Methods of indirect tagging of nucleic acids

Abstract

The present invention relates to preparation, sequencing and analysis of nucleic acids. The nucleic acids are prepared, by attaching a first tag to an input nucleic acid. The first tag comprises a binding partner of a first binding pair. A second tag comprises a. reciprocal binding partner of the first binding pair. The second tag is attached to the first tag to produce an input nucleic acid complex, thereby indirectly tagging the input nucleic acid with the second tag.

Description

20230183-02 / 027644.8509 METHODS OF INDIRECT TAGGING OF NUCLEIC ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 636,312, filed April 19, 2024, which is incorporated by reference herein in its entirety.. FIELD OF THE INVENTION

[0002] The present invention relates to preparation of nucleic acids for sequencing or other analysis. The present invention also relates to target enrichment of nucleic acids. BACKGROUND

[0003] Next-Generation Sequencing (NGS) methods and systems involve the parallel sequencing of a library of nucleic acids by a sequencing platform. Preparation of a sequencing library generally includes various steps such as amplification of the nucleic acids, attachment of adaptors, and / or other preparatory steps. Emerging polynucleotide sequencing platforms can enable direct detection and analysis of nucleic acid molecules without the need for amplification, though the nucleic acids often require an adaptor or other moiety to immobilize the nucleic acid for sequencing steps. An adaptor can be attached to one or both ends of nucleic acid molecules in order to add sites for primer binding and for immobilization of the nucleic acid on a surface such as a flowcell or a bead, and to add other functional sequences to the fragments. Various kinds of adaptors are used in sequencing preparation kits to add these sites or sequences to the nucleic acids from the sample. Adaptors can be attached in various ways, such as by ligation, primer extension, tagmentation, and other techniques.20230183-02 / 027644.8509

[0004] In order to obtain a suitable signal from sequencing a single DNA fragment, many sequencing systems use clonal amplification to generate many identical copies of individual DNA molecules on a solid support. These copies are segregated in individual clusters, or on beads which are loaded with an individual DNA molecule. Sequencing reactions proceed on the identical copies of the fragment in parallel, thereby producing detectable signals from the clusters or beads, with signals simultaneously detected from an enormous number of distinct clusters or beads.

[0005] A sequencing library can be generated in a variety of ways, with different objectives regarding the nucleic acids to be used as inputs. For instance, PCR can be used with target- specific primers to generate a library of amplicons covering regions of interest in the nucleic acid sample. Other methods of library preparation involve random fragmentation of the nucleic acid sample by enzymatic or physical shearing methods, followed by amplification using common adaptor sequences. Enrichment procedures are used to remove or separate sequences of interest from the rest of the sample.

[0006] The adaptors used in many target enrichment procedures are Y-shaped adaptors (or “Y-adaptors”) which can contain duplex-UMI (unique molecular identifier) sequences. Said adaptors are made full-length through subsequent PCR and typically include a sample identifier barcode. Alternatively, full-length adaptors are ligated directly (at lower efficiency than truncated adaptors). Many Y-adaptors and other adaptors do not include a tag which supports direct immobilization of the nucleic acid molecule on a surface, or analysis of specific strands from a nucleic acid molecule.

[0007] Nucleic acid target capture methods can allow specific genes, exons, and other genomic regions of interest to be enriched for targeted sequencing or other analysis. However, target20230183-02 / 027644.8509 capture-based sequencing methods can involve cumbersome lengthy protocols and costly processes, as well as a low on-target rate for a small capture panel (e.g., less than 500 probes). Moreover, current methods for nucleic acid target capture can be ill-suited for low input and damaged DNA because of a low recovery rate.

[0008] There is a need for improved methods of preparing nucleic acid molecules for immobilization on a substrate and for sequencing. There is also a need for a more efficient, easy, fast, flexible, and practical target enrichment methods. When a tag such as biotin is attached directly to a target nucleic acid, prior to target enrichment, the tag will interfere with target enrichment protocols that employ the same tag, such as biotinylated probes. There remains a need for improved methods of tagging nucleic acids for sequencing or other analysis and / or for target enrichment. SUMMARY

[0009] The present disclosure provides methods of preparing nucleic acid molecules for sequencing. As one aspect, the methods comprise attaching a first tag to an input nucleic acid, wherein the first tag comprises a binding partner of a first binding pair. The methods comprise attaching a second tag to the first tag to produce an input nucleic acid complex, wherein the second tag comprises a reciprocal binding partner of the first binding pair. For example, the first tag can comprise an adaptor conjugated with digoxigenin (DIG), and the second tag can comprise anti-DIG antibody. In this example, DIG and anti-DIG antibody constitute the first binding pair.20230183-02 / 027644.8509

[0010] The first tag can be attached to the input nucleic acid by a covalent bond or by non- covalent binding. The second tag can be attached to the first tag by a covalent bond or by non- covalent binding.

[0011] The second tag can also comprise a binding partner of a second binding pair, and the present methods can comprise attaching the second tag to a reciprocal binding pair of the second binding pair, which itself may be attached to the solid support. For example, the second tag can comprise a biotin moiety, and the reciprocal binding partner can be a streptavidin coated bead; in this example, biotin:streptavidin constitutes the second binding pair.

[0012] The present methods can further comprise one or more steps for capture or target enrichment of a tagged input nucleic acid construct. For instance, the input nucleic acid construct can be hybridized with a probe, such as a capture probe or a bridge probe which is hybridized with an anchor probe. The capture probe or anchor probe can be attached to an enrichment tag such as a biotin moiety.

[0013] In some embodiments, the present methods further comprise performing an amplification of the input nucleic acid construct to produce amplicons of the input nucleic acid, wherein the amplification is performed before attaching the second tag to the first tag. After the amplification is performed, the input nucleic acid complex can be separated from the amplicons by binding the binding partner of the second tag to a reciprocal binding partner of the second binding pair. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG.1A illustrates tagging of an input nucleic acid molecule by ligation of a first tag. The first tag comprises an adaptor that is ligated to the input molecule. The first tag also comprises a binding partner (DIG) of a first binding pair (DIG:anti-DIG antibody).20230183-02 / 027644.8509

[0015] FIG.1B illustrates a target enrichment procedure in which an input nucleic acid having an attached first tag is hybridized with bridge probes and the input nucleic acid:bridge probe complex is hybridized with biotinylated universal (or anchor) probes.

[0016] FIG.1C illustrates the attachment of a second tag to the first tag on the adaptor- ligated nucleic acid to form an input construct.

[0017] FIG.1D illustrates immobilization of the nucleic acid complex comprising the second tag (biotin) onto a flowcell.

