of invariant bases in the bead code by up to 50%, since only a single region of complementary bases is sufficient for assembly. Starting with a first polynucleotide bound to a bead that includes a first index, an adapter is added to the bead and hybridized that includes a double-stranded fragment with a 3' overhang that is complementary to the 3'-end of the first polynucleotide. The "top" fragment, which contains the index sequence and the 3' hybridization sequence for the next index level, is then ligated to the first polynucleotide by enzymatic or chemical ligation. Once ligation is complete, the bottom strand of the double-stranded index portion of the adapter can be removed by denaturation, and additional index levels can be added by the same procedure. In some embodiments, an additional single-stranded oligonucleotide may be present during ligation (see, e.g., FIG. 12, right panel, "Index 1' Oligo"). The additional oligonucleotide can hybridize to an index sequence of a polynucleotide bound to a bead. In some such embodiments, the additional oligonucleotide can further stabilize the ligation complex and facilitate ligation.

[0113] FIG. 5 shows an embodiment of a scheme for adding a second index (index B) and a third index (index C) to a first index (index A) attached to a substrate using an adaptor that includes a double-stranded region and a single-stranded overhang. As shown in FIG. 5, a first polynucleotide that includes a P5 sequence, index A, and a first linker (link 1a) is conjugated to a bead via a biotin / streptavidin binding pair. For example, the bead is coated with biotin and the first polynucleotide includes a 5' end linked to streptavidin, or the bead is coated with streptavidin and the first polynucleotide includes a 5' end linked to biotin. A first adaptor that includes a single-stranded 5' overhang that includes a region that can hybridize to the first linker, a double-stranded region that includes index B, and a region that includes a second linker (link 2a) is hybridized to the first linker. The first adaptor is covalently joined to the first polynucleotide by ligase ligation or by chemical ligation through the linker 1a and index B regions of the first polynucleotide and adaptor, respectively, to obtain an extended polynucleotide comprising indexes A and B. The strand of the first adaptor that is not covalently joined to the first polynucleotide is removed. The second adaptor, comprising a single-stranded 5' overhang comprising a region capable of hybridizing to a second linker, a double-stranded region comprising index C, and a capture probe (Hyb), is hybridized to the second linker. The second adaptor is covalently joined to the extended first polynucleotide by ligase ligation or by chemical ligation through the linker 1a and index B regions of the first polynucleotide and adaptor, respectively, to obtain an extended polynucleotide comprising indexes A, B, and C, and a capture probe (Hyb). The strand of the second adaptor that is not covalently attached to the extended polynucleotide, including indexes A, B, and C, and the capture probe (Hyb), is removed.

[0114] FIG. 19A shows an additional exemplary embodiment of combinatorial indexing in which a polynucleotide attached to a bead is extended by sequential addition of indexes by ligation of a second polynucleotide and an adaptor comprising a double-stranded region. As shown in FIG. 19A, an oligonucleotide comprising P5 sequence, index 1, and KS-3' sequence is linked to a bead via biotin (B) and streptavidin (SA). A partially double-stranded adaptor, one strand of which comprises KS-3 sequence, index 2', and MS-3 sequence, and the other strand of which comprises index 2 sequence and MS-3' sequence, is annealed to the bead-linked oligonucleotide via the KS-3 and KS-3' sequences. The adaptor sequence is ligated to the bead-linked sequence to form an extended bead-linked oligonucleotide hybridized to the adaptor strand. The adaptor strand is removed by denaturation. A second round of indexing is performed.

[0115] Some embodiments include extending the first polynucleotide with a second polynucleotide by ligation. Some embodiments also include (i) obtaining a double-stranded first adaptor comprising a second polynucleotide and a 3' single-stranded overhang capable of hybridizing to the first linker of the first polynucleotide, (ii) hybridizing the first adaptor to the first linker, and (iii) ligating the first polynucleotide to the second polynucleotide to obtain a secondary indexed polynucleotide. Some embodiments also include extending the second polynucleotide with a third polynucleotide by ligation. Some embodiments also include (i) obtaining a double-stranded second adaptor comprising a third polynucleotide and a 3' single-stranded overhang capable of hybridizing to a second linker of the second polynucleotide; (ii) hybridizing the second adaptor to the second linker; and (iii) ligating the second polynucleotide to the third polynucleotide to obtain a tertiary indexed polynucleotide. In some embodiments, the ligation comprises the use of a ligase. In some embodiments, the ligation comprises a chemical ligation reaction, such as a click chemistry reaction disclosed herein.

[0116] Combinatorial indexing including splint ligation Some embodiments of the methods and compositions provided herein include preparation of indexed beads by combinatorial indexing, where a polynucleotide attached to a bead is extended by sequential addition of indexes by splint ligation, where the splint comprises a nucleotide with increased hybridization specificity. In some embodiments, the splint comprises a nucleotide sequence that comprises a nucleotide with stronger binding to a complementary sequence, e.g., a nucleotide that results in an increased Tm for the nucleotide sequence. In some embodiments, the splint comprises one or more modified nucleotides or nucleotide analogs. In some embodiments, the splint comprises a locked nucleic acid (LNA). In some embodiments, the splint comprises one or more inosine nucleotides.

