Increasing spatial transcriptomics library yield by surface amplification of index-labeled cdna

Abstract

The present disclosure is generally directed to capture oligonucleotides and methods of making and using capture oligonucleotide to generate capture surfaces and spatial transcriptomic libraries.

Description

Docket No. 33080 / I P-2574INCREASING SPATIAL TRANSCRIPTOMICS LIBRARY YIELD BY SURFACE AMPLIFICATION OF INDEX-LABELED cDNACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 701 ,910, filed October 1 , 2025, which is incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “IP-2574_SeqListing.xml", which was created on October 1 , 2025 and is 13,329 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.BACKGROUND

[0003] Spatial transcriptomics enables highly multiplexed, spatially located gene expression analysis from fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples. An on-surface library preparation method must be used to spatially capture and barcode transcripts from a tissue sample. Captured transcripts can range up to several thousand nucleotides in size, while the ideal sequencing library size is about 200-700 base pairs. In addition, the libraries generated using polyT capture sequences always include at least a portion of the polyA tails, lengths of which can range up to serval hundreds of nucleotides [Liu et al. Nat Commun 10, 5292 (2019)]. Further, for spatial library preparation applications, final sequencing libraries must include index sequences and sample indices, while maintaining a desired fragment size for sequencing.SUMMARY

[0004] Current on-market spatial workflows contain index (e.g., a single molecule identifier (SMI)) information on a barcoded surface and include fragmentation to generate libraries of optimal fragment size for sequencing. Illumina bead-based tagmentation, solution tagmentation, and other methods all fragment the strands at random locations. Such random fragmenting leads to a portion of the library being too short or too long for Illumina sequencing and in the worst case, the fragmentation could happen in the barcode range, resulting in loss of the whole cDNA. These random fragmentations significantly reduce the library yield and sensitivity if there is only one copy of cDNA from each unique mRNA for fragmentation.Docket No. 33080 / 1 P-2574

[0005] Accordingly, in some aspects the disclosure provides a capture oligonucleotide comprising, from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a single molecule identifier (SMI) sequence; (e) a quality control sequence; and (f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T). In some embodiments, the capture oligonucleotide further comprises an index (IDX) sequence between the first clustering primer sequence and the SBC sequence. In some embodiments, the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence. In various embodiments, the capture nucleotide sequence comprises about 20 to about 30 nucleotides. In some embodiments, the capture nucleotide sequence comprises about 30 nucleotides. In various embodiments, the poly-T sequence consists of 30 thymine nucleotides. In some embodiments, the quality control sequence is a sequence that is complementary to a second sequencing primer. In some embodiments, the SMI is a unique molecular identifier (IIMI).

[0006] In further aspects, the disclosure provides a capture surface comprising: a plurality of moieties and a plurality of capture oligonucleotides immobilized on a surface, wherein each capture oligonucleotide in the plurality of capture oligonucleotides comprises, from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a single molecule identifier (SMI) sequence; (e) a sequence that is complementary to a second sequencing primer; (f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T). In some embodiments, each of the plurality of moieties comprises a first clustering primer sequence (e.g., P7). In some embodiments, each of the plurality of moieties is a biotin functionalized to a click chemistry handle. In some embodiments, a complementary oligonucleotide e.g., P7’) is hybridized to the first clustering primer sequence of one or more of the plurality of moieties and to the first clustering primer sequence of one or more of the plurality of capture oligonucleotides. Thus, in various embodiments there are a plurality of complementary oligonucleotides e.g., P7’ oligonucleotides), wherein a first complementary oligonucleotide in the plurality is hybridized to the first clustering primer sequence of one or more of the plurality of moieties, and a second complementary oligonucleotide in the plurality is hybridized to the first clustering primer sequence of one or more of the plurality of capture oligonucleotides. In some embodiments, one or more of the plurality of capture oligonucleotides comprises an index (IDX) sequence between the first clustering primer sequence and the SBC sequence. In various embodiments, the capture nucleotideDocket No. 33080 / I P-2574 sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence. In some embodiments, the capture nucleotide sequence comprises about 20 to about 30 nucleotides. In further embodiments, the capture nucleotide sequence comprises about 30 nucleotides. In some embodiments, the poly-T sequence consists of 30 thymine nucleotides. In further embodiments, the SMI is a unique molecular identifier (IIMI).

[0007] In further aspects, the disclosure provides a method of making a capture surface, comprising: (1) immobilizing a first plurality of oligonucleotides on a surface, wherein each oligonucleotide in the first plurality of oligonucleotides comprises from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a restriction endonuclease (RE) recognition sequence; (e) a quality control sequence; and (f) a sequence that is complementary to a second clustering primer sequence; (2) amplifying the first plurality of oligonucleotides, thereby generating clusters of one or more of the plurality of oligonucleotides on the surface; (3) contacting the surface with a restriction endonuclease capable of recognizing the RE recognition sequence of the first plurality of oligonucleotides, thereby generating a second plurality of oligonucleotides comprising from 5’ to 3’; (a) the first clustering primer sequence; (b) the spatial barcode (SBC) sequence; and (c) the first sequencing primer sequence; (4) hybridizing a third plurality of oligonucleotides to the second plurality of oligonucleotides, wherein the third plurality of oligonucleotides comprises from 5’ to 3’: (i) a sequence that is complementary to the first sequencing primer sequence; (ii) a sequence that is complementary to a single molecule identifier (SMI) sequence; (iii) a second sequencing primer sequence; (iv) an ABN sequence, wherein “A” is a sequence that is complementary to a capture nucleotide sequence, “B” is cytosine (C), guanine (G), or thymine (T), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T); (5) extending the first plurality of oligonucleotides having an oligonucleotide of the third plurality of oligonucleotides hybridized thereto, thereby generating a fourth plurality of oligonucleotides comprising from 5’ to 3’: (I) the first clustering primer sequence; (II) the spatial barcode (SBC) sequence; (III) the first sequencing primer sequence; (IV) a single molecule identifier (SMI) sequence; (V) a sequence that is complementary to the second sequencing primer sequence; and (VI) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T), thereby generating the capture surface. In some embodiments, the first plurality of oligonucleotides, the second plurality of oligonucleotides, and the fourth plurality of oligonucleotides further comprise an index (IDX) sequence between the first clustering primer sequence and the SBC sequence. In further embodiments, the capture nucleotide sequence is a poly-T sequence, a poly-ADocket No. 33080 / 1 P-2574 sequence, a gene-specific capture sequence, or a universal capture sequence. In some embodiments, the capture nucleotide sequence comprises about 20 to about 30 nucleotides. In further embodiments, the capture nucleotide sequence comprises about 30 nucleotides. In some embodiments, the poly-T sequence consists of 30 thymine nucleotides. In various embodiments, the SMI is a unique molecular identifier (IIMI). In some embodiments, the quality control sequence is a sequence that is complementary to a second sequencing primer. In further embodiments, the quality control sequence comprises a mosaic end (ME) sequence. In some embodiments, the method further comprises removing the third plurality of oligonucleotides from the surface after step (5). In some embodiments, removing the third plurality of oligonucleotides from the surface comprises denaturing the third plurality of oligonucleotides. In some embodiments, removing the third plurality of oligonucleotides from the surface comprises digesting the target nucleic acids. In some embodiments, synthesis of the first plurality of oligonucleotides comprises adding a click chemistry handle during the last cycle of synthesis. In various embodiments, the click chemistry handle is 5’ Hexynyl. In some embodiments, click chemistry is used to immobilize the first plurality of oligonucleotides on the surface through the click chemistry handle. In various embodiments, synthesis of the third plurality of oligonucleotides comprises adding a biotin-functionalized deoxynucleotide triphosphate (dNTP) during the last cycle of synthesis. In some embodiments, the third plurality of oligonucleotides is purified via streptavidin (SA) beads prior to step (4). In some embodiments, the method further comprises (6) immobilizing a fifth plurality of oligonucleotides on the surface, wherein one or more of the fifth plurality of oligonucleotides comprises or consists of the first clustering primer sequence. In some embodiments, a complementary oligonucleotide is hybridized to the first clustering primer sequence of the fifth plurality of oligonucleotides and to the first clustering primer sequence of one or more of the first plurality of oligonucleotides. Thus, in various embodiments there are a plurality of complementary oligonucleotides, wherein a first complementary oligonucleotide in the plurality is hybridized to the first clustering primer sequence of the fifth plurality of oligonucleotides, and a second complementary oligonucleotide in the plurality is hybridized to the first clustering primer sequence of one or more of the first plurality of oligonucleotides.