[0018] The present teachings are best understood from the following detailed description when read with the accompanying drawing figures. The features are not necessarily drawn to scale. Wherever practical, like reference numerals refer to like features. DETAILED DESCRIPTION

[0019] The present disclosure provides methods to indirectly attach a tag such as biotin to a nucleic acid and flexibly tag input molecules with biotin. The indirect tagging approach provides one or more advantages in its various implementations. For instance, the present methods can enable compatibility of tagging for target enrichment and tagging for immobilization. In some embodiments, the present disclosure enables the addition of a biotin tag to already-enriched nucleic acid material. In some embodiments, the present disclosure can also avoid the interference with target enrichment that would be caused if a biotin tag was attached to the input nucleic acid prior to target enrichment. In some embodiments, the present disclosure is compatible with a PCR-free workflow (e.g., target enrichment without pre-amplification).

[0020] Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such20230183-02 / 027644.8509 can, of course, vary. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0021] As one aspect, the present disclosure provides methods of preparing a nucleic acid for sequencing. The methods comprise attaching a first tag to an input nucleic acid molecule, wherein the first tag has a binding partner of a first binding pair. The methods also comprise attaching a second tag to the first tag to produce an input nucleic acid complex, wherein the second tag comprises binding partner of a second binding pair. The binding partners of the first binding pair do not bind with the binding partners of the second binding pair. In other words, the first tag and the second tag having binding partners from two different binding pairs.

[0022] Input nucleic acid molecules can be obtained in any suitable manner. For instance, the input molecules can be cfDNA or fragmented genomic DNA. The input nucleic acid can be DNA. The DNA can be genomic DNA (gDNA), mitochondrial DNA, viral DNA, cDNA, cfDNA, or synthetic DNA. The DNA can be double-stranded DNA, single-stranded DNA, fragmented DNA, or damaged DNA.

[0023] The input nucleic acid can be naturally occurring or synthetic. The input nucleic acid can have modified heterocyclic bases. The modification can be methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses, or other heterocycles. The input nucleic acid can have modified sugar moieties. The modified sugar moieties can include peptide nucleic acid. The input nucleic acid can comprise peptide nucleic acid. The input nucleic acid can comprise threose nucleic acid. The input nucleic acid can comprise locked nucleic acid. The input nucleic acid can comprise hexitol nucleic acid. The input nucleic acid can be flexible nucleic acid. The input nucleic acid can comprise glycerol nucleic acid.20230183-02 / 027644.8509

[0024] The input nucleic acid can be captured and enriched from low-input (e.g., 1 ng of nucleic acid materials) samples such as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), a single cell, or 10 or fewer cells. Examples of a single cell or other cells for which analysis may be desired include a neuron, a glial cell, a germ cell, a gamete, an embryonic stem cell, a pluripotent stem cell (including an induced pluripotent stem cell), an adult stem cell, a cell of the hematopoietic lineage, a differentiated somatic cell, a microbial cell, a cancer cell (including, for example a cancer stem cell), and a disease cell. In some embodiments, the input nucleic acid is captured from 10 or fewer cells (such as 1-10 cells, 2-10 cells, 5-10 cells, 1-2 cells, or 2-5 cells). The low-input samples can have 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, or more of nucleic acid materials. The low-input samples can have less than 10 ng, 9 ng, 8 ng, 7 ng, 6 ng, 5 ng, 4 ng, 3 ng, 2 ng, 1 ng, or less of nucleic acid materials. The low-input samples can have from 200 pg to 10 ng of nucleic acid materials. The low-input samples can have less than 10 ng of nucleic acid materials. The low-input sample can less than 10 ng, 5 ng, 1 ng, 100 pg, 50 pg, 25 pg, or less of the nucleic acid materials. In some cases, the input samples can have 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, or more of nucleic acid molecule. The input samples can have less than 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 1 ng, or less of nucleic acid materials.

[0025] The capture and enrichment can be done by target probe hybridization. The target probe can be a capture probe, bridge probe, and / or anchor probe. The target probe can comprise one or more binding moieties. The binding moiety can be a biotin. The binding moieties can be attached to a support. The support can be a bead. The bead can be a streptavidin coated bead.

[0026] The input nucleic acid can be damaged. The damaged nucleic acid can comprise altered or missing bases, and / or modified backbone. The input nucleic acid can be damaged by20230183-02 / 027644.8509 oxidation, radiation, or random mutation. The input nucleic acid can be damaged by bisulfite treatment.

[0027] Damaged dsDNA (with a nick) or ssDNA can be used as input nucleic acid for a library construction. For the damaged dsDNA, the dsDNA can be denatured so at least one undamaged strand can be used as an input nucleic acid. The input nucleic acid can then be hybridized and attached to a capture probe and amplified using various primers.

[0028] The input nucleic acid can be derived from cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). The cfDNA can be fetal or tumor in source. The input nucleic acid can be derived from liquid biopsy, solid biopsy, or fixed tissue of a subject. The input nucleic acid can be cDNA and can be generated by reverse transcription. The input nucleic acid can be derived from fluid samples, including not limited to plasma, serum, sputum, saliva, urine, or sweat. The input nucleic acid can be derived from liver, esophagus, kidney, heart, lung, spleen, bladder, colon, or brain.

[0029] The input nucleic acid can be derived from male or female subject. The subject can be an infant, a teenager, a young adult or an elderly person. The input nucleic acid can originate from human, rat, mouse, other animal, or specific plants, bacteria, algae, viruses, and the like. The input nucleic acid can originate from primates, such as chimpanzees or gorillas. Other animals include a rhesus macaque. The input nucleic acid can be from a mixture of genomes of different species including host-pathogen, bacterial populations, etc. In some embodiments, the input nucleic acid can be cDNA made from RNA expressed from genomes of one or more species.

[0030] The input nucleic acid can comprise a target sequence. The target sequence can be an exon, an intron, or a promoter. The target sequence can be previously known, partially known20230183-02 / 027644.8509 previously, or previously unknown. The target sequence can comprise a chromosome, chromosome arm, or a gene. The gene can be gene associated with a condition, e.g., cancer. Attaching A First Tag To A Nucleic Acid

[0031] In some embodiments, the first tag is attached to an adaptor, and the present method comprise attaching one or more adaptors to an input nucleic acid to form a nucleic acid construct. An adaptor can be attached to an input before or after amplification, and in some embodiments the adaptor is attached before amplification. The adaptor can be attached by any suitable technique, such as by ligation, use of a transposase, hybridization, and / or primer extension. In some embodiments, the input nucleic acid is ligated with an adaptor at one or both ends. In a ligation reaction, a covalent bond or linkage is formed between the termini of two or more nucleic acid molecules (such as an input and an adaptor). The nature of the bond or linkage may vary, and the ligation may be carried out enzymatically or chemically. Ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon of a terminal nucleotide of one polynucleotide or oligonucleotide with 3' carbon of another polynucleotide or oligonucleotide. In some embodiments, the adaptor is a Y-adaptor. Other examples of adaptors including linear adaptors, circular adaptors, and bubble adaptors.