[0117] Some embodiments include the use of a reduced number of bases in the splint, including chemically modified bases that increase the strength of hybridization between the splint and the index / capture oligonucleotide, and splinted ligation to link successive levels of the index (Figure 6). In their current form, the splint sequences are 8 nucleotides long with a denaturation midpoint (Tm) close to room temperature (about 25°C). This ensures that the splint is able to bind to both the capture and index oligonucleotides during a room temperature ligation reaction.

[0118] FIG. 6 shows an embodiment of a scheme for adding a second index (index B) and a third index (index C) to a first index (index A) attached to a substrate by sequential splinted ligation reactions using a short linker-sprint. As shown in FIG. 6, a first polynucleotide comprising a P5 sequence, index A and a first linker (link 1a) is conjugated to a bead via a biotin / streptavidin binding pair. For example, the bead is coated with biotin and the first polynucleotide comprises a 5' end linked to streptavidin, or the bead is coated with streptavidin and the first polynucleotide comprises a 5' end linked to biotin. The second polynucleotide comprises a second linker (link 1b), index B and a third linker (link 2a). A single-stranded first adaptor, such as a splint, that includes a region that can hybridize to the first linker and a region that can hybridize to the second linker, is hybridized to both the first linker of the first polynucleotide and the second linker of the second polynucleotide. The first polynucleotide is covalently joined to the second polynucleotide by ligation, such as by using a ligase, to obtain an extended polynucleotide that includes indexes A and B and a third linker (link 2a). The first adaptor is removed. The third polynucleotide includes a fourth linker (link 2b), index C, and a capture probe (Hyb). A single-stranded second adaptor, such as a splint, that includes a region that can hybridize to the third linker and a region that can hybridize to the fourth linker, is hybridized to both the third linker of the extended polynucleotide and the fourth linker of the third polynucleotide. A third polynucleotide is covalently joined to the extended polynucleotide by ligation, such as by use of a ligase, to obtain an extended polynucleotide comprising indexes A, B, and C and the capture probe. The second adaptor is removed.

[0119] Some embodiments include extending the first polynucleotide by ligation, where the first polynucleotide comprises a first linker and the second polynucleotide comprises a second linker. Some embodiments also include (i) obtaining a first adaptor comprising a region capable of hybridizing to the first linker and a region capable of hybridizing to the second linker, (ii) hybridizing the first adaptor to the first linker, (iii) hybridizing a second oligonucleotide to the region capable of hybridizing to the second linker, and (iv) ligating the first polynucleotide to the second polynucleotide to obtain a secondary indexed polynucleotide. In some embodiments, the first linker and / or the second linker have a length of less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive nucleotides. In some embodiments, the first linker and / or the second linker have a length of less than 5 consecutive nucleotides. In some embodiments, the first linker and / or the second linker is modified to have an increased Tm compared to an oligonucleotide having the same length as the first adaptor. In some embodiments, the first linker and / or the second linker comprises an increased G / C content or comprises modified nucleotides compared to an oligonucleotide having the same length. Some embodiments also include removing the first adaptor from the secondary indexed polynucleotide. In some embodiments, removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0120] In some embodiments, obtaining a population of tertiary indexed beads includes extending the second polynucleotide by ligation, where the second oligonucleotide includes a third linker, such that the secondary indexed polynucleotide includes the third linker and the third polynucleotide includes a fourth linker. Some embodiments also include (i) obtaining a second adaptor including a region capable of hybridizing to the third linker and a region capable of hybridizing to the fourth linker, (ii) hybridizing the second adaptor to the third linker, (iii) hybridizing the third polynucleotide to the second adaptor through a region capable of hybridizing to the fourth index, and (iv) ligating the secondary indexed polynucleotide to the third polynucleotide to obtain a tertiary indexed polynucleotide. In some embodiments, the third linker and / or the fourth linker have a length of less than 9, 8, 7, 6, 5, 4, 3, or 2 consecutive nucleotides. In some embodiments, the third linker and / or the fourth linker has a length of less than 5 consecutive nucleotides. In some embodiments, the third linker and / or the fourth linker is modified to have an increased Tm compared to an oligonucleotide having the same length as the second adaptor. In some embodiments, the third linker and / or the fourth linker comprises an increased G / C content or comprises modified nucleotides compared to an oligonucleotide having the same length. Some embodiments also include removing the second adaptor from the tertiary indexed polynucleotide. In some embodiments, removing includes denaturing the first adaptor by heat or base, or degrading the first adaptor by enzymatic degradation.

[0121] Sequencing and Analysis of Target Nucleic Acids Some embodiments include sequencing and / or analysis of the target nucleic acid. Certain methods and compositions useful with the embodiments provided herein are disclosed in U.S. Patent Application Publication No. 2021 / 0087613, the entirety of which is incorporated by reference. Some embodiments include decoding the position of a polynucleotide in an array according to the methods provided herein, hybridizing the target nucleic acid to a capture probe, extending the capture probe, and detecting the extension of the capture probe hybridized to the target nucleic acid at the position on the array. In some embodiments, the position of the polynucleotide on the array can be decoded before hybridizing the target nucleic acid to the polynucleotide. In some embodiments, the position of the polynucleotide on the array can be decoded after detecting the extension of the capture probe hybridized to the target nucleic acid. In some such embodiments, each polynucleotide can be associated with a capture probe via a common element. For example, the polynucleotide and the capture probe can each be attached to the same microfeature, such as a bead. In more such embodiments, each polynucleotide can include a capture probe.