[0008] In further aspects, the disclosure provides a method of preparing an immobilized library of target nucleic acids of a biological sample, comprising: (a) providing (i) a surface comprising a plurality of moieties and a plurality of the capture oligonucleotides of the disclosure immobilized thereon; (ii) a capture surface of the disclosure; or (iii) the capture surface produced by a method of the disclosure; (b) contacting the biological sample with the surface or the capture surface, the contacting resulting in hybridization of the target nucleicDocket No. 33080 / 1 P-2574 acids of the biological sample to the TVN sequence of the plurality of capture oligonucleotides to form hybridized capture oligonucleotides; (c) extending the TVN sequence of the hybridized capture oligonucleotides to form first complementary strands of the target nucleic acids, wherein the extending comprises addition of a plurality of non- templated nucleotides to the end of the first complementary strands; (d) hybridizing a plurality of template switching oligonucleotides (TSOs) to the plurality of non-templated nucleotides of the first complementary strands such that each of the plurality of template switching oligonucleotides that is hybridized to the plurality of non-templated nucleotides of the first complementary strands is positioned at the terminus of the target nucleic acids that is not hybridized to the TVN sequence; (e) extending the plurality of non-templated nucleotides on the first complementary strands using the template switching oligonucleotides as template, thereby generating a complementary template-switching oligonucleotide sequence (TSO’) on the first complementary strands, thereby preparing the immobilized library of target nucleic acids. In some embodiments, the method further comprises (f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands. In some embodiments, the method further comprises (f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands; and (g) amplifying the first complementary strands and the second complementary strands, thereby generating a plurality of clusters comprising hybridized first complementary strands and second complementary strands. In some embodiments, the method further comprises (h) performing tagmentation on the plurality of clusters comprising hybridized first complementary strands and second complementary strands to prepare tagged fragments. In some embodiments, the method further comprises (h) denaturing the plurality of clusters comprising hybridized first complementary strands and second complementary strands; (i) hybridizing an oligonucleotide comprising, from 5’ to 3’, the second sequencing primer and a capture nucleotide sequence to the one or more second complementary strands; (j) extending the oligonucleotide in the presence of a dideoxynucleotide triphosphate (ddNTP) comprising a click chemistry handle; (k) hybridizing a primer comprising the first clustering primer sequence to the one or more second complementary strands, extending the hybridized primer comprising the first clustering primer sequence, wherein the extending comprises a gap fill, and ligating the 3’ end of the extended primer comprising the first clustering primer sequence to the 5’ end of the extended oligonucleotide of step (j); (I) attaching an adapter oligonucleotide comprising, from 5’ to 3’, a third sequencing primerDocket No. 33080 / 1 P-2574 sequence, a sequence complementary to a unique index sequence (i5’), and a second clustering primer sequence, wherein the adapter oligonucleotide is attached by a click chemistry reaction using the click chemistry handle. In some embodiments, the click chemistry handle is an azide, a tetrazine, a strained alkene, or an alkyne. In some embodiments, the gap fill comprises using a reverse transcriptase or a DNA polymerase. In some embodiments, each of the plurality of moieties comprises a first clustering primer sequence. In some embodiments, each of the plurality of moieties is a biotin functionalized to a click chemistry handle. In some embodiments, the method further comprises contacting the surface with an exonuclease after step (d), thereby digesting single-stranded capture oligonucleotides. In further embodiments, the plurality of moieties is immobilized on the surface after the exonuclease is contacted with the surface. In some embodiments, the method further comprises adding streptavidin and an additional plurality of moieties to the surface, wherein each of the additional plurality of moieties is a biotin functionalized to a first clustering primer sequence. In some embodiments, prior to step (b) a complementary oligonucleotide is hybridized to the first clustering primer sequence of the capture oligonucleotides and to the first clustering primer sequence of one or more of the plurality of moieties. In some embodiments, the complementary oligonucleotide is removed prior to step (g). In some embodiments, the amplifying of the first complementary strands and the second complementary strands in step (g) is performed via one or more cycles of hybridizing the first clustering primer sequence of the plurality of moieties to the complement of the first clustering primer in the second complementary strands and extending the hybridized first clustering primer sequence to generate the plurality of clusters comprising hybridized first complementary strands and second complementary strands. In various embodiments, the amplification is Exclusion-Amplification (ExAmp). In some embodiments, the method further comprises removing the target nucleic acids from the surface after step (e). In some embodiments, removing the target nucleic acids from the surface comprises denaturing the target nucleic acids. In some embodiments, removing the target nucleic acids from the surface comprises digesting the target nucleic acids. In some embodiments, the method further comprises removing the biological sample from the surface after step (c). In some embodiments, each of the plurality of capture oligonucleotides comprises the same capture nucleotide sequence. In various embodiments, the plurality of capture oligonucleotides comprises multiple, different capture nucleotide sequences. In various embodiments, the multiple, different capture nucleotide sequences comprise one or more gene-specific capture sequences, one or more universal capture sequences, or a combination thereof. In various embodiments, the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence. In some embodiments, the universal capture sequence is a random nucleotide sequence or a non-selfDocket No. 33080 / I P-2574 complementary semi-random sequence. In further embodiments, the target nucleic acids are mRNA, gDNA, rRNA, tRNA, or a combination thereof. In some embodiments, the target nucleic acids are RNA, mRNA, or a combination thereof. In various embodiments, the extending of the capture nucleotide sequence in step (c) is carried out using a reverse transcriptase. In some embodiments, the target nucleic acids are polyadenylated prior to hybridization of the target nucleic acids to the capture nucleotide sequences. In various embodiments, the target nucleic acids are polyadenylated using a poly(A) polymerase. In some embodiments, the target nucleic acids are polyadenylated using chemical ligation or enzymatic ligation.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows the synthesis and oligonucleotide design of a capture oligonucleotide of the disclosure.

[0010] Figure 2 depicts purification and quality control steps during synthesis of a capture oligonucleotide of the disclosure.

[0011] Figure 3 shows ExAmp surface amplification of indexed cDNA.

[0012] Figure 4 shows in-solution library preparation - click chemistry adapter (e.g., P5) ligation.

[0013] Figure 5A shows results of experiments demonstrating ExAMP on a surface (qPCR). Figure 5B shows results of experiments demonstrating ExAMP on a surface (sensitivity determined by sequencing).DETAILED DESCRIPTION

[0014] The present disclosure is generally directed to capture oligonucleotides and methods of making and using capture oligonucleotide to generate capture surfaces and spatial transcriptomic libraries. The design of the capture oligonucleotides provided herein minimize variation of polyA length in the final library, which reduces waste when sequencing a long polyA sequence of a target analyte e.g., target nucleic acid). In various embodiments, capture oligonucleotides of the disclosure anchored on a surface include both a spatial barcode and a single molecule identifier (SMI), thereby enabling the amplification of cDNA from each unique target analyte e.g., target nucleic acid such as mRNA) on the surface. After amplification there are multiple copies of cDNA with the same SMI barcode available for fragmentation, which significantly increases the chance to get the optimal insert size (which, in various embodiments, is about 200-700 bp) during the subsequent library preparation steps. Further, steric hindrance from the surface increases library yield by reducing unfavorable tagmentation in range of a capture oligonucleotide described herein.Docket No. 33080 / I P-2574

[0015] The combination of the capture oligonucleotide designs described herein, the amplification of SMI labeled cDNA, and on-substrate tagmentation significantly increases the library yield and spatial transcriptomics sensitivity.TERMS

[0016] As used in this specification and the enumerated paragraphs herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0017] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), for example, within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range of values.

[0018] As used herein, the term “adapter” refers generally to any linear nucleic acid molecule that can be added (e.g., through synthesis or ligation) to an oligonucleotide of the disclosure. In some embodiments, adapters are copied onto the library molecules using templated polymerase synthesis e.g., second strand cDNA synthesis as described herein). In some embodiments, adapters are ligated to a first complementary strand of the disclosure. In some embodiments, oligonucleotides of the disclosure comprise adapters (“adapter oligonucleotides”). In some embodiments, an adapter oligonucleotide comprises from 5’ to 3’, a third sequencing primer sequence e.g., SBS3), a sequence complementary to a unique index sequence {e.g., i5’), and a second clustering primer sequence {e.g., P5). In some embodiments, an adapter comprises a sequence that is complementary to a primer. In further embodiments, an adapter comprises a sequence that is complementary to a P5 primer or a P5’ primer. In some embodiments, an adapter comprises a sequence complementary to a P7 primer or a P7’ primer. In some embodiments, an adapter comprises a sequence complementary to a B15 primer or a B15’ primer.

[0019] The terms “P5”,”P7”, “B15”, “P5”’ (P5 prime), “P7”’ (P7 prime), “B15”’ (B15 prime), “P15”, and “P17” may be used when referring to examples of oligonucleotide sequences of primers, e.g., clustering primers, and / or oligonucleotide sequences that are complementary to primers. The terms "P5"' (P5 prime), "P7"' (P7 prime), and “B15”’ (B15 prime) refer to the complement of P5, P7, and B15, respectively. It will be understood that any suitable primer can be used in the methods presented herein, and that the use of P5, P5’, P7, P7’, P15, P17, B15, and B15’ are exemplary embodiments only. Uses of primers such as P5, P5’, P7, P7’, P15, P17, B15, and B15’ or their complements on flow cells are known in the art, as exemplified by the disclosures of WO 2019 / 222264, WO 2007 / 010251 , WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941 , WO 1998 / 044151 , and WO 2000 / 018957, each ofDocket No. 33080 / I P-2574 which is incorporated herein by reference in its entirety. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. One of skill in the art will understand how to design and use primer sequences that are suitable for capture and / or amplification of nucleic acids as presented herein. In some embodiments, a “first clustering primer” as described herein is a P5 primer. In some embodiments, a “first clustering primer” as described herein is a P7 primer. In some embodiments, a “first clustering primer” as described herein is a P5' primer. In some embodiments, a “first clustering primer” as described herein is a P7' primer. In some embodiments, a “second clustering primer” as described herein is a P5 primer. In some embodiments, a “second clustering primer” as described herein is a P7 primer. In some embodiments, a “second clustering primer” as described herein is a P5' primer. In some embodiments, a “second clustering primer” as described herein is a P7' primer. In some embodiments, P5 comprises or consists of the polynucleotide sequence 5’ AAT GATACG GCG ACC ACC GA 3’ (SEQ ID NO: 1), or a variant thereof. In some embodiments, P5 comprises or consists of the polynucleotide sequence 5’ AAT GAT ACG GCG ACC ACC GAG ATC TAC AC 3’ (SEQ ID NO: 2), or a variant thereof. In some embodiments, P7 comprises or consists of the polynucleotide sequence 5’ CAA GCA GAA GAC GGC ATA CG 3’ (SEQ ID NO. 3), or a variant thereof. In some embodiments, P7 comprises or consists of the polynucleotide sequence 5’ CAA GCA GAA GAC GGC ATA CGA GAT 3’ (SEQ ID NO. 4), or a variant thereof. In some embodiments, P5' comprises or consists of the polynucleotide sequence 5’ TCG GTG GTC GCC GTA TCA TT 3’ (SEQ ID NO: 5), or a variant thereof. In some embodiments, P5' comprises or consists of the polynucleotide sequence 5’ GTG TAG ATC TCG GTG GTC GCC GTA TCA TT 3’ (SEQ ID NO: 6), or a variant thereof. In some embodiments, P7' comprises the polynucleotide sequence 5’ CGT ATG CCG TCT TCT GCT TG 3’ (SEQ ID NO. 7), or a variant thereof. In some embodiments, P7' comprises or consists of the polynucleotide sequence 5’ ATC TCG TAT GCC GTC TTC TGC TTG 3’ (SEQ ID NO. 8), or a variant thereof. In some embodiments, B15 comprises or consists of the polynucleotide sequence 5’ GTCTCGTGGGCTCGG 3’ (SEQ ID NO: 9), or a variant thereof. In some embodiments, B15’ comprises or consists of the polynucleotide sequence 5’ CCGAGCCCACGAGAC 3’ (SEQ ID NO: 10), or a variant thereof. In some embodiments, P15 comprises or consists of the polynucleotide sequence 5’ TTTTTTAATG ATACGGCGAC CACCGAGANC TACAC 3’ (SEQ ID NO: 11 ), or a variant thereof. In some embodiments, P17 comprises or consists of the polynucleotide sequence 5’ TTTTTTNNNC AAGCAGAAGA CGGCATACGA GAT 3’ (SEQ ID NO: 12), or a variant thereof. The termDocket No. 33080 / I P-2574“variant” as used herein with reference to any of the sequences recited herein refers to a variant nucleic acid that is substantially identical, i.e., has only some nucleotide sequence variations, for example to the non-variant sequence. In some embodiments, a variant has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall nucleotide sequence identity to the non-variant nucleic acid sequence. It will be understood that reference to P5 and P7 herein could refer to different primer sequences. Any suitable primer sequence combinations are encompassed by the present disclosure.