[0032] In some embodiments, the first tag comprises a binding partner selected from the group consisting of digoxigenin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris- Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine (e.g., a primary amine), an aldehyde, an alkyne or an azide or other groups reacting by click chemistry, and mixtures thereof. Accordingly, examples of first binding pairs include DIG:anti-DIG antibody, BrdU:anti-BrdU20230183-02 / 027644.8509 antibody, DNP:anti-DNP antibody, Ni-NTA:poly-Histidine (His-tag), Tyramide:tyrosine residues, Thiol:Thiol (disulfide bonds), Amine:Aldehyde conjugation, alkyne:azide, or other click chemistry reactants. Examples of tyramines include Tyramine, N-Methyltyramine, N,N- Dimethyltyramine, and N,N,N-Trimethyltyramine. Examples of alkynes and azides binding via click chemistry include copper-catalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1, 3-dipolar cycloaddition) and strain-promoted azide alkyne cycloaddition (SPAAC).

[0033] Digoxigenin is advantageous as the binding partner included in the first tag, due to its very low non-specific binding and the availability of high-affinity anti-digoxigenin antibodies. Examples of anti-DIG antibodies include Perkin Elmer’s Anti-Digoxigenin biotin conjugate. Binding A Second Tag To Produce A Nucleic Acid Construct

[0034] In some embodiments, the second tag comprises a reciprocal binding partner of the first tag. For example, the first tag can comprise an antigen or hapten and the second tag can comprise an antibody that selectively binds that antigen or hapten. The attachment of the second tag to the nucleic acid constructs can facilitate enrichment or isolation of an input. The binding partners of the second binding pair do not bind with the binding partners of the first binding pair. A target nucleic acid can be attached to an adaptor comprising a binding partner, or a target nucleic acid can be amplified using one or more primers comprising a binding partner. In some embodiments, the present methods comprise forming a complex between reciprocal binding partners, such as a biotinylated target nucleic acid and solid-supported avidin or streptavidin.

[0035] In some embodiments, the binding partner of the second binding pair comprises a biotin moiety such as biotin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris-20230183-02 / 027644.8509 Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine (e.g., a primary amine), an aldehyde, an alkyne or an azide or other groups reacting by click chemistry, and mixtures thereof. The reciprocal binding partner of the second binding pair can comprise an avidin moiety such as avidin or streptavidin(Avidin, Streptavidin, NeutrAvidin and CaptAvidin, etc.), or antibodies that specifically bind the binding partner, His-tags, tyrosine residues, thiols, amines, aldehydes, alkynes, azides, etc. Accordingly, examples of second binding pairs include biotin:streptavidin, BrdU:anti-BrdU antibody, DNP:anti-DNP antibody, Ni-NTA:His-tag, Tyramide:tyrosine residues, Thiol:Thiol (disulfide bonds), Amine:Aldehyde conjugation, alkyne:azide, or other click chemistry reactants. The proteins avidin and streptavidin form exceptionally tight complexes with biotin moieties. In general, when a biotin moiety is coupled to a second molecule through its carboxyl side chain, the resulting conjugate is still tightly bound by avidin or streptavidin. The second molecule is said to be "biotinylated" when such conjugates are prepared.

[0036] Emerging nucleic-acid sequencing platforms can enable direct detection and analysis of nucleic acid molecules without the need for traditional sample preparation and amplification steps. In one example of such emerging platforms, input nucleic acid molecules can be tagged with a biotin molecule through a simple enzymatic addition step. The biotinylated nucleic acid molecule can then be bound to a flowcell containing avidin or streptavidin moieties. Subsequent analysis can then be performed, e.g. using fluorescently labelled probes.

[0037] If a biotin moiety is tagged to input nucleic acid molecules, as is done in some sample preparation procedures, it would interfere with certain probe-based capture and enrichment protocols that uses biotin-tagged oligonucleotide probes. However, addition of a20230183-02 / 027644.8509 biotin tag to nucleic acids after target enrichment is challenging due to the single stranded nature of the enriched nucleic acid molecules. In addition, residual components from the target enrichment protocol could also be processed, impacting subsequent analysis.

[0038] The present disclosure provides an indirect approach for nucleic acid sequences tagging that is compatible with existing target enrichment procedures and supports subsequent biotin-tag addition, enabling compatibility with direct nucleic acid sequence analysis platforms.

[0039] FIG.1A illustrates the tagging of an input nucleic acid molecule by ligation of a first tag. The first tag comprises an adaptor that is ligated to the input molecule, thereby forming a nucleic acid construct. More particularly, FIG.1A shows an input nucleic acid construct 102 in which an input nucleic acid 101 is ligated at its ends to first tags 103. First tag 103 comprising an adaptor 104 and a binding partner 105 of a first binding pair. For example, the first tag 103 can comprise a Y-adaptor as adaptor 104 and DIG as the binding partner 105 of a first binding pair (DIG:anti-DIG antibody). In FIG.1A, both of the first tags 103 ligated to the input nucleic acid 101 are the same, but in some embodiments, the first tags 103 of the input nucleic acid construct 102 can have different adaptors 104 and / or different binding partners 105.

[0040] In the present methods, any desired adaptor may be attached to an input nucleic acid molecule to form an input nucleic acid construct. Generally an adaptor is attached to at least one strand of a double-stranded DNA molecule, and usually an adaptor can be a molecule that is at least partially double-stranded. An adaptor may be 40 to 150 bases in length, e.g., 50 to 120 bases. An adaptor can be joined to a 5' end and / or a 3' end of a nucleic acid molecule. A Y- adaptor is an adaptor that contains a double-stranded region and a single-stranded region in which the opposing sequences are not complementary. The end of the double-stranded region may be or can be joined to target molecules such as double-stranded fragments of genomic DNA,20230183-02 / 027644.8509 e.g., by via a transposase-catalyzed reaction. Each strand of a double-stranded DNA molecule that has been joined to a Y adaptor is asymmetrically tagged in that it has the sequence of one strand of the Y-adaptor at one end and the other strand of the Y-adaptor at the other end. Amplification of nucleic acid molecules that have been joined to Y-adaptors at both ends results in an asymmetrically tagged nucleic acid, i.e., a nucleic acid that has a 5' end containing one tag sequence and a 3' end that has another tag sequence.

[0041] When a first tag comprises an adaptor, the binding partner of the first binding pair can be attached to the adaptor in any suitable manner, such as by conjugation.

[0042] Input nucleic acid molecules can be prepared or treated in other ways that cooperated with the present methods. For instance, the input nucleic acid molecules are prepared by a procedure that includes traditional end-repair, A-tailing, and ligation with Y-adaptors 104 conjugated to digoxigenin (DIG) as the binding partner 105. In some embodiments, the adaptor 104 also comprises a sample specific index and / or molecular barcode to enable strand-pairing of sequence data or to facilitate other analysis.