[0122] Some embodiments include single base extension (SBE) of a capture probe. In some embodiments, SBE can be used to detect alleles, mutations, or other features in a target nucleic acid. Briefly, SBE utilizes a capture probe that hybridizes to a target genome fragment at a position proximal or adjacent to a detection position, which indicates a specific locus. A polymerase can be used to extend the 3' end of the capture probe with a nucleotide analog labeled with a detection label. Based on the fidelity of the enzyme, a nucleotide is incorporated into the capture probe only if it is complementary to the detection position in the target nucleic acid. Optionally, the nucleotide can be derivatized using a blocking group (including a reversible blocking group) so that further extension cannot occur, and therefore only a single nucleotide is added. The presence of the labeled nucleotide in the extended capture probe can be detected, for example, at a specific position in an array, and the added nucleotide can be identified to determine the identity of the locus or allele. SBE can be carried out under known conditions, such as those described in US Pat. Nos. 9,441,267 and 9,045,796, each of which is incorporated by reference in its entirety.

[0123] Some embodiments include allele specific primer extension (ASPE). In some embodiments, ASPE may involve extension of capture probes that differ in nucleotide composition at the 3' end. The ASPE method can be performed using nucleosides or nucleotides that contain a cleavable linker, so that the label can be removed after the probe is detected. This allows for further use of the probe or verification that the detected signal was due to the label that was just removed. Briefly, ASPE can be performed by hybridizing a target nucleic acid to a capture probe that has a 3' sequence portion that is complementary to the detection position and a 5' portion that is complementary to a sequence adjacent to the detection position. Template-directed modification of the 3' portion of the capture probe, for example by addition of a labeled nucleotide by a polymerase, produces a labeled extension product, but only if the template contains the target sequence. The presence of such a labeled primer extension product can then be detected based on its location in the array, for example to indicate the presence of a particular allele. In some embodiments, ASPE can be performed with multiple capture probes that have similar 5' ends to anneal adjacent to the same detection position in the target nucleic acid, but different 3' ends so that only the capture probes with 3' ends that are complementary to the detection position are modified by polymerase. A capture probe with a 3' end base that is complementary to a particular detection position is referred to as a perfect match (PM) probe for the position, while a capture probe that has a 3' end mismatch base and cannot be extended in the ASPE reaction is a mismatch (MM) probe for the position. The presence of a labeled nucleotide in the PM probe can be detected, and the 3' sequence of the capture probe can be determined to identify the specific allele at the detection position.

[0124] Kits and Systems Some embodiments provided herein include kits and systems, which may include reagents for carrying out certain methods provided herein, including beads, multi-well plates, enzymes such as ligases and polymerases, polynucleotides, binding pairs such as biotin and streptavidin or derivatives thereof, and click chemistry reagents. EXAMPLES

[0125] Example 1 - Beadcode synthesis by click chemistry ligation A plurality of beads are coated with streptavidin and distributed into wells of a first 96-well plate. A different first polynucleotide is distributed into each well. Each first polynucleotide comprises a 5' end linked to biotin such that the first polynucleotide is conjugated to the bead via a biotin / streptavidin binding pair. The first polynucleotide comprises a P5 sequence, an index A, and a 3' end having a propargyl moiety. The first polynucleotide in each well has a different index A than the first polynucleotide in a different well. The beads are pooled and distributed into wells of a second 96-well plate.

[0126] A second polynucleotide is added to each well of a second 96-well plate. The second polynucleotide comprises an index B and a 5' end with an azide moiety. Index B is different for each well. A copper-catalyzed click reaction is performed in each well, resulting in the conjugation of the first and second polynucleotides. The bead-linked polynucleotides are treated with TdT to add a single nucleotide containing a propargyl moiety. The beads are pooled and distributed into the wells of a third 96-well plate.

[0127] An additional polynucleotide is added to the 3' end of the second polynucleotide. The bead-linked polynucleotide can be further extended via an additional click reaction using a third polynucleotide that contains an azide moiety at its 5' end and an index C. Index C is different for each well.

[0128] Example 2 - Combinatorial Indexing Including Polymerase Extension A plurality of beads are coated with streptavidin and distributed into wells of a first 96-well plate. A different first polynucleotide is distributed into each well. Each first polynucleotide comprises a 5' end linked to biotin such that the first polynucleotide is attached to the bead via a biotin / streptavidin binding pair. The first polynucleotide comprises a P5 sequence, an index A, and a first linker of 8 nucleotides. The first polynucleotide in each well has a different index A than the first polynucleotide in a different well. The beads are pooled and distributed into wells of a second 96-well plate.

[0129] A first adaptor is added to each well of a second 96-well plate. The first adaptor comprises a region capable of hybridizing to a first linker, an index B' sequence, and a linker 2a'. The index B' sequence is different for each well. The first adaptor is hybridized to the first linker, and the first polynucleotide is extended in the presence of a polymerase to obtain an extended polynucleotide comprising indexes A and B. The first adaptor is removed from the beads. The beads are pooled and distributed into the wells of a third 96-well plate.