[0020] The term “bead” refers to a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions. Example materials that are useful for beads include, without limitation, glass; plastic such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or polytetrafluoroethylene (TEFLON®, from Chemours); polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resin; silica or silica-based materials including silicon and modified silicon; carbon-fiber, metal; inorganic glass; optical fiber bundle, or a variety of other polymers. Example beads include, without limitation, controlled pore glass beads, paramagnetic beads, thoria sol, Sepharose beads, nanocrystals and others known in the art as described, for example, in Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. Beads may also be coated with a polymer that has a functional group that can attach to an oligonucleotide.

[0021] As used herein, “surface” can refer to a part of a substrate or support structure that is accessible to contact with reagents, beads, or analytes. The surface can be substantially flat or planar. Alternatively, the surface can be rounded or contoured. Example contours that can be included on a surface are wells (e.g., microwells or nanowells), depressions, pillars, ridges, channels or the like. Example materials that can be used as a substrate or support structure include glass such as modified or functionalized glass; plastic such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or TEFLON; polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resin; silica or silica-based materials including silicon and modified silicon, carbon-fibre; metal; inorganic glass; optical fibre bundle, or a variety of other polymers. A single material or mixture of several different materials can form a surface useful in certain examples. In some examples, a surface comprises wells e.g., microwells or nanowells). In some aspects, the surface comprises an array of wells e.g., microwells or nanowells) on glass, silicon, plastic or other suitable solidDocket No. 33080 / I P-2574 supports with patterned, covalently-linked gel such as poly(N-(5- azidoacetamidylpentyl)acrylamide-coacrylamide) (PAZAM, see, for example, U.S. Pat. App. Pub. No. 2014 / 0079923 A1 , which is incorporated herein by reference). In some embodiments, each nanowell comprises a unique oligonucleotide (e.g., an oligonucleotide with a unique spatial barcode). In some examples, a support structure can include one or more layers. Non-limiting examples of a surface include a bead array, a spotted array, clustered particles arranged on a surface of a chip, a film, a multi-well plate, and a flow cell.

[0022] Exemplary flow cells include but are not limited to those used in a nucleic acid sequencing apparatus such as flow cells for the Genome Analyzer®, MiSeq®, NextSeq® or HiSeq® platforms commercialized by Illumina, Inc. (San Diego, Calif.); or for the SOLiD™ or Ion Torrent™ sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Exemplary flow cells and methods for their manufacture and use are also described, for example, in WO 2014 / 142841 A1 ; U.S. Pat. App. Pub. No. 2010 / 0111768 A1 and U.S. Pat. No. 8,951 ,781 , each of which is incorporated herein by reference.

[0023] A bead array comprising oligonucleotide barcodes can also be used in a sequencing procedure, such as a sequencing-by-synthesis (SBS) technique. Briefly, SBS can be initiated by contacting the barcodes with one or more labeled nucleotides, DNA polymerase, etc. Those features where a primer is extended using the sequences comprising the barcode as a template will incorporate a labeled nucleotide that can be detected. Optionally, the labeled nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for embodiments that use reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; WO 91 / 06678; WO 07 / 123744; U.S. Pat. Nos. 7,057,026; 7,329,492; 7,211 ,414; 7,315,019 or 7,405,281 , and US Pat. App. Pub. No. 2008 / 0108082 A1 , each of which is incorporated herein by reference.

[0024] As used herein, the term "barcode" is intended to mean a series of nucleotides in an oligonucleotide that can be used to identify the oligonucleotide, a spatial address on a surface ( / .e., a “spatial barcode”), a characteristic of the oligonucleotide, or a manipulationDocket No. 33080 / 1 P-2574 that has been carried out on the oligonucleotide. The barcode can be a naturally occurring nucleotide sequence or a nucleotide sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained. A barcode sequence can be unique to a single nucleic acid species in a population, or a barcode sequence can be shared by several different nucleic acid species in a population. For example, each capture oligonucleotide in a plurality on a surface for spatial capture of nucleic acids in a biological sample (e.g., a permeabilized biological sample e.g., tissue sample), a cell suspension) can include different barcode sequences from all other nucleic acid capture oligonucleotides in the population. Alternatively, each capture oligonucleotide in a plurality can include different barcode sequences from some or most other nucleic acid capture oligonucleotides in a plurality. For example, each capture oligonucleotide in a plurality can have a barcode that is present for several different capture oligonucleotides in the plurality even though the capture oligonucleotides with the common barcode differ from each other at other sequence regions along their length. In various embodiments, one or more barcode sequences that are used with a biological tissue are not present in the genome, transcriptome or other nucleic acids of the biological specimen. For example, barcode sequences can have less than 80%, 70%, 60%, 50% or 40% sequence identity to the nucleic acid sequences in a particular biological tissue.

[0025] As used herein, the term "array" refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. Exemplary features include without limitation, beads (or other particles) in or on a substrate, droplets, wells in a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.

[0026] As used herein, a "biological sample" may include one or more biological or chemical substances, such as nucleic acids, oligonucleotides, proteins, cells, tissues, organisms, and / or biologically active chemical compound(s), such as analogs or mimetics ofDocket No. 33080 / I P-2574 the aforementioned species. As used herein, the term "tissue" is intended to mean an aggregation of cells, and, optionally, intercellular matter. Typically the cells in a tissue are not free floating in solution and instead are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermal and connective tissues. In some instances, the biological sample may include whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, viruses including viral pathogens, liquids containing multi-celled organisms, biological swabs and biological washes. In further examples, the sample can be derived from an organ, including for example, an organ of the musculoskeletal system such as muscle, bone, tendon or ligament; an organ of the digestive system such as salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder or pancreas; an organ of the respiratory system such as larynx, trachea, bronchi, lungs or diaphragm; an organ of the urinary system such as kidney, ureter, bladder or urethra; a reproductive organ such as ovary, fallopian tube, uterus, vagina, placenta, testicle, epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum; an organ of the endocrine system such as pituitary gland, pineal gland, thyroid gland, parathyroid gland, or adrenal gland; an organ of the circulatory system such as heart, artery, vein or capillary; an organ of the lymphatic system such as lymphatic vessel, lymph node, bone marrow, thymus or spleen; an organ of the central nervous system such as brain, brainstem, cerebellum, spinal cord, cranial nerve, or spinal nerve; a sensory organ such as eye, ear, nose, or tongue; or an organ of the integument such as skin, subcutaneous tissue or mammary gland. In various embodiments, the tissue can be derived from a multicellular organism. In some embodiments, a tissue section can be contacted with a surface, for example, by laying the tissue on the surface. The tissue can be freshly excised from an organism, or it may have been previously preserved for example by freezing (e.g., fresh frozen tissue), embedding in a material such as paraffin e.g., formalin fixed paraffin embedded (FFPE) samples), formalin fixation, infiltration, dehydration or the like. Optionally, a tissue section can be attached to a surface, for example, using techniques and compositions described in, for example, U.S. Patent No. 11 ,390,912, incorporated by reference herein in its entirety. In some embodiments, a tissue can be permeabilized and the cells of the tissue lysed when the tissue is in contact with a surface. Any of a variety of treatments can be used such as those set forth above in regard to lysing cells. Target proteins and / or nucleic acids that are released from a tissue that is permeabilized can be captured by capture oligonucleotides on the surface. Thus, in various embodiments, the biological sample is a tissue sample. TheDocket No. 33080 / I P-2574 thickness of a tissue sample or other biological sample that is contacted with a surface in a method set forth herein can be any suitable thickness desired. In representative embodiments, the thickness will be at least 0.1 pm, 0.25 pm, 0.5 pm, 0.75 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm or thicker. Alternatively or additionally, the thickness of a biological sample that is contacted with a surface will be no more than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, 0.25 pm, 0.1 pm or thinner.

[0027] As used herein, a "capture oligonucleotide" is generally an oligonucleotide comprising a nucleotide sequence capable of hybridizing or otherwise associating with an analyte (e.g., target nucleic acid). In various embodiments, a capture oligonucleotide comprises a clustering primer sequence and a capture nucleotide sequence that is configured to bind to target nucleic acids of a biological sample. In some embodiments, a capture oligonucleotide comprises a clustering primer sequence e.g., a P7 sequence), a spatial barcode (SBC) sequence, a sequencing primer sequence e.g., a sequencing by synthesis (SBS) sequence such as SBS12), a single molecule identifier (SMI) sequence, a quality control sequence, and a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T). In various embodiments, a capture oligonucleotide is between about 30 bases to about 100 bases in length, or between about 30 bases to about 90 bases, or between about 30 bases and 80 bases, or between about 30 bases and 70 bases, or between about 30 bases and 60 bases, or between about 30 bases and 55 bases, or between about 30 bases and 50 bases in length. In further embodiments, a capture oligonucleotide of the disclosure is about 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 55 bases, 60 bases, 65 bases, 70 bases, 75 bases, 80 bases, 85 bases, 90 bases, 95 bases, or 100 bases in length. The capture nucleotide sequence capable of hybridizing or otherwise associating with an analyte {e.g., a target nucleic acid) is, for example and without limitation, a universal sequence {e.g., a poly T sequence, a random nucleotide sequence, or a semi-random nucleotide sequence), or a target-specific {e.g., a genespecific) sequence. In various embodiments, a capture nucleotide sequence {e.g., a poly T nucleotide sequence or a random nucleotide sequence) is, is about, or is at least about 2, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 45, 50, or more bases in length.Alternatively or additionally, a capture nucleotide sequence can include less than or equal to about 50, 45, 40, 38, 35, 32, 30, 28, 25, 22, 20, 18, 15, 12, 10, 8, 5, or 2 bases. A capture oligonucleotide can comprise additional elements, including but not limited to a single molecule identifier (SMI) {e.g., a unique molecular identifier (UMI)), an index sequence, a sequence that is complementary to a sequencing primer {e.g., SBS12), or a combination thereof. In some embodiments, beads are packed onto a solid support {e.g., a planarDocket No. 33080 / I P-2574 support or flow cell), wherein the beads comprise a plurality of capture oligonucleotides immobilized thereon, wherein one or more of the plurality of capture oligonucleotides comprises, from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a single molecule identifier (SMI) sequence; (e) a quality control sequence; and (f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T), and wherein the spatial barcode sequence of the plurality of capture oligonucleotides is unique to each bead.