[0043] FIG.1B illustrates how an input nucleic acid construct 102 can be further processed in a target enrichment procedure. In FIG.1B, the input nucleic acid construct 102 has been denatured to form a single-stranded nucleic acid construct 102’, which is hybridized with one or more bridge probes 110, 111. Bridge probes 110, 111 are hybridized with universal probe 112 (also referred to as an anchor probe) conjugated with a binding partner 113 (for example, biotin) of a second binding pair (for example, the binding pair of biotin:streptavidin). In the illustrated embodiment, the hybridizations proceed at the same time; in other embodiments, the hybridizations can be sequential. The binding partners of the first binding pair (DIG:anti-DIG antibody) do not bind with the binding partners of the second binding pair (biotin:streptavidin).20230183-02 / 027644.8509 In FIG.1B, target enrichment of the input nucleic acid construct 102 is performed by binding the binding partner 113 (biotin) to its reciprocal binding partner 114 (streptavidin) which is on a solid support 115 (e.g., a magnetic bead).

[0044] Although the present methods can employ a capture probe that direct hybridizes with a target sequence of an input nucleic acid, the use of bridge probes and anchor probes offer several advantages. Bridge probes can be used to hybridize an input nucleic acid molecule and can further allow indirect association between an anchor probe and the input nucleic acid. The bridge probe can comprise target specific region (TSR) that hybridizes to target sequence. The bridge probe can comprise anchor-probe-landing sequence (ALS) that hybridizes to bridge-binding- sequence of anchor probe. The bridge probe can comprise a linker connecting TSR and ALS. The TSR can be located in the 3’-portion of the bridge probe. The TSR can be located in the 5’- portion of the bridge probe.

[0045] The bridge probe can comprise DNA. The bridge probe can comprise of RNA. The bridge probe can comprise of uracil and methylated cytosine. The bridge probe might not comprise of uracil. The bridge probe can comprise about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 120 nucleotides, about 100 nucleotides, about 90 nucleotides, about 80, about 70 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, or about 10 nucleotides. The bridge probe can comprise one or more molecular barcodes. The bridge probe can comprise one or more binding moieties. The binding moiety can be a biotin. The binding moieties can be attached to a support. The support can be a bead. The bead can be a streptavidin bead.

[0046] Multiple bridge probes can be used to anneal to multiple target sequences in a sample. The bridge probes can be designed to have similar melting temperatures. The melting20230183-02 / 027644.8509 temperatures for a set of bridge probes can be within about 15oC, within about 10°C, within about 5oC, or within about 2oC. The melting temperature for one or more bridge probes can be about 75oC, about 70oC, about 65oC, about 60oC, about 55oC, about 50oC, about 45oC, or about 40oC. The melting temperature for the bridge probe can be about 40oC to about 75oC, about 45oC to about 70oC, 45oC to about 60oC, or about 52oC to about 58oC.

[0047] Use of an anchor probe along with one or more bridge probe around a particular bridge probe can help to stabilize the hybridization of the particular bridge probe to its target sequence through synergistic effect. A hybridization temperature to form the multiple bridge probe assembly can be higher than the melting temperature of a single bridge probe. The higher temperature can result in a better capture specificity by reducing nonspecific hybridization that can occur at lower temperature. The hybridization temperature can be about 5oC, about 10oC, about 15oC, or about 20oC higher than the melting temperature of individual bridge probe. The hybridization temperature can be about 5oC to about 20oC higher than the melting temperature of a bridge probe, or about 5oC to about 20oC higher than an average melting temperature of a plurality of bridge probes.

[0048] The hybridization temperature for multiple bridge probes can be about 75oC, about 70oC, about 65oC, about 60oC, about 55oC, or about 50oC. The hybridization temperature for multiple bridge probes can be about 50oC to about 75oC, 55oC to about 75oC, 60oC to about 75oC, or 65oC to about 75oC.

[0049] The bridge probe can further comprise a label. The label can be fluorescent. The fluorescent label can be organic fluorescent dye, metal chelate, carbon nanotube, quantum dot, gold particle, or fluorescent mineral. The label can be radioactive. The label can be biotin. The20230183-02 / 027644.8509 bridge probe can bind to labeled nucleic acid binder molecule. The nucleic acid binder molecule can be antibody, antibiotic, histone, antibody, or nuclease.

[0050] The bridge probe can comprise a linker. In some embodiments, the linker comprises about 30 nucleotides, about 25 nucleotides, about 20 nucleotides, about 15 nucleotides, about 10 nucleotides, or about 5 nucleotides; any of those numbers can be combined to form a range for the number of nucleotides in a linker. The linker can comprise non-nucleic acid polymers (e.g., string of carbons). The linker non-nucleotide polymer can comprise about 30 units, about 25 units, about 20 units, about 15 units, about 10 units, or about 5 units; any of those numbers can be combined to form a range for the number of units in a linker.

[0051] The bridge probe can be blocked at the 3’ and / or 5’ end. The bridge probe can lack a 5’ phosphate. The bridge probe can lack a 3’ OH. The bridge probe can comprise a 3’ddC, 3’inverted dT, 3’C3 spacer, 3’ amino, or 3’ phosphorylation.

[0052] The anchor probe or universal anchor probe can comprise one or more bridge-binding- sequences (BBS) that hybridize to anchor-probe-landing sequence of the one or more bridge probes.

[0053] The anchor probe can comprise spacers in between the BBSs. The presence of the one or more spacers can improve the efficiency of the hybridization capture and increase the specificity of the capture.

[0054] The anchor probe can comprise a molecular barcode (MB). The anchor probe can comprise a BBS to which the one or more bridge probes can hybridize to. The anchor probe can comprise from 1 to 100 BBSs. The anchor probe can comprise an index for distinguishing samples. The molecular barcode or index can be 5’ of the adaptor sequence and 5’ of the BBS.20230183-02 / 027644.8509

[0055] The anchor probe can comprise about 400 nucleotides, about 200 nucleotides, about 120 nucleotides, about 100 nucleotides, about 90 nucleotides, about 80 nucleotides, about 70 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, or about 10 nucleotides. The anchor probe can be about 20 to about 70 nucleotides.

[0056] The melting temperature of anchor probe to the bridge probe can be about 65oC, about 60oC, about 55oC, about 50oC, about 45oC, or about 45oC to about 70oC.

[0057] The anchor probe can comprise a label. The label can be fluorescent. The fluorescent label can be an organic fluorescent dye, metal chelate, carbon nanotube, quantum dot, gold particle, or fluorescent mineral. The label can be radioactive. The label can be biotin. The anchor probe can bind to labeled nucleic acid binder molecule. The nucleic acid binder molecule can be antibody, antibiotic, histone, antibody, or nuclease.

[0058] FIG.1C illustrates the attachment of a second tag to the first tag 103 of the single- stranded nucleic acid construct 102’ to form an input nucleic acid complex 120. In FIG.1C, the single-stranded nucleic acid construct 102’ has been released from bridge probes 110, 111. A second tag 116 comprises an anti-DIG antibody 117 conjugated to biotin (118) is attached to the binding partner 105 (DIG) of the first tag 103. Again, the binding partners of the first binding pair (DIG:anti-DIG antibody) do not bind with the binding partners of the second binding pair (biotin:streptavidin). In other words, the first tag 103 and the second tag 116 have binding partners from two different binding pairs.