[0130] A second adaptor is added to each well of a third 96-well plate. The second adaptor comprises a region capable of hybridizing to linker 2a, index C', and capture probe' (Hyb'). In some embodiments, the capture probe is useful for certain applications where the universal transposome is attached to the 3' end of the bead-linked oligo, and can be irrelevant to indexing (i.e., it can be any sequence depending on the application). The index C' sequence is different for each well. The second adaptor is hybridized to the linker 2a region of the extended polynucleotide comprising indexes A and B, and the extended polynucleotide comprising indexes A and B is further extended in the presence of a polymerase to obtain an extended polynucleotide comprising indexes A, B, and C, and capture probe (Hyb). The second adaptor is removed from the beads.

[0131] Example 3 - Combinatorial indexing involving ligation of double-stranded fragments A plurality of beads are coated with streptavidin and distributed into wells of a first 96-well plate. A different first polynucleotide is distributed into each well. Each first polynucleotide comprises a 5' end linked to biotin such that the first polynucleotide is attached to the bead via a biotin / streptavidin binding pair. The first polynucleotide comprises a P5 sequence, an index A, and a first linker of 8 nucleotides. The first polynucleotide in each well has a different index A than the first polynucleotide in a different well. The beads are pooled and distributed into wells of a second 96-well plate.

[0132] A first adaptor is added to each well of a second 96-well plate. The first adaptor comprises a single-stranded 5' overhang that comprises a region capable of hybridizing to a first linker, a double-stranded region that comprises index B, and a region that comprises a second linker (link 2a). Index B is different for each well. The first adaptor is hybridized to the first linker. The first adaptor is covalently joined to the first polynucleotide by ligation with a ligase or by chemical ligation via the linker 1a and index B regions of the first polynucleotide and adaptor, respectively, to obtain an extended polynucleotide that comprises indexes A and B. The strand of the first adaptor that is not covalently joined to the first polynucleotide is removed. The beads are pooled and distributed into the wells of a third 96-well plate.

[0133] A second adaptor is added to each well of a third 96-well plate. The second adaptor comprises a single-stranded 5' overhang comprising a region capable of hybridizing to a second linker, a double-stranded region comprising index C, and a capture probe (Hyb). The second adaptor is hybridized to the second linker. The second adaptor is covalently joined to the extended first polynucleotide by ligation with a ligase or by chemical ligation through the linker 1a and index B regions of the first polynucleotide and the adaptor, respectively, to obtain an extended polynucleotide comprising indexes A, B, and C, and a capture probe (Hyb). The strand of the second adaptor that is not covalently joined to the extended polynucleotide comprising indexes A, B, and C, and a capture probe (Hyb) is removed from the beads.

[0134] Example 4 - Combinatorial Indexing Including Splint Ligation A plurality of beads are coated with streptavidin and distributed into wells of a first 96-well plate. A different first polynucleotide is distributed into each well. Each first polynucleotide comprises a 5' end linked to biotin such that the first polynucleotide is attached to the bead via a biotin / streptavidin binding pair. The first polynucleotide comprises a P5 sequence, an index A, and a first linker. The first polynucleotide in each well has a different index A than the first polynucleotide in a different well. The beads are pooled and distributed into wells of a second 96-well plate.

[0135] A second polynucleotide is added to each well of a second 96-well plate. The second polynucleotide comprises a second linker (Link 1b), an index B, and a third linker (Link 2a). Index B is different for each well. A single-stranded first adaptor, such as a splint, is added to each well, comprising a region capable of hybridizing to the first linker and a region capable of hybridizing to the second linker. The first adaptor hybridizes to both the first linker of the first polynucleotide and the second linker of the second polynucleotide. The first polynucleotide is covalently joined to the second polynucleotide by ligation, such as by use of a ligase, to obtain an extended polynucleotide comprising indexes A and B and a third linker (Link 2a). The first adaptor is removed. The beads are pooled and distributed into the wells of a third 96-well plate.

[0136] A third polynucleotide is added to each well of a third 96-well plate. The third polynucleotide comprises a fourth linker (Link 2b), an index C, and a capture probe (Hyb). Index B is different for each well. A single-stranded second adaptor, such as a splint, is added to each well, comprising a region capable of hybridizing to the third linker and a region capable of hybridizing to the fourth linker. The second adaptor is hybridized to both the third linker of the extended polynucleotide and the fourth linker of the third polynucleotide. The third polynucleotide is covalently joined to the extended polynucleotide by ligation, such as by using a ligase, to obtain an extended polynucleotide comprising indexes A, B, and C, and a capture probe. The second adaptor is removed from the beads.

[0137] Example 5 - Combinatorial Indexing Including Polymerase Extension Combinatorial indexing with polymerase extension (see, e.g., FIG. 4) was compared with combinatorial indexing with splint ligation (see, e.g., FIG. 2). A two-level indexing protocol with polymerase extension was performed using beads with capture oligonucleotides (P5-IndexA-Link1a) and extension templates (Link1a'-IndexB'-Hyb'). Polymerase extension was performed with different amounts of template and T4 polymerase with and without exonuclease activity. The number of capture probes and the number of full-length products were measured using quantitative PCR (qPCR) with primers at the positions shown in FIG. 7A. As shown in FIG. 7B, (1) T4 polymerase (exo+) degraded the capture probe and extension template, (2) T4 polymerase (exo-) showed reduced degradation of the capture oligonucleotide, and (3) a 50× excess of extension template over the capture oligo (330 fmol / ug) was sufficient for full-length extension with T4 polymerase (exo-).