[0028] As used herein, the term “different”, when used in reference to nucleic acids, means that the nucleic acids have nucleotide sequences that are not the same as each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more nucleic acids can have target nucleotide sequence portions that are different for the two or more molecules while also having a universal sequence portion that is the same on the two or more molecules. The term can be similarly applied to proteins which are distinguishable as different from each other based on amino acid sequence differences.

[0029] As used herein, the term “clustering primer sequence” refers to a nucleotide sequence in solution and / or immobilized on a surface that is used for amplifying the template polynucleotides to create identical copies of the same templates (i.e., clusters). Examples of clustering primer sequences may include but are not limited to P5 primer, P5’ primer, P7 primer, P7’ primer, P15 primer, P15’ primer, P17 primer, P17’ primer, B15 primer, and B15’ primer as described herein. Clustering primers, their sequences, and their uses are also described, e.g., in WO2019222264, incorporated by reference herein in its entirety.

[0030] As used herein, the term “quality control sequence” refers to a known nucleotide sequence that may be part of a capture oligonucleotide of the disclosure. The quality control sequence is , in some embodiments, a known sequence that is added for its utility during quality control measurements. For example and without limitation, in some embodiments a fluorescently labeled oligonucleotide is hybridized to the "quality control sequence" on a capture oligonucleotide of the disclosure and provides a fluorescent readout of percent conversion of the capture oligonucleotide to a SMI-containing capture oligonucleotide. Detection of a signal following the hybridization is indicative of successful conversion of a capture oligonucleotide to a SMI-containing capture oligonucleotide. In some embodiments, the quality control sequence is a sequence that is complementary to a second sequencing primer.Docket No. 33080 / I P-2574

[0031] By “complementary” is meant that a first oligonucleotide sequence can form a double-stranded structure by matching base-pairs with a second oligonucleotide sequence or portion thereof. In various embodiments, “complementary” oligonucleotides are 100% complementary to each other, while in other embodiments, a first oligonucleotide sequence is at least (meaning greater than or equal to) about 95% complementary to a second oligonucleotide sequence over the length of the first oligonucleotide, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, or at least about 50% complementary to the second oligonucleotide over the length of the first oligonucleotide to the extent that the oligonucleotides are able to hybridize to each other under the conditions being utilized. The percent complementarity is determined over the length of the oligonucleotide. For example, given a first oligonucleotide in which 18 of 20 nucleotides of the first oligonucleotide are complementary to a 20-nucleotide region in a second oligonucleotide of 100 nucleotides total length, the oligonucleotides would be 90 percent complementary. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleotides.

[0032] As used herein, “hybridize” is intended to mean noncovalently associating a first oligonucleotide to a second oligonucleotide along the lengths of those polymers to form a double-stranded “duplex.” For example, two DNA oligonucleotide strands may associate through complementary base pairing. The strength of the association between the first and second oligonucleotides increases with the complementarity between the sequences of nucleotides within those oligonucleotides. The strength of hybridization between oligonucleotides may be characterized by a temperature of melting (Tm) at which 50% of the duplexes have oligonucleotide strands that disassociate from one another. Oligonucleotides that are “partially” hybridized to one another means that they have sequences that are complementary to one another, but such sequences are hybridized with one another along only a portion of their lengths to form a partial duplex. Oligonucleotides with an “inability” to hybridize include those that are physically separated from one another such that an insufficient number of their bases may contact one another in a manner so as to hybridize with one another.

[0033] As used herein the term “analyte” is intended to include any of a variety of target nucleic acids (e.g., DNA, RNA, or analogs thereof) that are to be detected, characterized, modified, synthesized, or the like. A target nucleic acid, in various embodiments, is a RNA molecule, such as a mRNA. In further embodiments, the target nucleic acids are mRNA, gDNA, rRNA, tRNA, or a combination thereof. In some embodiments, the target nucleicDocket No. 33080 / I P-2574 acids are RNA, mRNA, or a combination thereof. An array can include multiple different species from a library of analytes {e.g., nucleic acids having different sequences from a library of nucleic acids).

[0034] As used herein, a “primer” is a nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to an oligonucleotide {e.g., a first complementary strand as described herein) . As one example, a clustering primer can serve as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template) strand may include a site to which a primer {e.g., a sequencing primer) can hybridize in order to prime synthesis of a new strand that is complementary to the synthesized nucleic acid strand. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In various embodiments, the sequencing primer is a short strand, ranging from 10 to 60 bases, or from 20 to 40 bases.

[0035] As used herein, the term “single molecular identifier” or “SMI” refers to a molecular tag, either random, non-random, or semi-random, that may be attached to a nucleic acid. In various embodiments, a SMI is a unique molecular identifier (IIMI). When incorporated into a nucleic acid, a SMI can be used to correct for subsequent amplification bias by directly counting single molecular identifiers (SMIs) that are sequenced after amplification. A SMI {e.g., a UM I) can be attached to similar nucleic acids, e.g., adapters, making each nucleic acid unique. SMIs e.g., UMIs) may also be used to uniquely tag individual molecules e.g., individual mRNA molecules) in a sample {e.g., individual mRNA molecules in a tissue sample, cell sample, or sample library).

[0036] As used herein, the term “universal sequence” refers to a series of nucleotides that is common to two or more nucleic acid molecules even if the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of molecules can allow capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to the universal sequence. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to the universal sequence. Thus, a universal capture nucleic acid or a universal primer includes a sequence that can hybridize specifically to a universal sequence. Target nucleic acid molecules may be modified to attach universal adapters, for example, at one or both ends of the different target sequences. Universal capture oligonucleotides are applicable for interrogating a plurality of different oligonucleotides without necessarily distinguishing the different species whereas target-Docket No. 33080 / I P-2574 specific capture sequences are applicable for distinguishing the different species. A nonlimiting example of a universal sequence is a polyT nucleotide sequence.

[0037] As used herein, the term “plurality” is intended to mean a population of two or more members, which may all be the same or two or more members may be different. Pluralities may range in size from small, medium, large, to very large. The size of small plurality may range, for example, from a few members to tens of members. Medium sized pluralities may range, for example, from tens of members to about 100 members or hundreds of members. Large pluralities may range, for example, from about hundreds of members to about 1000 members, to thousands of members and up to tens of thousands of members. Very large pluralities may range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions and up to or greater than hundreds of millions of members. Therefore, a plurality may range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality may be set, for example, by the theoretical diversity of bead types in an array.

[0038] As used herein, a "semi-random" nucleotide sequence comprises or consists of a partially pre-determined nucleotide sequence combined with a random nucleotide sequence.OLIGONUCLEOTIDES

[0039] An oligonucleotide is a polymer comprised of nucleotides. Oligonucleotides of the disclosure may be of any length and include, in various embodiments, DNA oligonucleotides, RNA oligonucleotides, analogs thereof, or a combination thereof. In any aspects or embodiments described herein, an oligonucleotide is single-stranded, double-stranded, or partially double-stranded.

[0040] Nucleotides may include naturally occurring nucleotides and functional analogs thereof. Examples of functional analogs are those that are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar e.g., found in RNA). An analog structure can have an alternate sugar moiety including any of a variety known in the art. Nucleotides can include native or non-native bases. A native DNA can include one or more of adenine, thymine, cytosine and / or guanine, and a native RNA can include one or more of adenine, uracil, cytosine and / or guanine. Any non-native baseDocket No. 33080 / I P-2574 may be used, such as a locked nucleic acid (LNA) and a bridged nucleic acid (BNA). Example modified nucleotides include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6- methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7- methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5'-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.

[0041] Oligonucleotides contemplated by the disclosure also include those having at least one modified internucleotide linkage. In some embodiments, the oligonucleotide is all or in part a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those comprising one or more universal bases. "Universal base" refers to molecules capable of substituting for binding to any one of A, C, G, T and U in nucleic acids by forming hydrogen bonds without significant structure destabilization. Examples of universal bases include but are not limited to 5’-nitroindole-2’-deoxyriboside, 3-nitropyrrole, inosine and hypoxanthine.

[0042] In various aspects, an oligonucleotide of the disclosure, or a modified form thereof, is generally about 5 nucleotides to about 150 nucleotides in length. In further embodiments, an oligonucleotide of the disclosure is about 5 to about 125 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 150 nucleotides in length, about 10 to about 125 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90 about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, and all oligonucleotides intermediate in length of the sizes specifically disclosed to the extent that the oligonucleotideDocket No. 33080 / I P-2574 is able to achieve the desired result. Accordingly, in various embodiments, an oligonucleotide of the disclosure is or is at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65,66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89,90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110,1 11 , 1 12, 1 13, 1 14, 1 15, 1 16, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128,129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146,147, 148, 149, 150 or more nucleotides in length. In further embodiments, an oligonucleotide of the disclosure is less than 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43,44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67,68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 1 ,1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129,130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147,148, 149, 150, or more nucleotides in length. In various embodiments, the length of an oligonucleotide (such as a primer) of the disclosure is between about 5 base pairs (bp) and 40 bp, or between about 5 bp and 35 bp, or between about 5 bp and 30 bp, or between about 10 bp and 35 bp, or between about 10 bp and 30 bp, or between about 20 bp and 40 bp, or between about 20 bp and 35 bp, or between about 20 bp and 30 bp, or between about9 and 20 bp or between about 5 and 15 bp, or between about 9 and 15 bp in length. In some embodiments, the length of an oligonucleotide (such as a primer) of the disclosure is about10 bp, 13 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, or 40 bp. As described herein, in various embodiments the oligonucleotide may be a P5 primer, a P5’ primer, a P7 primer, or a P7’ primer.