[0059] FIGs.1A to 1C demonstrate how the present methods can be used for target enrichment on a sample containing the input nucleic acid construct tagged with a first tag by binding to a reciprocal binding partner of the first binding pair. Preparation of nucleic acids for sequencing- by-synthesis or other analysis often employs target enrichment, and one or more target20230183-02 / 027644.8509 enrichment procedures can be included in the present methods. By enriching for one or more desired targets, sequencing or other analysis can be more focused with reduced effort and expense and / or with high coverage depth. Examples of target enrichment procedures include hybridization-based capture protocols such as SureSelect Hybrid Capture from Agilent and TruSeq Capture from Illumina. Other examples include PCR-based protocols such as HaloPlex from Agilent; AmpliSeq from ThermoFisher; TruSeq Amplicon from Illumina; and emulsion / digital PCR from Raindance.

[0060] In some embodiments, the present methods also comprise capture of input nucleic acid constructs comprising target sequences. The present methods allow efficient capture and enrichment of input nucleic acid constructs having a first tag attached thereto. Target enrichment can be performed after library construction by attachment of a first tag comprising an adaptor to input nucleic acid molecules. Target enrichment can be performed after attaching an adaptor to a 3ʹ end of an input nucleic acid molecule. The present methods can be used to handle low input samples such cell-free DNA (cfDNA).

[0061] In some embodiments, the present methods comprise target enrichment by indirect hybridization of the input nucleic acid construct with an anchor probe through hybridization of one or more bridge probes to the input nucleic acid construct. The one or more bridge probes can be designed to hybridize to particular target sequences in the input nucleic acid. An anchor probe in turn can be designed to hybridize to the one or more bridge probes, thereby creating an assembly of three or more hybridized nucleic acid molecules. The multi-structure hybridization assembly can act synergistic to provide more stability to the assembly.

[0062] Enrichment of an input nucleic acid containing a target sequence can be facilitated by interaction of the input nucleic acid and two or more probes that form a hybridization assembly.20230183-02 / 027644.8509 The multi-complex assembly can stabilize the hybridization interaction between the input and the enrichment such as bridge probes. A bridge probe can comprise a target specific region that hybridizes to a target region of the input nucleic acid and anchor-probe-landing sequence (ALS) that hybridizes to bridge-binding-sequence (BBS) of an anchor probe. The hybridizations between the input nucleic acid and the bridge probe and between the bridge probe and the anchor probe can form a multi-complex assembly.

[0063] In some embodiments, the present methods comprise hybridizing a first target specific region of a first bridge probe to a first target sequence of a molecule with a sequence corresponding to the genome region, wherein a first anchor-probe-landing sequence of the first bridge probe is bound to a first bridge-binding-sequence of an anchor probe; and hybridizing a second target specific region of a second bridge probe to a second target sequence of the molecule with a sequence corresponding to the genome region, wherein a second anchor-probe- landing sequence of the second bridge probe is bound to a second bridge-binding-sequence of the anchor probe. As described herein the anchor probe may comprise a binding moiety. The method generally comprises attaching adaptors to the 5’ end or the 3’ ends of nucleic acid molecules of the plurality of nucleic acid molecules, thereby generating a library of nucleic acid molecules comprising adaptors.

[0064] More than two bridge probes per input nucleic acid molecule can be used in the methods disclosed herein. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, or more bridge probes can be used to bridge the input nucleic acid and the anchor probe. The target enrichment can further comprise hybridizing a second target specific region of a second bridge probe to a second target sequence of the input nucleic acid molecule, wherein a second anchor-probe- landing sequence of the second bridge probe can be bound to a second bridge-binding-sequence20230183-02 / 027644.8509 of the anchor probe. In some cases, the target enrichment can be conducted after attachment of adaptors or other first tags to the input nucleic acid molecules.

[0065] The bridge probes can further comprise linkers that connect the target specific region and the anchor-probe-landing sequence. The adaptor anchor can comprise one or more spacers in between the bridge-binding-sequences. The presence of the one or more spacers can improve the efficiency of the hybridization capture and increase the specificity of the capture.

[0066] The input nucleic acid can be captured and enriched from low-input samples such as cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA). The capture and enrichment can be done by the indirect association with anchor probe through hybridization with bridge probe. The bridge probe and / or anchor probe can comprise one or more binding moieties. The binding moiety can be a biotin. The binding moieties can be attached to a support. The support can be a bead. The bead can be a streptavidin bead.

[0067] The present methods of capture and enrichment can further include solid phase extraction of the input nucleic acid. The bridge probe or anchor probe can be bound to a solid support. The bridge probe, or anchor probe can comprise a label. The disclosed methods can further comprise capturing to the bridge probe, the anchor probe, or the hybridization complex comprising input nucleic acid molecule, bridge probe, and anchor probe by the label. The label can be biotin. The label can be a nucleic acid sequence, such as poly A or Poly T, or specific sequence. The nucleic acid sequence can be about 5 to 30 bases in length. The nucleic acid sequence can comprise DNA and / or RNA. The label can be at the 3’ end of the bridge probe, or anchor probe. The label can be a peptide, or modified nucleic acid that can be recognized by antibody such as 5-Bromouridine, and biotin. The label can be conjugated to the bridge probe, or anchor probe by reactions such as “click” chemistry. “Click” chemistry can allow for the20230183-02 / 027644.8509 conjugation of a reporter molecule like fluorescent dye to a biomolecule like DNA. Click Chemistry can be a reaction between and azide and alkyne that can yield a covalent product (e.g., 1,5-disubstituted 1,2,3-triazole). Copper can serve as a catalyst.

[0068] The label can be captured on a solid support. The solid support can be magnetic. The solid support can comprise a bead, flowcell, glass, plate, device comprising one or more microfluidic channels, or a column. The solid support can be a magnetic bead.

[0069] The solid support (e.g., bead) can comprise (e.g., by coated with) one or more capture moieties that can bind the label. The capture moiety can be streptavidin, and the streptavidin can bind biotin. The capture moiety can be an antibody. The antibody can bind the label. The capture moiety can be a nucleic acid, e.g., a nucleic acid comprising DNA and / or RNA. The nucleic acid capture moiety can bind a sequence on, e.g., an anchor probe or bridge probe. In some cases, an anti-RNA / DNA hybrid antibody bound to a solid surface can be used as a capture moiety.

[0070] The label and the capture moiety can bind through one or more covalent or non-covalent bonds. Following capture of the bridge probe, anchor probe, or the hybridization complex on the solid support, the solid support can be washed to remove, e.g., unbound template from the sample. In some cases, no wash step is performed. The wash can be stringent or gentle. The capture probe or anchor probe that are hybridized to an input nucleic acid molecule can be eluted, e.g., by adding free biotin to the sample when the label is biotin and the capture moiety is streptavidin.