[0138] Figures 8A and 8B summarize the various conditions tested in the three-level indexing protocol. The volume was 25 μl / condition in duplicate. Polymerase extension was performed with NaOH extension oligo denaturation at 20°C (no rotation) for 15 min. Splint ligation was performed with 60°C splint denaturation overnight and for 4 h (no rotation). Readouts included quantitative PCR (qPCR) assays and direct bead-coded sequencing. For qPCR, chemically synthesized full-length control oligos (sequence identical to the corresponding three-level PE or SL oligos) were used to generate standard curves for quantification. Concentrations of the second and third level amplicons of splint ligation (SL) were adjusted for size differences.

[0139] Figure 9 summarizes the qPCR results and shows that PE (8) gives less full-length oligos than SL (12). The efficiency of synthesis was lower with PE versus SL, e.g., 20% of the oligos were full-length with PE and 40% with SL, which could have been due to the second ligation (12) being much less efficient than the first ligation, and / or the 20°C incubation without rotation. Samples that received PE showed lower capture oligo (CO) molecules / bead (3-8) compared to full length (FL) PE (1) and SL (10-12). Each extension reduced the CO molecules / bead (3-5 vs. 6-8). Some loss was due to stripping of Bio-oligo from the beads by the NaOH wash. Some loss may have been due to T4 polymerase backbiting the oligo, as in (6) vs. (7 and 8) and (3) vs. (5), but this was not observed in (3) vs. (4).

[0140] The effect of 0.2N NaOH in the denaturation step was tested. Beads with 6.6 fmol of first level oligos (B1 index) bound to the beads via either 5'biotin or 5'ddbiotin were used without extension / ligation in five different denaturation treatments and controls: none (-control); no denaturation, only washing steps (2 washes per level, 4 in total); 2x heat denaturation at 60°C + wash; 2x heat denaturation at 80°C + wash; 1x 0.2N NaOH denaturation + wash; and 2x 0.2N NaOH denaturation + wash. The remaining oligos were measured by qPCR using the first level primers. The results are summarized in Figure 10, which shows that 0.2N NaOH denaturation caused biotin-specific CO loss.

[0141] Direct bead-coded sequencing analysis was performed for three-level indexing. Briefly, the assay involves amplifying synthetic bead-coded oligos from beads by PCR using P5 forward and reverse primers targeting the Hyb region. The reverse primer introduces the sample index, the binding region for the ME_V2_B15 sequencing primer, and the P7' sequence that allows for clustering. Figure 11A outlines the read orientation for the analysis. The analysis involves trimming and pairwise alignment of each read to the expected sequence with error calling including identification of deletions and putative "SNPs" that indicate base change errors in the sequence. Fully accurate indexes contained no errors (SNPs, insertions or deletions) across the index region, for example in the three-level index, index 1, index 2, and index 3. All usable indexes included those indexes that could be correctly decoded by index error correction implementation. Figures 11B and 11C show the levels of fully accurate sequences, usable indexes, and error rates per base. Splint ligation was more efficient than polymerase extension, although both had similar levels of errors.

[0142] From the aforementioned studies, three-level polymerase extension produced fewer full-length molecules than splint ligation. The 0.2N NaOH denaturation and addition of T4 polymerase exonuclease activity likely contributed to the reduction in total captured oligos. Polymerase extension appeared to be less efficient than splint ligation in this experiment. 0.2N NaOH denaturation resulted in a loss of biotinylated oligos from the beads, but this can be mitigated by replacing the single 5' biotin with a 5' double desthiobiotin. The polymerase-extended oligos synthesized had similar levels of errors compared to the splint ligation method.

[0143] Example 6 - Two-level indexing by double-stranded splint ligation The two-level indexing with double-stranded splint ligation was compared with the two-level indexing with splint ligation (standard). The outline of standard combinatorial indexing with splint ligation and double-stranded splint ligation is shown in Figure 12A. Compared with standard splint ligation, in the double-stranded method, the 5' half of the splint can be complementary to the second level index, reducing the common sequence length between indexes by half. The outline of the protocol and conditions tested is shown in Figure 12B.

[0144] qPCR assays were performed and standard curves were generated using full-length splint ligation and double-stranded ligation oligos. For combined indexing with splint ligation (standard) and combined indexing with double-stranded splint ligation (ds index ligation), primer positions are shown in FIG. 12C and products were determined using primer combinations including F2 and R2 or F3 and R2. Concentrations were determined using the appropriate standard curve for each sample. FIG. 13A shows the qPCR results, and FIG. 13B and FIG. 13C show normalized qPCR results, where the synthesized ds index ligation oligos were similar to the splint ligation oligos. After normalizing for differential amplification between the SL full-length control oligo and the DS full-length control oligo, the DS ligation and splint ligation oligos performed similarly.