[0043] As used herein, the term "poly T" or "poly A," when used in reference to a nucleic acid sequence (e.g., a capture nucleotide sequence), is intended to mean a series of two or more thiamine (T) or adenine (A) bases, respectively. A poly T or poly A can include at least about 2, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, or more of the T or A bases, respectively. Alternatively or additionally, a poly T or poly A can include at most about 40, 38, 35, 32, 30, 28, 25, 22, 20, 18, 15, 12, 10, 8, 5, or 2 of the T or A bases, respectively. In some embodiments, the disclosure contemplates use of a "TVN" sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T) The TVN sequence is used, in someDocket No. 33080 / I P-2574 embodiments, to bias reverse transcription to the base of the poly A tail on a mRNA molecule.

[0044] As used herein, the term “immobilized” refers to the state of two things being joined, fastened, adhered, attached, connected, or bound to each other. For example, an analyte, such as a nucleic acid, can be immobilized on a material, such as a bead, gel, or surface, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions. In various embodiments, covalent attachment can be used, but all that is required is that the oligonucleotides remain stationary or attached to a surface under conditions in which it is intended to use the surface, for example, in applications requiring nucleic acid capture, amplification, and / or sequencing. Oligonucleotides to be used as capture oligonucleotides can be immobilized such that a 3'-end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence.

[0045] Exemplary covalent linkages include, for example, those that result from the use of click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, non-specific interactions (e.g., hydrogen bonding, ionic bonding, van der Waals interactions etc.) or specific interactions e.g., affinity interactions, receptor-ligand interactions, antibodyepitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectincarbohydrate interactions, etc.). Exemplary linkages are set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234 and 7,427,678; and US Pat. Pub. No. 2011 / 0059865 A1 , each of which is incorporated herein by reference.

[0046] As used herein, the term "extend," when used in reference to a nucleic acid, is intended to mean addition of at least one nucleotide to the nucleic acid. In particular embodiments one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis e.g. DNA polymerase, RNA polymerase or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid. An extension reaction, in which nucleotides are added to the 3' end of an oligonucleotide {e.g., a primer) is performed in the presence of a polymerase, such as a DNA or RNA polymerase. In some embodiments, the polymerase is a non-thermostable isothermal strand displacement polymerase. Suitable non-thermostable strand displacement polymerases according to the present disclosure can be found, for example, through New England BioLabs, Inc. and include phi29, Bsu, Klenow, DNA Polymerase I (E. coli), and Therminator. In some embodiments, the extension reaction is carried out by recombinase polymerase amplification (RPA). RPA comprises three coreDocket No. 33080 / I P-2574 enzymes - a recombinase, a single-stranded DNA binding protein (SSB) and a stranddisplacing polymerase. As described in Daher etal. (Rana K Daher, Gale Stewart, Maurice Boissinot, Michel G Bergeron, Recombinase Polymerase Amplification for Diagnostic Applications, Clinical Chemistry, Volume 62, Issue 7, 1 July 2016). One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended.METHODS

[0047] The present disclosure is generally directed to capture oligonucleotides and methods of making and using capture oligonucleotide to generate capture surfaces and spatial transcriptomic libraries. In various aspects the disclosure also provides methods for spatially capturing target nucleic acids of a tissue sample. Use of methods of the disclosure provides the ability to spatially preserve the location of target nucleic acids in a biological sample (e.g., tissue sample). In some aspects, the disclosure provides a capture oligonucleotide comprising, from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a single molecule identifier (SMI) sequence; (e) a quality control sequence; and (f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T). In some embodiments, the capture oligonucleotide further comprises an index (IDX) sequence between the first clustering primer sequence and the SBC sequence. In further aspects, the disclosure provides a capture surface comprising a plurality of moieties and a plurality of capture oligonucleotides immobilized on a surface, wherein each capture oligonucleotide in the plurality of capture oligonucleotides comprises, from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a single molecule identifier (SMI) sequence; (e) a sequence that is complementary to a second sequencing primer; (f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T). In some embodiments, each of the plurality of moieties comprises a first clustering primer sequence. In some embodiments, each of the plurality of moieties is a biotin functionalized to a click chemistry handle. In some embodiments, a complementary oligonucleotide is hybridized to the first clustering primer sequence of one or more of the plurality of moieties and to the first clustering primer sequence of one or more of the plurality of capture oligonucleotides. In further embodiments, one or more of the pluralityDocket No. 33080 / I P-2574 of capture oligonucleotides comprises an index (IDX) sequence between the first clustering primer sequence and the SBC sequence.

[0048] The disclosure also provides, in further aspects, methods of making a capture surface. In various aspects, methods of making a capture surface comprise: (1 ) immobilizing a first plurality of oligonucleotides on a surface, wherein each oligonucleotide in the first plurality of oligonucleotides comprises from 5’ to 3’: (a) a first clustering primer sequence; (b) a spatial barcode (SBC) sequence; (c) a first sequencing primer sequence; (d) a restriction endonuclease (RE) recognition sequence; (e) a quality control sequence; and (f) a sequence that is complementary to a second clustering primer sequence; (2) amplifying the first plurality of oligonucleotides, thereby generating clusters of one or more of the plurality of oligonucleotides on the surface; (3) contacting the surface with a restriction endonuclease capable of recognizing the RE recognition sequence of the first plurality of oligonucleotides, thereby generating a second plurality of oligonucleotides comprising from 5’ to 3’; (a) the first clustering primer sequence; (b) the spatial barcode (SBC) sequence; and (c) the first sequencing primer sequence; (4) hybridizing a third plurality of oligonucleotides to the second plurality of oligonucleotides, wherein the third plurality of oligonucleotides comprises from 5’ to 3’: (i) a sequence that is complementary to the first sequencing primer sequence; (ii) a sequence that is complementary to a single molecule identifier (SMI) sequence; (iii) a second sequencing primer sequence; (iv) an ABN sequence, wherein “A” is a sequence that is complementary to a capture nucleotide sequence, “B” is cytosine (C), guanine (G), or thymine (T), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T); (5) extending the first plurality of oligonucleotides having an oligonucleotide of the third plurality of oligonucleotides hybridized thereto, thereby generating a fourth plurality of oligonucleotides comprising from 5’ to 3’: (I) the first clustering primer sequence; (II) the spatial barcode (SBC) sequence; (III) the first sequencing primer sequence; (IV) a single molecule identifier (SMI) sequence; (V) a sequence that is complementary to the second sequencing primer sequence; and (VI) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T), thereby generating the capture surface. In some embodiments, the first plurality of oligonucleotides, the second plurality of oligonucleotides, and the fourth plurality of oligonucleotides further comprise an index (IDX) sequence between the first clustering primer sequence and the SBC sequence, the quality control sequence is a sequence that is complementary to a second sequencing primer. In some embodiments, the quality control sequence comprises a mosaic end (ME) sequence. In some embodiments, a method of making a capture surface according to the disclosure further comprises removing the third plurality of oligonucleotides from the surface after stepDocket No. 33080 / I P-2574(5). In some embodiments, removing the third plurality of oligonucleotides from the surface comprises denaturing the third plurality of oligonucleotides. In some embodiments, removing the third plurality of oligonucleotides from the surface comprises digesting the target nucleic acids. In some embodiments, synthesis of the first plurality of oligonucleotides comprises adding a click chemistry handle during the last cycle of synthesis. In some embodiments, the click chemistry handle is 5’ Hexynyl. In further embodiments, click chemistry is used to immobilize the first plurality of oligonucleotides on the surface through the click chemistry handle. In some embodiments, synthesis of the third plurality of oligonucleotides comprises adding a biotin-functionalized deoxynucleotide triphosphate (dNTP) during the last cycle of synthesis. In some embodiments, the third plurality of oligonucleotides is purified via streptavidin (SA) beads prior to step (4). In some embodiments, a method of making a capture surface according to the disclosure further comprises (6) immobilizing a fifth plurality of oligonucleotides on the surface, wherein one or more of the fifth plurality of oligonucleotides comprises or consists of the first clustering primer sequence. In some embodiments, a complementary oligonucleotide is hybridized to the first clustering primer sequence of the fifth plurality of oligonucleotides and to the first clustering primer sequence of one or more of the first plurality of oligonucleotides. In some aspects, and as depicted in Figure 2, methods of making a capture surface comprise: seeding capture oligonucleotides comprising, from 5’ to 3’: a first clustering primer sequence (e.g., P7), an index sequence (IDX), a spatial barcode (SBC) sequence, a first sequencing primer sequence e.g., SBS12), and a restriction enzyme binding site (RE), a sequence complementary to a mosaic end sequence (ME’), a sequence complementary to a second sequencing primer {e.g., A14’), and a sequence complementary to a second clustering primer {e.g., P5’) on a surface; performing bridge amplification of P7-IDX-SBC-SBS12-RE - ME'-A14'-P5 on the surface to generate clusters that are approximately 1 pm in size; contacting the surface with a restriction endonuclease to generate an oligonucleotide comprising the index and spatial barcode {e.g., P7-IDX-SBC-SBS12); hybridizing an additional oligonucleotide {e.g., SBS12'-UMr-SBS8-A30BN-Biotin) that comprises a purification handle {e.g., biotin) to the oligonucleotide; extending to generate the capture oligonucleotide (P7-IDX-SBC-SBS12-UMI-SBS8'-T30-VN). By way of non-limiting example, the efficiency of synthesizing the additional oligonucleotide that comprises a purification handle is approximately 99.6% at each cycle. A significant percentage of the oligonucleotides will be truncated (>30% for oligonucleotides having a length of 100 nucleotides), and therefore the oligonucleotides need to be purified so that only full-length additional oligonucleotides {e.g., SBS12’-UMI-SBS8-A30BN-Biotin) will be used. Because the UM Is are a random sequence, it is not possible to purify the additional oligonucleotides by traditional methods e.g., PAGE, HPLC). In some embodiments, during the final cycle ofDocket No. 33080 / I P-2574 synthesis of the additional oligonucleotides that comprise a purification handle, a biotin functionalized dNTP is be used instead of a normal dNTP. In some embodiments, the additional oligonucleotides are purified by streptavidin (SA) beads and only the additional oligonucleotides possessing the full length (SBS12’-UMI-SBS8-A30BN-Biotin) will be recovered. The additional oligonucleotides may be released from SA beads to IDTE buffer with 5mM biotin at 95° C.