[0071] Cleanup can be performed using streptavidin beads after the input nucleic acid, bridge probe, and anchor probe hybridization, wherein the 3’ end of the anchor probe is biotinylated. The input nucleic acid complex hybridized to the bridge probes (and indirectly with the anchor20230183-02 / 027644.8509 probe) is bound to the bead. The input nucleic acid that has not hybridized to the bridge probe can be washed away. The 5’ end or the 3’ end of a first and or second bridge probe can be biotinylated. In this manner, streptavidin beads can be used to remove and separate the unhybridized input nucleic acid from input nucleic acid having the target sequence.

[0072] FIG.1D illustrates how input nucleic acid complex 120 can be immobilized on a substrate 122, such as a flowcell of a sequencing system. The input nucleic acid complex 120 is loaded onto a flowcell or contacted with another substrate 122 which has a reciprocal binding partner 123 (streptavidin) of a second binding pair disposed on its surface. The binding partner 118 (biotin) acts as a handle to the complex which includes a strand from the input nucleic acid 101 on a flow-cell or microarray surface coated with its reciprocal binding partner 123 (streptavidin).

[0073] In some embodiments, a first tag is attached to an input nucleic acid molecule without an adaptor. For instance, a nucleic acid can be direct tagged using digoxigenin 3’ end oligonucleotide labeling kits which might improve tagging efficiency over traditional adaptor ligation. Such labeling kits are commercially available. However, such an approach potentially limits inclusion of identifiers (sample index / UMI) to support pairing of sequences obtained from original DNA strands.

[0074] As illustrated in FIG.1D, in some embodiments, the nucleic acid complexes comprising a second tag are immobilized on a solid support such as a flowcell or a bead. Immobilization of the nucleic acid can facilitate washing of the nucleic acids to remove any undesired species (e.g., deoxynucleotides). In some embodiments, a nucleic acid construct comprises one or more adaptors which attach to the solid support, rendering the nucleic acid immobilized on the support. In some embodiments, the nucleic acid construct is immobilized on the surface of a20230183-02 / 027644.8509 flowcell or a glass slide. In some embodiments, the nucleic acid construct is immobilized on a well or magnetic bead. In some embodiments, the solid support may be coated with a polymer attached to a functional group or moiety. In some embodiments, the solid support may carry functional groups such as amino, hydroxyl, or carboxyl groups, or other moieties such as avidin or streptavidin for attachment of adaptors.

[0075] As another aspect, a method is provided for tagging input nucleic acids for further analysis after amplification. This method enables selection and later isolation of original input nucleic acid molecules (which may include base modifications) as part of a sample preparation workflow for nucleic acid sequencing. For example, DNA molecules could be tagged with DIG- conjugated adaptors. Following subsequent enrichment, the original input nucleic acid with DIG-conjugated adaptors could be separated from amplicons which do not have a DIG tag. The original input nucleic acid molecules or fraction could then be analyzed directly (e.g. on a nucleic acid sequencing platform capable of detecting base modifications) or processed through target enrichment / base modification conversion (e.g. bisulfite conversion). The amplicons or amplified fraction could then be processed through a target enrichment workflow and analyzed separately.

[0076] In some embodiments, the present method comprises amplifying a nucleic acid, before and / or after it is attached to an adaptor. In some embodiments, an adaptor is located at a 5'-end of a target sequence in the input nucleic acid, and the adaptor provides a priming site for amplification of the target sequence. The nucleic acid construct with a ligated adaptor can be amplified using a first amplification primer and a second amplification primer. In some embodiments, the first amplification primer has sequence specificity for a target sequence in the20230183-02 / 027644.8509 nucleic acid, and is capable of hybridizing to a portion of the target sequence (a nucleic acid of interest). The second amplification primer is capable of hybridizing to a priming site of the adaptor or to a target-specific priming site of the input nucleic acid. During the amplification step, the first amplification primer hybridizes to the target sequence and the second primer hybridizes to the sequence priming site on the adaptor. In some embodiments, the first amplification primer hybridizes at the 5'-end of the nucleic acid construct. The primers should be sufficiently large to provide adequate hybridization with the target sequence or other primer binding site.

[0077] An input nucleic acid may be amplified using any suitable method. In some embodiments, the input nucleic acid is amplified using polymerase chain reaction (PCR). In general, PCR comprises denaturation of polynucleotide strands (e.g., DNA melting), annealing of primers to the denatured polynucleotide strand, and extension of primers with a polymerase to synthesize the complementary polynucleotide. The process generally requires a DNA polymerase, forward and reverse primers, deoxynucleoside triphosphates, bivalent cations, and a buffer solution. In some embodiments, the input nucleic acid is amplified by linear amplification. In some embodiments, the input nucleic acid is amplified using Emulsion PCR, Bridge-PCR, or Rolling Circle amplification. The amplicons of the input nucleic acid may be analyzed to determine the order of base pairs using a suitable sequencing method. Sequencing The Nucleic Acid

[0078] The present methods may be used as part of a high-throughput sequencing method such as a Next Generation Sequencing (NGS) method. In some embodiments, a high-throughput20230183-02 / 027644.8509 sequencing method comprises three steps: library preparation, immobilization, and sequencing. A nucleic acid sample generally is subjected to fragmentation, and adaptors are attached to one or both ends of the fragments to form a sequencing library. The sequencing library molecules are immobilized on a solid support, and sequencing reactions are performed to identify the nucleic acid sequence. The high-throughput sequencing method may employ Emulsion PCR, Bridge-PCR, or Rolling Circle amplification to provide colonies or copies of the original nucleic acid construct.

[0079] In some embodiments, the input nucleic acid complex is sequenced without amplification. The input nucleic acid complex is attached or immobilized to a solid substrate which has a reciprocal binding partner for the second tag of the complex. For example, when the input nucleic acid complex is biotinylated (such as at its 5′ end or 3′ end), it can be immobilized on a streptavidin-coated surface of a flowcell or bead. The biotinylation can be achieved at the 3′ end by tailing with biotinylated nucleotide using a terminal transferase. After immobilization, the input nucleic acid can be sequenced by any suitable technique such as sequencing-by-synthesis or sequencing-by-hybridization. In some embodiments, the immobilized the input nucleic acid is sequences using a single-molecule sequencing platform, such as the methods discussed in Wöhrstein et al. US Patent 10,851,411.