[0145] Double-stranded splint ligations were performed with increasing amounts of ds index oligos and products were measured by qPCR. As shown in FIG. 13D, there was no substantial increase in the amount of product. Double-stranded splint ligations were performed in the presence of an oligo complementary to index 1 and products were measured by qPCR. As shown in FIG. 13E, there was no substantial increase in the amount of product when double-stranded splint ligations were performed in the presence of an oligo complementary to index 1. Double-stranded splint ligations were performed with an oligo with a 3'ddC and products were measured by qPCR. As shown in FIG. 13F, there was no substantial increase in the amount of product when double-stranded splint ligations were performed with an oligo with a 3'ddC. Double-stranded splint ligations were performed with pre-ligation incubation at either room temperature or 75° C. and products were measured by qPCR. As shown in FIG. 13G, there was no substantial increase in the amount of product when double-stranded splint ligation was performed with pre-ligation incubation at either room temperature or 75° C.

[0146] Direct bead-coded sequencing for two-level double-stranded index ligation was performed, and the results are summarized in Figures 14A and 14B.

[0147] From the two-level experiments described above, double-stranded index ligation gave similar results to splint ligation as measured by qPCR. For double-stranded index amounts above 33 fmol / μg beads, there was no substantial increase in full-length product with single ligation or in errors in the bead code. The following manipulations had little effect: (1) adding an oligo complementary to index 1, (2) removing the 3′ddC modification, or (3) replacing the 75°C pre-ligation incubation with a room temperature incubation. In addition, bead code sequencing showed fewer errors with double-stranded ligation compared to splint ligation.

[0148] Example 7 - Three-level indexing by double-stranded splint ligation Three-level indexing with double-stranded splint ligation was compared to three-level indexing with splint ligation (standard). The experimental design is outlined in Figures 15A and 15B.

[0149] qPCR was performed on the 3-level indexed products using either double-stranded splint ligation or splint ligation. The full-length double-stranded ligation index control was used as a standard curve (higher purity) to size-adjust the concentration of the splint ligation amplicon. The results are summarized in Figure 16A. For splint ligation, denaturation at 80°C reduced the total number of oligos (2 vs. 3), while annealing at 50°C slightly increased the total number of oligos (3 vs. 4). For splint ligation vs. ds ligation, annealing at 50°C and denaturing at 80C gave a comparable number of full-length oligos to the splint and ds ligation methods (4 vs. 7), the oligos were full-length (3rd level vs. bio oligos), and ds ligation showed a lower number of 2nd level oligos. For the full-length control, the total number of molecules was lower, likely due to purity issues.

[0150] Direct bead-coded sequencing for three-level double-stranded index ligation was performed, and the results are summarized in Figures 16B and 16C.

[0151] From the double-stranded splint ligation studies described above, splint ligation and ds ligation gave comparable numbers of full-length oligos, and denaturation at 80° C. resulted in the loss of some capture oligonucleotides (COs), while annealing at 50° C. slightly increased the number of COs. COs synthesized by ds ligation had fewer errors than those synthesized by splint ligation.

[0152] Example 8 - LNA-containing splint oligo Design a truncated first splint (Kangaroo splint) containing a locked nucleic acid nucleotide (LNA). The Kangaroo splint is based on the sequence: ATGCTCTAGACAAGT / 3ddC (SEQ ID NO: 03), where "3ddC" is a 3' dideoxycytidine. Generated splints include splints truncated from both the 5' and 3' ends, all of which keep the last base to dideoxy-cytidine (ddC), and splints containing all possible LNA substitutions. Analyze generated oligos with IDT's OligoAnalyzer (Integrated DNA Technologies™, Coralville, Iowa). Select generated oligos that follow Qiagen's design guidelines for LNA oligos, which include 30-60% GC content, less than 4 consecutive LNA bases, and less than 3 consecutive Gs or Cs. The generated oligos are selected that have a Tm similar to the original splint and do not have any LNA at the base position complementary to the ligation site. The generated oligos are selected that do not have secondary structure or self-hybridization at room temperature and are not complementary to the P5 or Hyb sequences. Table 2 lists three exemplary oligos.

[0153] [Table 2]

[0154] 5' and 3' TM are calculated using an IDT OligoAnalyzer with the following settings (based on ligation reactions): 1.98 μM oligo, 100 mM Na + , 7.9 mM Mg ++ Full length TM is calculated using an IDT OligoAnalyzer with the following settings (based on splint denaturation): 0.05 µM oligo, 100 mM Na + The experimental outline is shown in Figure 17.

[0155] Example 9 - Modified splint oligonucleotides to reduce linker sequence length Further splints were designed in which the phos-oligo splint hybridization sequence was reduced to 6 nucleotides and the G / C content was increased. Multiple splints were designed for the new phos-oligos. An overview of the splints designed is shown in Figure 18A, which shows the sequences listed in Table 3, where X is an inosine.