[0049] Thus, in various aspects the disclosure provides capture oligonucleotides and methods of making a capture surface. The disclosure further provides, in various aspects, methods of preparing an immobilized library of target nucleic acids of a biological sample, comprising: (a) providing (i) a surface comprising a plurality of moieties and a plurality of the capture oligonucleotides of the disclosure immobilized thereon; (ii) a capture surface of the disclosure; or (iii) the capture surface produced by a method of the disclosure; (b) contacting the biological sample with the surface or the capture surface, the contacting resulting in hybridization of the target nucleic acids of the biological sample to the TVN sequence of the plurality of capture oligonucleotides to form hybridized capture oligonucleotides; (c) extending the TVN sequence of the hybridized capture oligonucleotides to form first complementary strands of the target nucleic acids, wherein the extending comprises addition of a plurality of non-templated nucleotides to the end of the first complementary strands; (d) hybridizing a plurality of template switching oligonucleotides (TSOs) to the plurality of non- templated nucleotides of the first complementary strands such that each of the plurality of template switching oligonucleotides that is hybridized to the plurality of non-templated nucleotides of the first complementary strands is positioned at the terminus of the target nucleic acids that is not hybridized to the TVN sequence; (e) extending the plurality of non- templated nucleotides on the first complementary strands using the template switching oligonucleotides as template, thereby generating a complementary template-switching oligonucleotide sequence (TSO’) on the first complementary strands, thereby preparing the immobilized library of target nucleic acids. In some embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise: (f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands. In further embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise: (f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands; and (g)Docket No. 33080 / 1 P-2574 amplifying the first complementary strands and the second complementary strands, thereby generating a plurality of clusters comprising hybridized first complementary strands and second complementary strands. In some embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise: (h) performing tagmentation on the plurality of clusters comprising hybridized first complementary strands and second complementary strands to prepare tagged fragments. Tagmentation refers to the modification of DNA by a transposome complex comprising a transposase enzyme complexed with adaptors comprising transposon end sequence, and results in the simultaneous fragmentation of the DNA and ligation of the adaptors to the 5' ends of both strands of duplex fragments. Tagmentation is described in detail in, for example, U.S. Patent Publication No. 2022 / 0403376, incorporated by reference herein in its entirety. In some embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise: (h) denaturing the plurality of clusters comprising hybridized first complementary strands and second complementary strands; (i) hybridizing an oligonucleotide comprising, from 5’ to 3’, the second sequencing primer and a capture nucleotide sequence to the one or more second complementary strands; (j) extending the oligonucleotide in the presence of a dideoxynucleotide triphosphate (ddNTP) comprising a click chemistry handle; (k) hybridizing a primer comprising the first clustering primer sequence to the one or more second complementary strands, extending the hybridized primer comprising the first clustering primer sequence, wherein the extending comprises a gap fill, and ligating the 3’ end of the extended primer comprising the first clustering primer sequence to the 5’ end of the extended oligonucleotide of step (j); (I) attaching an adapter oligonucleotide comprising, from 5’ to 3’, a third sequencing primer sequence, a sequence complementary to a unique index sequence (i5’), and a second clustering primer sequence, wherein the adapter oligonucleotide is attached by a click chemistry reaction using the click chemistry handle. In some embodiments, the click chemistry handle is an azide, a tetrazine, a strained alkene, or an alkyne. In further embodiments, the gap fill comprises using a reverse transcriptase or a DNA polymerase. In various embodiments, the reverse transcriptase is Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, each of the plurality of moieties comprises a first clustering primer sequence. In some embodiments, each of the plurality of moieties is a biotin functionalized to a click chemistry handle. In some embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise contacting the surface with an exonuclease (e.g., Exonuclease I) after step (d), thereby digesting single-stranded capture oligonucleotides. Exonuclease digestion is performed immediately after synthesis of the first complementary strand to remove single stranded capture oligonucleotides on the surface that did not capture or extend a targetDocket No. 33080 / 1 P-2574 nucleic acid {e.g., mRNA). After exonuclease digestion, in various embodiments, digestion of the biological sample and removal of the target nucleic acids from the surface is performed, followed by synthesis of second complementary strands via, in various embodiments, template switching oligonucleotides (TSOs). In some embodiments, the plurality of moieties is immobilized on the surface after the exonuclease is contacted with the surface. In various embodiments, methods of preparing an immobilized library of target nucleic acids of a biological sample further comprise adding streptavidin and an additional plurality of moieties to the surface, wherein each of the additional plurality of moieties is a biotin functionalized to a first clustering primer sequence. In some embodiments, prior to step (b) a complementary oligonucleotide is hybridized to the first clustering primer sequence of the capture oligonucleotides and to the first clustering primer sequence of one or more of the plurality of moieties. In various embodiments, the step of hybridizing a complementary oligonucleotide to the first clustering primer sequence of the capture oligonucleotides and to the first clustering primer sequence of one or more of the plurality of moieties is performed after capture oligonucleotide generation and before the step of contacting the surface with an exonuclease. In some embodiments, the complementary oligonucleotide is removed prior to step (g). In some embodiments, the amplifying of the first complementary strands and the second complementary strands in step (g) is performed via one or more cycles of hybridizing the first clustering primer sequence of the plurality of moieties to the complement of the first clustering primer in the second complementary strands and extending the hybridized first clustering primer sequence to generate the plurality of clusters comprising hybridized first complementary strands and second complementary strands. In some embodiments, the amplification is Exclusion-Amplification (ExAmp). In further embodiments, ExAmp (or clustering) is performed in the presence of an ExAmp mix, which in various embodiments comprises a recombinase, a ssDNA binding protein e.g., gp32), a polymerase, creatine kinase, ATP, Phosphocreatine, dNTPs, MgOAc, and polyethylene glycol (PEG). During the amplification of the first complementary strands and the second complementary strands, a clustering primer sequence of the first complementary strand is hybridized to the complementary clustering primer sequence of the second complementary strand. Next, a new oligonucleotide that is immobilized on the surface and comprises or consists of the clustering primer sequence e.g., a moiety as described herein) will, in the presence of a recombinase, “invade” the hybridized first and second complementary strands and will hybridize to the complementary clustering primer sequence of the second complementary strand. The foregoing amplification process is a subtype of ExAmp clustering referred to as “non-bridging clustering” or “isothermal amplification”. The "invasion" mechanism described above is applicable to all ExAmp clustering, while non-bridge clustering is a subset of ExAmp clustering that includes thisDocket No. 33080 / I P-2574 invasion property. Non-bridging clustering is described, for example, in PCT / 2013 / 045491 (WO2013188582) “Kinetic Exclusion Amplification of Nucleic Acid Libraries” and PCT / US2022 / 053005 (WO2023114397) “Hybrid Clustering”, each of which is incorporated by reference herein in its entirety. In some embodiments, there may also be TSOs present in solution that will hybridize to any newly made amplified strands ( / .e., the strands comprising the first complementary oligonucleotides and original SMI), and upon hybridization of the TSO to its complementary sequence, it will extend and produce new double stranded complexes. In these embodiments in which TSOs are present in solution that will hybridize to any newly made amplified strands ( / .e., the strands comprising the first complementary oligonucleotides and original SMI), and upon hybridization of the TSO to its complementary sequence, extend and produce new double stranded complexes, the amplification is exponential amplification.

[0050] Methods of the disclosure further provide, in various embodiments, that the biological sample is digested. The digestion of the biological sample can occur, in various embodiments, after generation of the first complementary strands. In some embodiments, digestion of the biological sample occurs after generation of the first complementary strands but prior to generation of second complementary strands. The disclosure also provides methods in which the target nucleic acids (e.g., RNA) are removed from the surface. Removal of target nucleic acids from the surface can occur, in various embodiments, after generation of the first complementary strands. In some embodiments, removal of the target nucleic acids occurs after generation of the first complementary strands but prior to generation of second complementary strands. Removal of the target nucleic acids from the surface is achieved, in various embodiments, by changing a condition. In further embodiments, the condition is temperature, pH, formamide concentration, or a combination thereof.

[0051] Aspects of the disclosure include those in which a plurality of capture oligonucleotides is immobilized on a surface. The capture oligonucleotides, in various embodiments, hybridize to target nucleic acids of a biological sample. In some embodiments, each of the plurality of capture oligonucleotides comprises the same capture nucleotide sequence. In further embodiments, the plurality of capture oligonucleotides comprises multiple, different capture nucleotide sequences. In still further embodiments, the multiple, different capture nucleotide sequences comprise one or more gene-specific capture sequences, one or more universal capture sequences, or a combination thereof. In various embodiments, the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence. In further embodiments, the universal capture sequence is a random nucleotide sequence or a non-selfDocket No. 33080 / I P-2574 complementary semi-random sequence. Aspects of the disclosure also include those in which capture nucleotide sequences are extended following hybridization of the capture oligonucleotide to the target nucleic acid. In some embodiments, the extending of the capture nucleotide sequence is carried out using a reverse transcriptase. In some implementations of a method of the disclosure, the target nucleic acids are polyadenylated prior to hybridization of the target nucleic acids to the capture nucleotide sequences. In some embodiments, the target nucleic acids are polyadenylated using a poly(A) polymerase. In further embodiments, the target nucleic acids are polyadenylated using chemical ligation or enzymatic ligation.

[0052] In various embodiments, a primer (e.g., a primer comprising the first clustering primer sequence) used in a method of the disclosure is used at a concentration in the range of 0.1 pM to 100 pM, 1 pM to 100 pM or 3 pM to 75 pM or 5 pM to 30 pM or 5 to 50 pM or 10 pM to 20 pM or 10 pM to 30 pM or 10 pM to 40 pM or 10 to 50 pM. In some embodiments, the primer is used at a concentration of 0.25 pM, 0.5 pM or 1 .1 pM or 2.2 pM. In still further embodiments, the primer is used at a concentration of 1 pM 5 pM, 10 pM, 25 pM or 50 pM. In various embodiments, such a primer is a P5 primer, a P5’ primer, a P7 primer, or a P7’ primer.

[0053] To further illustrate the present disclosure, examples are given herein. It is to be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure.EXAMPLESEXAMPLE 1

[0054] Methods of preparing an immobilized library of target nucleic acids of a biological sample e.g., tissue sample) are provided herein. The present disclosure provides capture oligonucleotides and methods of making and using capture oligonucleotide to generate capture surfaces and spatial transcriptomic libraries. The design of the capture oligonucleotides provided herein minimize variation of polyA length in the final library, which reduces waste when sequencing a long polyA sequence of a target analyte e.g., target nucleic acid).