[0080] In some embodiments, the present methods comprise aligning sequence reads of the input nucleic acids. The sequence reads may be processed and grouped in any suitable way. In some embodiments, the sequence reads may be initially grouped by the fragment sequence and / or the identifier(s). In some implementations, initial processing of the sequence reads may include identification of molecular barcodes (including sample identifier sequences or sub- sample identifier sequences), and / or trimming reads to remove low quality or adaptor sequences.20230183-02 / 027644.8509 In addition, quality assessment metrics can be run to ensure that the dataset is of an acceptable quality. With sequencing platforms that require clonal amplification of input nucleic acid molecules, there is a concern a potential sequence variation is a PCR or amplification error rather than a true variation. An advantage from identifying and sequencing an input nucleic acid molecule without amplification is that it avoids such errors.

[0081] The amplified products generated using methods described herein can be further analyzed using various methods including southern blotting, polymerase chain reaction (PCR) (e.g., real-time PCR (RT-PCR), digital PCR (dPCR), droplet digital PCR (ddPCR), quantitative PCR (Q-PCR), nCounter analysis (Nanostring technology), gel electrophoresis, DNA microarray, mass spectrometry (e.g., tandem mass spectrometry, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), chain termination sequencing (Sanger sequencing), or next generation sequencing.

[0082] The next generation sequencing can comprise 454 sequencing (ROCHE) (using pyrosequencing), sequencing using reversible terminator dyes (ILLUMINA sequencing), semiconductor sequencing (THERMOFISHER ION TORRENT), single molecule real time (SMRT) sequencing (PACIFIC BIOSCIENCES), nanopore sequencing (e.g., using technology from OXFORD NANOPORE or GENIA), microdroplet single molecule sequencing using pyrophosphorolyis (BASE4), single molecule electronic detection sequencing, e.g., measuring tunnel current through nanoelectrodes as nucleic acid (DNA / RNA) passes through nanogaps and calculating the current difference (QUANTUM SEQUENCING from QUANTUM BIOSYSTEMS), GenapSys Gene Electornic Nano-Integrated Ultra-Sensitive (GENIUS) technology (GENAPYS), GENEREADER from QIAGEN, sequencing using sequential hybridization and ligation of partially random oligonucleotides with a central determined base20230183-02 / 027644.8509 (or pair of bases) identified by a specific fluorophore (SOLiD sequencing). The sequencing can be paired-end sequencing.

[0083] The performance of a panel or method for capturing targets or preparing a NGS library may be defined by a number of different metrics describing efficiency, accuracy, and precision. Such metrics can be obtained by sequencing the captured nucleic acid molecules or amplicons thereof. For example, coverage percentage region-wide (0.2X or 0.5X), coverage percentage base-wide, target coverage, depth of coverage, fold enrichment, percent mapped, percent on- target, AT or GC dropout rate, fold 80 base penalty, percent zero coverage targets, PF reads, percent selected bases, percent duplication, or other variables can be used to characterize a library.

[0084] The number of target sequences from a sample that can be sequenced using methods described herein can be about 5, 10, 15, 25, 50, 100, 1000, 10,000, 100,000, or 1,000,000, or about 5 to about 100, about 100 to about 1000, about 1000 to about 10,000, about 10,000 to about 100,000, or about 100,000 to about 1,000,000.

[0085] Nucleic acid libraries generated using methods described herein can be generated from more than one sample. Each library can have a different index associated with the sample. For example, a capture probe or an anchor probe can comprise an index that can be used to identify nucleic acids as coming from the same sample (e.g., a first set of capture probes or anchor probes comprising the same first index can be used to generate a first library from a first sample from a first subject, and a second set of capture probes or anchor probes comprising the same second index can be used to generate a second library from a second sample from a second subject, the first and second library can be pooled, sequenced, and an index can be used to discern from which sample a sequenced nucleic acid was derived). Amplified products generated using the20230183-02 / 027644.8509 methods described herein can be used to generate libraries from at least 2, 5, 10, 25, 50, 100, 1000, or 10,000 samples, each library with a different index, and the libraries can be pooled and sequenced, e.g., using a next generation sequencing technology.

[0086] The sequencing can generate at least 100, 1000, 5000, 10,000, 100,000, 1,000,000, or 10,000,000 sequence reads. The sequencing can generate between about 100 sequence reads to about 1000 sequence reads, between about 1000 sequence reads to about 10,000 sequence reads, between about 10,000 sequence reads to about 100,000 sequence reads, between about 100,000 sequence reads and about 1,000,000 sequence reads, or between about 1,000,000 sequence reads and about 10,000,000 sequence reads.

[0087] The depth of sequencing can be about 1x, 5x, 10x, 50x, 100x, 1000x, or 10,000x. The depth of sequencing can be between about 1x and about 10x, between about 10x and about 100x, between about 100x and about 1000x, or between about 1000x and about 10000x.

[0088] The present disclosure provides methods in which separate fractions of a nucleic acid sample can be prepared for sequencing or treated with different procedures. Because the present methods facilitate sequencing and analysis without amplification and enable the separation of different fractions, the enriched input nucleic acids may be analyzed by sequencing. In some cases, a first fraction and a second fraction are both assessed by straightforward sequencing to access genomic alteration; however the samples may be sequenced at different depths. In some cases, an analysis of a first fraction may be performed prior to performing a second target enrichment step. The results of the analysis of the first fraction sample may be used to select a second panel for the second enrichment step.

[0089] The target specific sequence or target specific region (TSR) of a capture probe or a bridge probe can be designed based on the target sequence of the input nucleic acid molecule.20230183-02 / 027644.8509 Kits for Indirect Tagging of Nucleic Acids

[0090] As another aspect of the present invention, kits are provided which comprise first and second tagging reagents for making nucleic acid constructs as described herein. A first tagging reagent comprises a first tag (according to any of the embodiments described herein) in a composition that comprises a solvent or other components. Likewise, a second tagging reagent comprises a first tag (according to any of the embodiments described herein) in a composition. The kits can comprise the first and second tagging reagents in one or more vessels, such as vials, tubes, etc.

[0091] In some embodiments, the present kits comprise one or more first tags comprising adaptor configured to be attached to an end of the input nucleic acid molecule. The first tags also comprise a binding partner of a first binding pair. The adaptors can comprise one or more identifiers such as UMI sequences. The present kits can also comprise one or more second tags comprising a reciprocal binding partner of the first binding pair.

[0092] In some embodiments, the present kits comprise one or more bridge probes that comprises a target specific region which hybridizes to a target sequence of an input nucleic acid molecule; and an anchor probe that comprises a bridge-binding-sequence which hybridizes to an anchor-probe-landing sequence of the bridge probe. In some embodiments, the kit comprises two, three or more bridge probes.

[0093] In addition to above-mentioned components, the kits may further include instructions for using the components of the kit to practice the present methods, i.e., to prepare nucleic acids for sequencing. The instructions for practicing the present methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as20230183-02 / 027644.8509 paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, portable drive, or cloud-based storage, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate. TERMINOLOGY

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0095] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.20230183-02 / 027644.8509

[0001] Numeric ranges are inclusive of the numbers defining the range. Unless otherwiseindicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0002] The present technology may employ, unless otherwise indicated, techniques anddescriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label.