[0156] [Table 3]

[0157] An overview of the experimental conditions is shown in Figure 18B. Conditions tested included splint ligation at 20°C vs. 12°C, which was performed overnight in a thermocycler (no rotation). DS oligo annealing was performed using either a fast cooling protocol (incubation at 75°C, immediately transferred to ice) or slow annealing (samples were incubated at 75°C in a thermocycler, then the temperature was ramped down at a ramp rate of 1°C / 30 sec). Capture and full-length oligos were measured by qPCR. Results are summarized in Figure 18C, which show that ligation with the redesigned 6 nucleotide p-oligo / splint was approximately 10-20% less. Ligation with the redesigned 4 nucleotide splint was approximately 40-50% less. Results with the "6bp_New_6bp-K splint" shown in FIG. 18A, which has the shortest 3' portion of the splint among the splints tested, suggest that reducing the 3' half of the splint to only 6 nucleotides reduced ligation efficiency by about 90%. Slow annealing had little effect on the number of full-length oligos. Addition of inosine bases may not improve ligation efficiency. Substitution of bases with inosine may have reduced ligation efficiency. In summary, redesign of the splint sequence and shortening of the 5' end of the splint to 6 and 4 nucleotides reduced ligation efficiency, even when ligation was performed below the splint Tm. Addition of inosine to the 5' end of the splint did not improve ligation efficiency. Substitution of the base at the 5' end of the splint with inosine reduced ligation efficiency. Slow annealing was found to have little effect on ligation efficiency. The reduction in ligation efficiency when preparing bead-linked indexed oligonucleotides with shortened linker sequences would likely outweigh the increased efficiency of the sequencing workflow involving the use of linker sequences with reduced length.

[0158] Example 10 - Workflow for indexing by double-stranded splint ligation An overview of indexing by double-stranded splint ligation is shown in Figure 19A, and an exemplary workflow is shown in Figure 19B. In the scheme shown in Figure 19A, annealing includes 60°C for 3 minutes and transfer to ice, ligation includes 1st ligation: O / N, 2nd ligation: 5 hours, and denaturation includes 80°C for 2 minutes with immediate removal of buffer. Below, the steps for preparing an indexed bead pool by double-stranded splint ligation are summarized.

[0159] [Table 4]

[0160] [Table 5]

[0161] [Table 6]

[0162] [Table 7]

[0163] [Table 8]

[0164] [Table 9]

[0165] [Table 10]

[0166] [Table 11]

[0167] [Table 12]

[0168] [Table 13]

[0169] [Table 14]

[0170] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0171] The above description discloses some methods and materials of the present invention. The present invention is susceptible to modifications of the methods and materials, and changes in the manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from consideration of this disclosure or the disclosure or practice of the invention disclosed herein. Therefore, the present invention is not intended to be limited to the specific embodiments disclosed herein, but rather to cover all modifications and alternatives falling within the true scope and spirit of the invention.

[0172] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or supersede such conflicting material.

Claims

1. A method for preparing beads with multiple combinatorial indices, comprising: (a) obtaining a population of primary-indexed beads comprising a first polynucleotide comprising a first index, wherein the population of primary-indexed beads comprises a plurality of sub-populations of first-indexed beads, and the sub-populations of first-indexed beads comprise different first indices; (b) dividing the population of primary-indexed beads into a plurality of sub-populations of second beads; (c) obtaining a population of secondary-indexed beads, comprising: (i) extending the first polynucleotide of the plurality of sub-populations of second beads with a second polynucleotide comprising a second index to obtain a sub-population of second-indexed beads, wherein the sub-population of second-indexed beads comprises different second indices; (ii) combining the sub-populations of second-indexed beads to obtain the population of secondary-indexed beads; and optionally: (d) dividing the population of secondary-indexed beads into a plurality of sub-populations of third beads; (e) obtaining a population of tertiary-indexed beads, comprising: (i) extending the second polynucleotide of the plurality of sub-populations of third beads with a third polynucleotide comprising a third index to obtain a sub-population of third-indexed beads, wherein the sub-population of third-indexed beads comprises different third indices; (ii) combining the sub-populations of third-indexed beads to obtain the population of tertiary-indexed beads; and further comprising the method.