[0055] A schematic workflow of a method of the disclosure is shown in Figure 1 and Figure 2. Figure 1 shows the design of an anchored capture oligonucleotide of the disclosure, while Figure 2 depicts oligonucleotide purification and quality control. The spatial barcode was introduced to the substrate by seeding template (P7-IDX-SBC-SBS12-RE-ME’- A14’-P5) followed by bridge amplification to form clusters. The clustered strands were then cleaved with a restriction enzyme to expose SBS12 at the 3’ end. The UMI barcode wasDocket No. 33080 / I P-2574 added to each capture oligo by hybridization and extension of an oligonucleotide containing SBS12’-UMr-SBS8-A30BN-Biotin. The extension oligonucleotide was then denatured off the surface-bound strand. The extended capture oligonucleotide ends with T30-VN (V is any base other than T; N is any base A, G, C, T) which enables the hybridization to the mRNA polyT region adjacent the 3’ UTR. The use of an anchored oligonucleotide advantageously reduces the waste of sequencing the long polyA tail in the library.

[0056] Another schematic workflow of a method of the disclosure is shown in Figure 3. Figure 3 depicts surface amplification of UMI labeled cDNA. The mRNA capture, reverse transcription (RT), tissue digestion, Exonuclease I (Exol) treatment, and RNA removal step conditions are standard workflow conditions. For example, mRNA capture is performed overnight at 42° C. The following day the reverse transcriptase (RT) mix is removed, Exo1 digestion mix is added and incubated at 37° C for 45 minutes to remove un-used capture oligonucleotide from the surface. The Exo1 mix is then removed, and tissue digestion mix is added and incubated at 37° C for 40 minutes to remove traces of tissue. The tissue digestion mix is removed and NaOH is incubated three times for 5 minutes to remove RNA and P7'. The surface is then washed. The clustering only consumes less than 10% of P7. The remaining P7 oligonucleotides are protected from Exol digestion by pre-hybridization with P7’. After the removal of the RNA, the P7’ is typically washed away from the surface. The P7 is used to amplify the UMI-labeled cDNA by ExAmp. Alternatively, the P7 for cDNA ExAmp amplification may be added to the surface after Exol treatment by pre-grafting alkyne-biotin and then, after Exol treatment, P7 may be immobilized by adding streptavidin followed by biotin-P7. After the amplification of the cDNA, there are multiple copies of the UMI-labeled cDNA and second strand DNA from each unique target nucleic acid (e.g., each unique mRNA strand). Because the amplification step is finished on the surface, it is advantageously possible to perform the tagmentation step on the surface. With the steric hindrance from the substrate, the chance of bead based tagmentation in the cDNA range, instead of the P7-IDX-SBC-UMI-SBS8’-T30 range, is increased. It will also increase the chance to convert cDNA to a final library for sequencing.

[0057] Another schematic workflow of a method of the disclosure is shown in Figure 3. Figure 3 depicts a workflow of in-solution library preparation after amplification of UMI labeled cDNA. After the amplification of the cDNA, there are multiple copies of the UMI- labeled cDNA and second strand DNA from each unique mRNA strand. In some embodiments, the following library preparation is performed in solution. In this workflow, the second strand DNA is denatured and eluted from the surface. Without the surface hindrance, an alternative way to prepare the library is provided herein. By hybridization and extension from SBS8’-T30 with dNTPs, with an optimized percentage of azide-dNTPs that isDocket No. 33080 / I P-2574 determined experimentally, the extension will stop at a random location with a functional azide group at the 3’ end for the attachment of SBS-i5’-P5-biotin. The remaining portion of the library will also be copied by the hybridization and extension from P7 followed by gap filling. The final library may be purified by streptavidin beads. Using a 3’ azide-ddATP serves as a “fragmentation step” by shortening the spatial library to desirable sequencing size. With amplification occurring on surface, and sample index added at the ligation step, this workflow may be performed PCR-free.EXAMPLE 2

[0058] The following experiments demonstrate ExAMP amplification of a library generated from mouse kidney tissue on a substrate surface.

[0059] Fresh frozen sections (10um) from mouse kidney were mounted onto a substrate containing spatially barcoded capture oligonucleotides with polyT capture sites. Tissues were methanol-fixed at -20C for 30 minutes, after which they were stained with hematoxylin and eosin, air-dried, and imaged with an optical microscope. The substrate was then placed in a proprietary device with sealable wells, which allowed heated incubations on a thermal cycler. Each well had an approximate surface area of 28mm2overlying each tissue section. Tissue sections were then permeabilized with a proprietary permeabilization reagent at 37C for 7 minutes, followed by three room temperature washes in a proprietary spatial wash buffer. The samples were incubated at 42C overnight in first strand cDNA synthesis mix with a Rd1 -containing template switch oligonucleotide. Following first strand cDNA synthesis, RT mix was discarded, and the samples were treated with a proprietary exonuclease mix for 45 minutes at 37C. The mix was then discarded, and the samples were treated with a proprietary tissue digestion mix for 40 minutes at 37C. The wells were then washed 3 times with water. NaOH was added to the wells and incubated at room temperature for 5 minutes three times. The solution was discarded, and the wells were washed with proprietary wash buffer and then three times with water. A proprietary second strand mix containing randomers with SBS3 adapters was added to control wells and incubated for 2 hours at 37C. To +EXAMP wells, a proprietary isothermal amplification mix was added to the samples incubated for 15 minutes at 38C. From the control wells, second strand cDNA was eluted with KOH at room temp for 10 minutes. For the ExAMP wells, supernatant was collected, and the wells were treated with KOH to remove excess second strand and pooled with the ExAMP supernatant. A proprietary neutralizing solution was added to the samples, and they were 1 ,5X SPRI-purified. A 1 :10 dilution of second strand product was amplified with a proprietary probe-based qPCR method targeting a highly expressed kidney transcript. Cqs were plotted to determine fold comparison between -i-ExAMP and control library prep methods. See Figure 5A.Docket No. 33080 / I P-2574

[0060] Second strand cDNA material generated in Figure 5A was amplified with a proprietary PCR master mix with P7 and SBS3 primers using the following cycling parameters: 95C 3 minutes, 95C 30 second followed by (11 cycles for +ExAMP libraries and 13 cycles for control random prime libraries) of 95C 30 seconds, 60C 1 minute, 72C 1 minute, then a final extension at 72C for 2 minutes and held at 4C. After amplification, libraries were SPRI-purified with 1 ,2X SPRI and run on a HSD5000 screen tape to determine concentration. Sul of 2nM library was then subjected to indexed PCR using a proprietary master mix containing P7 and D5XX primers using the following PCR parameters: 95C 3 minutes, 95C 30 seconds followed by 9 cycles of 95C 30 seconds, 60C 1 minute, 72C 1 minute, then a final extension at 72C for 2 minutes and held at 4C. Libraries were 0.7X SPRI-purified and sequenced. Using a proprietary sequencing analysis pipeline mean UMI / bin 100 was plotted. High sensitivity for the ExAMP samples showed the feasibility of ExAMP library amplification on a surface. See Figure 5B.

Claims

Docket No. 33080 / I P-2574WHAT IS CLAIMED IS:1 . A capture oligonucleotide comprising, from 5’ to 3’:(a) a first clustering primer sequence;(b) a spatial barcode (SBC) sequence;(c) a first sequencing primer sequence;(d) a single molecule identifier (SMI) sequence;(e) a quality control sequence; and(f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T).

2. The capture oligonucleotide of claim 1 , further comprising an index (IDX) sequence between the first clustering primer sequence and the SBC sequence.

3. The capture oligonucleotide of claim 1 or claim 2, wherein the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence.

4. The capture oligonucleotide of claim 3, wherein the capture nucleotide sequence comprises about 20 to about 30 nucleotides.

5. The capture oligonucleotide of claim 4, wherein the capture nucleotide sequence comprises about 30 nucleotides.

6. The capture oligonucleotide of any one of claims 3-5, wherein the poly-T sequence consists of 30 thymine nucleotides.

7. The capture oligonucleotide of any one of claims 1 -6, wherein the quality control sequence is a sequence that is complementary to a second sequencing primer.

8. The capture oligonucleotide of any one of claims 1 -6, wherein the SMI is a unique molecular identifier (IIMI).

9. A capture surface comprising: a plurality of moieties and a plurality of capture oligonucleotides immobilized on a surface, wherein each capture oligonucleotide in the plurality of capture oligonucleotides comprises, from 5’ to 3’:(a) a first clustering primer sequence;(b) a spatial barcode (SBC) sequence;(c) a first sequencing primer sequence;Docket No. 33080 / I P-2574(d) a single molecule identifier (SMI) sequence;(e) a sequence that is complementary to a second sequencing primer;(f) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T).

10. The method of claim 9, wherein each of the plurality of moieties comprises a first clustering primer sequence.11 . The method of claim 9, wherein each of the plurality of moieties is a biotin functionalized to a click chemistry handle.

12. The capture surface of claim 9 or claim 10, wherein a complementary oligonucleotide is hybridized to the first clustering primer sequence of one or more of the plurality of moieties and to the first clustering primer sequence of one or more of the plurality of capture oligonucleotides.

13. The capture surface of any one of claims 9-12, wherein one or more of the plurality of capture oligonucleotides comprises an index (IDX) sequence between the first clustering primer sequence and the SBC sequence.