2. (c) is (I) extending the first polynucleotide with the second polynucleotide by chemical ligation; (II) extending the first polynucleotide by polymerase extension, wherein the first polynucleotide comprises a first linker; (III) extending the first polynucleotide with the second polynucleotide by ligation, or (IV) extending the first polynucleotide by ligation, wherein the first polynucleotide comprises a first linker and the second polynucleotide comprises a second linker The method according to claim 1, comprising: **Claim 3** The step (C) includes (I), the first polynucleotide comprises a terminal 3'-modified deoxynucleotide (dNTP) containing a 3'-functional moiety capable of participating in a click chemistry reaction, the second polynucleotide comprises a terminal 5'-modified dNTP containing a compatible 5'-functional moiety capable of participating in a click chemistry reaction with the 3'-functional moiety, the 3'-functional moiety and the 5'-functional moiety are capable of reacting with each other to form a modified backbone bond The method according to claim 2 **Claim 4** extending the first polynucleotide with the second polynucleotide to obtain a polynucleotide with a secondary index, the method further comprising modifying the polynucleotide with the secondary index to obtain a modified polynucleotide comprising a terminal 3'-modified deoxynucleotide (dNTP) containing a 3'-functional moiety capable of participating in a click chemistry reaction, according to the method of claim 3 **Claim 5** The method according to claim 4, wherein the modifying step comprises contacting the polynucleotide with the secondary index with a template-independent polymerase **Claim 6** The method according to claim 5, wherein the template-independent polymerase is selected from terminal deoxynucleotidyl transferase (TdT), poly A polymerase, or CCA-adding RNA polymerase **Claim 7** The following: (A) the method further comprises extending the modified polynucleotide with the third polynucleotide by a chemical ligation reaction, the third polynucleotide comprising a terminal 5'-modified dNTP containing a compatible 5'-functional moiety capable of participating in a click chemistry reaction with the 3'-functional moiety, (B) the 3'-functional moiety is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone, (C) the 5'-functional moiety is different from and compatible with the 3'-functional moiety and is selected from the group consisting of azide, alkynyl, alkenyl, thiol, and nitrone, and (D) The click chemistry reaction includes copper-catalyzed azide-alkyne cycloaddition (CuAAC) to form a modified backbone bond containing triazolyl. The method according to claim 3, comprising one or more of the above. **Claim 8**: Step (C) includes (II), and the method (i) obtaining a first adapter, comprising a region capable of hybridizing to the first linker and a region comprising the second index or a complement of the second index; (ii) hybridizing the first adapter to the first linker; (iii) further comprising extending the first polynucleotide to obtain a polynucleotide with a secondary index. The method according to claim 2. **Claim 9** The method according to claim 8, wherein the first adapter comprises a complement of a second linker, such that the polynucleotide with the secondary index comprises the second linker. **Claim 10** (i) obtaining a second adapter, comprising a region capable of hybridizing to the second linker and a region comprising the third index or a complement of the third index; (ii) hybridizing the second adapter to the second linker; (iii) further comprising extending the polynucleotide with the secondary index to obtain a polynucleotide with a tertiary index. The method according to claim 9. **Claim 11**: Step (C) includes (III), and the method (i) obtaining a double-stranded first adapter, comprising the second polynucleotide and a 3'-single-stranded overhang capable of hybridizing to the first linker of the first polynucleotide; (ii) hybridizing the first adapter to the first linker, and optionally hybridizing an additional oligonucleotide to the first index; (iii) further comprising ligating the first polynucleotide to the second polynucleotide to obtain a polynucleotide with a secondary index. The method according to claim 2. **Claim 12** The method according to claim 11, further comprising extending the second polynucleotide with a third polynucleotide by ligation. **Claim 13** (i) obtaining a double-stranded second adapter comprising the third polynucleotide and a 3' single-stranded overhang capable of hybridizing to the second linker of the second polynucleotide; (ii) hybridizing the second adapter to the second linker; (iii) further comprising ligating the second polynucleotide to the third polynucleotide to obtain a polynucleotide with a tertiary index, the method according to claim 12.

14. Step (C) includes (IV), and the method is (i) obtaining a first adapter comprising a region capable of hybridizing to the first linker and a region capable of hybridizing to the second linker; (ii) hybridizing the first adapter to the first linker; (iii) hybridizing the second oligonucleotide to the region capable of hybridizing to the second linker; (iv) further comprising ligating the first polynucleotide to the second polynucleotide to obtain a polynucleotide with a secondary index, the method according to claim 2.

15. Step (e) includes extending the second polynucleotide by ligation, the second oligonucleotide comprises a third linker, as a result, the polynucleotide with a secondary index comprises the third linker, and the third polynucleotide comprises a fourth linker, the method according to claim 14.

16. (i) obtaining a second adapter comprising a region capable of hybridizing to the third linker and a region capable of hybridizing to the fourth linker; (ii) hybridizing the second adapter to the third linker; (iii) hybridizing the third polynucleotide to the second adapter via the region capable of hybridizing to a fourth index; (iv) further comprising ligating the polynucleotide with a secondary index to the third polynucleotide to obtain a polynucleotide with a tertiary index, the method according to claim 15.

17. Step (a) is (i) A step of attaching the first polynucleotide to a sub-population of a plurality of first beads to obtain a sub-population of the first indexed beads, wherein the first polynucleotide includes a first index different for each sub-population of the first beads; (ii) A step of combining the sub-population of the first indexed beads to obtain a population of the primary-indexed beads, the method according to any one of claims 1 to 16, including the above steps.

18. The following: (A) The method further includes a step of repeating steps (d) and (e), and a step of adding an additional index to a sub-population of the indexed beads; (B) The method further includes a step of distributing a plurality of beads with combined indexes onto an array; (C) The method further includes a step of sequencing the beads with combined indexes on the array; (D) The method further includes a step of hybridizing a plurality of target nucleic acids to the capture probe, and optionally further includes a step of extending the capture probe; (E) Step (b) includes a step of randomly distributing a population of the primary-indexed beads into a plurality of compartments; (F) Step (d) includes a step of randomly distributing a population of the secondary-indexed beads into a plurality of compartments, and (G) Each bead of the plurality of beads with combined indexes includes a capture probe. The method according to any one of claims 1 to 16, including one or more of the above.

19. The method according to claim 18, wherein the step of sequencing the beads with combined indexes on the array further includes a step of decoding the position of one bead among the plurality of beads with combined indexes on the array based on the combined index.

20. The method according to claim 18, wherein each bead of the plurality of beads with combined indexes includes a capture probe, and the first polynucleotide, the second polynucleotide, or the third polynucleotide includes the capture probe.

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