14. The capture surface of any one of claims 9-13, wherein the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence.

15. The capture surface of claim 14, wherein the capture nucleotide sequence comprises about 20 to about 30 nucleotides.

16. The capture surface of claim 15, wherein the capture nucleotide sequence comprises about 30 nucleotides.

17. The capture surface of any one of claims 14-16, wherein the poly-T sequence consists of 30 thymine nucleotides.

18. The capture surface of any one of claims 9-17, wherein the SMI is a unique molecular identifier (IIMI).

19. A method of making a capture surface, comprising:(1 ) immobilizing a first plurality of oligonucleotides on a surface, wherein each oligonucleotide in the first plurality of oligonucleotides comprises from 5’ to 3’:(a) a first clustering primer sequence;(b) a spatial barcode (SBC) sequence;Docket No. 33080 / 1 P-2574(c) a first sequencing primer sequence;(d) a restriction endonuclease (RE) recognition sequence;(e) a quality control sequence; and(f) a sequence that is complementary to a second clustering primer sequence;(2) amplifying the first plurality of oligonucleotides, thereby generating clusters of one or more of the plurality of oligonucleotides on the surface;(3) contacting the surface with a restriction endonuclease capable of recognizing the RE recognition sequence of the first plurality of oligonucleotides, thereby generating a second plurality of oligonucleotides comprising from 5’ to 3’;(a) the first clustering primer sequence;(b) the spatial barcode (SBC) sequence; and(c) the first sequencing primer sequence;(4) hybridizing a third plurality of oligonucleotides to the second plurality of oligonucleotides, wherein the third plurality of oligonucleotides comprises from 5’ to 3’:(i) a sequence that is complementary to the first sequencing primer sequence;(ii) a sequence that is complementary to a single molecule identifier (SMI) sequence;(iii) a second sequencing primer sequence;(iv) an ABN sequence, wherein “A” is a sequence that is complementary to a capture nucleotide sequence, “B” is cytosine (C), guanine (G), or thymine (T), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T);(5) extending the first plurality of oligonucleotides having an oligonucleotide of the third plurality of oligonucleotides hybridized thereto, thereby generating a fourth plurality of oligonucleotides comprising from 5’ to 3’:(I) the first clustering primer sequence;(II) the spatial barcode (SBC) sequence;(III) the first sequencing primer sequence;(IV) a single molecule identifier (SMI) sequence;Docket No. 33080 / I P-2574(V) a sequence that is complementary to the second sequencing primer sequence; and(VI) a TVN sequence, wherein “T” is a capture nucleotide sequence, “V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T), thereby generating the capture surface.

20. The method of claim 19, wherein the first plurality of oligonucleotides, the second plurality of oligonucleotides, and the fourth plurality of oligonucleotides further comprise an index (IDX) sequence between the first clustering primer sequence and the SBC sequence.21 . The method of claim 19 or claim 20, wherein the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence.

22. The method of claim 21 , wherein the capture nucleotide sequence comprises about 20 to about 30 nucleotides.

23. The method of claim 22, wherein the capture nucleotide sequence comprises about 30 nucleotides.

24. The method of any one of claims 21 -23, wherein the poly-T sequence consists of 30 thymine nucleotides.

25. The method of any one of claims 19-24, wherein the SMI is a unique molecular identifier (IIMI).

26. The method of any one of claims 19-25, wherein the quality control sequence is a sequence that is complementary to a second sequencing primer.

27. The method of any one of claims 19-26, wherein the quality control sequence comprises a mosaic end (ME) sequence.

28. The method of any one of claims 19-27, further comprising removing the third plurality of oligonucleotides from the surface after step (5).

29. The method of claim 28, wherein removing the third plurality of oligonucleotides from the surface comprises denaturing the third plurality of oligonucleotides.

30. The method of claim 28, wherein removing the third plurality of oligonucleotides from the surface comprises digesting the target nucleic acids.Docket No. 33080 / I P-257431 . The method of any one of claims 19-30, wherein synthesis of the first plurality of oligonucleotides comprises adding a click chemistry handle during the last cycle of synthesis.

32. The method of claim 31 , wherein the click chemistry handle is 5’ Hexynyl.

33. The method of claim 31 or claim 32, wherein click chemistry is used to immobilize the first plurality of oligonucleotides on the surface through the click chemistry handle.

34. The method of any one of claims 19-33, wherein synthesis of the third plurality of oligonucleotides comprises adding a biotin-functionalized deoxynucleotide triphosphate (dNTP) during the last cycle of synthesis.

35. The method of claim 34, wherein the third plurality of oligonucleotides is purified via streptavidin (SA) beads prior to step (4).

36. The method of any one of claims 19-35, further comprising (6) immobilizing a fifth plurality of oligonucleotides on the surface, wherein one or more of the fifth plurality of oligonucleotides comprises or consists of the first clustering primer sequence.

37. The method of claim 36, wherein a complementary oligonucleotide is hybridized to the first clustering primer sequence of the fifth plurality of oligonucleotides and to the first clustering primer sequence of one or more of the first plurality of oligonucleotides.

38. A method of preparing an immobilized library of target nucleic acids of a biological sample, comprising:(a) providing (i) a surface comprising a plurality of moieties and a plurality of the capture oligonucleotides of any one of claims 1-8 immobilized thereon; (ii) the capture surface of any one of claims 9-18; or (iii) the capture surface produced by the method of any one of claims 19-37;(b) contacting the biological sample with the surface or the capture surface, the contacting resulting in hybridization of the target nucleic acids of the biological sample to the TVN sequence of the plurality of capture oligonucleotides to form hybridized capture oligonucleotides;(c) extending the TVN sequence of the hybridized capture oligonucleotides to form first complementary strands of the target nucleic acids, wherein the extending comprises addition of a plurality of non-templated nucleotides to the end of the first complementary strands;Docket No. 33080 / I P-2574(d) hybridizing a plurality of template switching oligonucleotides (TSOs) to the plurality of non-templated nucleotides of the first complementary strands such that each of the plurality of template switching oligonucleotides that is hybridized to the plurality of non- templated nucleotides of the first complementary strands is positioned at the terminus of the target nucleic acids that is not hybridized to the TVN sequence;(e) extending the plurality of non-templated nucleotides on the first complementary strands using the template switching oligonucleotides as template, thereby generating a complementary template-switching oligonucleotide sequence (TSO’) on the first complementary strands, thereby preparing the immobilized library of target nucleic acids.

39. The method of claim 38, further comprising:(f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands.

40. The method of claim 38, further comprising:(f) hybridizing the complementary template-switching oligonucleotide sequence (TSO’) on one or more of the first complementary strands to the template switching oligonucleotides (TSOs), and extending the TSOs thereby generating one or more second complementary strands; and(g) amplifying the first complementary strands and the second complementary strands, thereby generating a plurality of clusters comprising hybridized first complementary strands and second complementary strands.41 . The method of claim 40, further comprising:(h) performing tagmentation on the plurality of clusters comprising hybridized first complementary strands and second complementary strands to prepare tagged fragments.

42. The method of claim 40, further comprising:(h) denaturing the plurality of clusters comprising hybridized first complementary strands and second complementary strands;(i) hybridizing an oligonucleotide comprising, from 5’ to 3’, the second sequencing primer and a capture nucleotide sequence to the one or more second complementary strands;Docket No. 33080 / I P-2574(j) extending the oligonucleotide in the presence of a dideoxynucleotide triphosphate (ddNTP) comprising a click chemistry handle;(k) hybridizing a primer comprising the first clustering primer sequence to the one or more second complementary strands, extending the hybridized primer comprising the first clustering primer sequence, wherein the extending comprises a gap fill, and ligating the 3’ end of the extended primer comprising the first clustering primer sequence to the 5’ end of the extended oligonucleotide of step (j);(l) attaching an adapter oligonucleotide comprising, from 5’ to 3’, a third sequencing primer sequence, a sequence complementary to a unique index sequence (i5’), and a second clustering primer sequence, wherein the adapter oligonucleotide is attached by a click chemistry reaction using the click chemistry handle.

43. The method of claim 42, wherein the click chemistry handle is an azide, a tetrazine, a strained alkene, or an alkyne.

44. The method of claim 42 or claim 43, wherein the gap fill comprises using a reverse transcriptase or a DNA polymerase.

45. The method of any one of claims 38-44, wherein each of the plurality of moieties comprises a first clustering primer sequence.

46. The method of any one of claims 38-44, wherein each of the plurality of moieties is a biotin functionalized to a click chemistry handle.

47. The method of claim 45 or claim 46, further comprising contacting the surface with an exonuclease after step (d), thereby digesting single-stranded capture oligonucleotides.

48. The method of claim 47, wherein the plurality of moieties is immobilized on the surface after the exonuclease is contacted with the surface.

49. The method of claim 48, further comprising adding streptavidin and an additional plurality of moieties to the surface, wherein each of the additional plurality of moieties is a biotin functionalized to a first clustering primer sequence.

50. The method of any one of claims 38-49, wherein prior to step (b) a complementary oligonucleotide is hybridized to the first clustering primer sequence of the capture oligonucleotides and to the first clustering primer sequence of one or more of the plurality of moieties.51 . The method of claim 47, wherein the complementary oligonucleotide is removed prior to step (g).Docket No. 33080 / I P-257452. The method of any one of claims 40-51 , wherein the amplifying of the first complementary strands and the second complementary strands in step (g) is performed via one or more cycles of hybridizing the first clustering primer sequence of the plurality of moieties to the complement of the first clustering primer in the second complementary strands and extending the hybridized first clustering primer sequence to generate the plurality of clusters comprising hybridized first complementary strands and second complementary strands.

53. The method of claim 52, wherein the amplification is Exclusion-Amplification (ExAmp).

54. The method of any one of claims 38-53, further comprising removing the target nucleic acids from the surface after step (e).

55. The method of claim 54, wherein removing the target nucleic acids from the surface comprises denaturing the target nucleic acids.

56. The method of claim 54, wherein removing the target nucleic acids from the surface comprises digesting the target nucleic acids.

57. The method of any one of claims 38-56, further comprising removing the biological sample from the surface after step (c).

58. The method of any one of claims 38-57, wherein each of the plurality of capture oligonucleotides comprises the same capture nucleotide sequence.

59. The method of any one of claims 38-57, wherein the plurality of capture oligonucleotides comprises multiple, different capture nucleotide sequences.

60. The method of claim 59, wherein the multiple, different capture nucleotide sequences comprise one or more gene-specific capture sequences, one or more universal capture sequences, or a combination thereof.61 . The method of any one of claims 38-59, wherein the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence.

62. The method of claim 60 or claim 61 , wherein the universal capture sequence is a random nucleotide sequence or a non-self complementary semi-random sequence.

63. The method of any one of claims 38-62, wherein the target nucleic acids are mRNA, gDNA, rRNA, tRNA, or a combination thereof.

64. The method of any one of claims 38-62, wherein the target nucleic acids are RNA, mRNA, or a combination thereof.Docket No. 33080 / 1 P-257465. The method of any one of claims 38-64, wherein the extending of the capture nucleotide sequence in step (c) is carried out using a reverse transcriptase.

66. The method of any one of claims 38-65, wherein the target nucleic acids are polyadenylated prior to hybridization of the target nucleic acids to the capture nucleotide sequences.

67. The method of claim 66, wherein the target nucleic acids are polyadenylated using a poly(A) polymerase.

68. The method of claim 66, wherein the target nucleic acids are polyadenylated using chemical ligation or enzymatic ligation.

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