[0003] As used herein, the singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. A “plurality” contains at least 2 members. In certain cases, a plurality may have at least 10, at least 100, at least 100, at least 10,000, at least 100,000, at least 106, at least 107, at least 108or at least 109or more members.

[0004] It is further noted that the claims can be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0005] As used in the specification and appended claims, and in addition to their ordinarymeanings, the terms "substantial" or "substantially" mean to within acceptable limits or degree to one having ordinary skill in the art. For example, "substantially inactive" means that one skilled in the art considers the level of activity to be negligible.

[0096] The term “target” as used herein refers to a nucleic acid of interest, or which is desired for sequencing and / or other analysis. One or more targets may be present within an input nucleic20230183-02 / 027644.8509 acid, or a construct or complex made from an input nucleic acid. A target may be single- stranded or double-stranded, and often is double-stranded DNA when attached to an adaptor to form a nucleic acid construct. Target as used herein can refer to a specific sequence or the complement thereof or to both. The term target encompasses any nucleic acid molecule of biological or synthetic origin whose sequence or other characteristic is of interest. The target sequence does not include identifiers, primer binding regions, or adaptors sequences which may be added to the input nucleic acid molecule to prepare an input nucleic acid construct for sequencing or other analysis. A target may be within a nucleic acid in vitro or in vivo within the genome of a cell, or within the cytoplasm of a cell (such as RNA), or with a biological fluid (such as blood, plasma, amniotic fluid, or other biological sample).

[0097] The term “input” refers to a nucleic acid molecule to be processed in accordance with the present methods. For example, an input nucleic acid molecule may be present in a nucleic acid sample. The input may include one or more target sequences of interest, or it may include other sequences from which a target is desired to be separated. In some embodiments, an input nucleic acid comprises one or more sequences complementary to sequences of one or more capture probes, bridge probes, or other types of probes.

[0098] The terms “amplifying” and “amplification” as used herein refer to synthesizing nucleic acid molecules that are complementary to one or both strands of an input nucleic acid. Amplifying a nucleic acid molecule may include denaturing a double-stranded input nucleic acid, annealing primers the input nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product. The terms “amplicon” or “amplification product” refer to the nucleic acid sequences which are produced from an amplifying process, including the nucleic acid molecules20230183-02 / 027644.8509 synthesized by amplifying the input nucleic acid or its complementary sequence, as well as the nucleic acid molecules synthesized from other amplicons. The denaturing, annealing and elongating steps each can be performed one or more times. Amplification generally does not change the target or input nucleic acid sequence unless errors arise during the amplification.

[0099] Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme and an appropriate buffer and / or co-factors for optimal activity of the polymerase enzyme. Reverse transcription is a linear amplification reaction that employs a specialized DNA polymerase (reverse transcriptase) to copy RNA into cDNA (complementary DNA) using deoxyribonucleoside triphosphates.

[0100] The term “adaptor” generally refers to a nucleic acid molecule that is attached to an input nucleic acid molecule to add a desired structure or function. The term “tag” also generally refers to a moiety that can add a desired structure or function, though it is contemplated that a tag may be a nucleic acid molecule, a molecule other than a nucleic acid, or a combination thereof. For example, a “tag” as used herein can comprise an adaptor conjugated to a non-nucleic acid binding partner such as DIG. As another example, a “tag” as used herein can comprise an antibody conjugated to a biotin moiety. As another example, an adaptor can be attached to an input fragment or an amplicon thereof to add a binding site for a NGS platform. In some embodiments, an adaptor refers to molecules that are at least partially double-stranded. An adaptor or a tag may be any desired length, including but not limited to 40 to 150 bases in length, e.g., 50 to 120 bases, although adaptors and tags outside of this range are envisioned.

[0101] The terms “identifier” or “barcode” refers to a sequence of nucleotides used to identify the origin of a sequence. Identifiers may comprise sample indices or sample barcodes, where the same sequence is shared for all nucleic acids from a particular source, organism, or20230183-02 / 027644.8509 sample. Sample barcodes enable the mixing of nucleic acids from different samples in one sequencing run, as the different sample barcode sequences enable the correct assignment of sequencing reads to each sample. One, two, or more sample barcodes may be used. Identifiers also comprise molecular barcodes (MBCs) or unique molecular identifier (UMI) sequences, which function to identify copies of individual input nucleic acid molecules. UMIs may comprise random nucleotides, known nucleotides, or a mixture of random and known nucleotides. UMIs enable more accurate sequencing by allowing error correction of sequences and more accurate estimation of the original number of input nucleic acids. In some embodiments, a large number of UMIs is used (e.g., 100,000, 1 million, 1 billion, or more possible sequences) such that each input nucleic acid has a unique molecular barcode. Molecular barcodes called degenerate base regions (DBR) are disclosed in US Patent 8,481,292 (Population Genetics Technologies Ltd.). The DBRs are random sequence tags that are attached to molecules that are present in the sample. DBRs and other molecular barcodes allow one to distinguish PCR errors during sample preparation from mutations and other variants that were present in the original input nucleic acid.

[0102] In other embodiments, a smaller number of molecular barcodes is used, and the beginning or ending positions (or both) of the sequence read are used together with the molecular barcode to identify copies arising from a unique input nucleic acid. Molecular barcodes may be combined with sample barcodes, on the same or different portions of the target nucleic acid. Molecular barcodes may be added to one end of a nucleic acid template (e.g., the 5’ end of the + strand, and the 3’ end of the – strand in a duplex), or to both ends of an input nucleic acid (e.g., to both the 5; and the 3’ ends of both the + and the – strands of the duplex).20230183-02 / 027644.8509

[0006] The term “sample” as used herein relates to a material or mixture of materials containingone or more nucleic acids of interest. In some embodiments, the term refers to any plant, animal or viral material containing DNA, RNA, or other nucleic acid, such as, for example, tissue or fluid isolated from a patient (including without limitation plasma, serum, amniotic fluid, cerebrospinal fluid, lymph, tears, saliva and tissue sections), from preserved tissue (such as FFPE sections) or from in vitro cell culture constituents, as well as samples from the environment. Any sample containing nucleic acid, e.g., genomic DNA from tissue culture cells or from a sample of tissue, may be employed in the present technology.

[0007] The term “nucleic acid sample” as used herein denotes a sample containing nucleicacids. The nucleic acid samples may be complex in that they contain multiple different molecules that contain sequences. Nucleic acid samples from a mammal (e.g., mouse or human) are types of complex samples. Complex samples may have more than 104, 105, 106or 107different nucleic acid molecules. Also, a complex sample may comprise only a few molecules, where the molecules collectively have more than 104, 105, 106or 107or more nucleotides. The term “complexity” generally refers the total number of different sequences in a population, such as in a population of fragments, adaptors, or adaptor-ligated fragments. For example, if a population has 4 different sequences, then that population has a complexity of 4. A population may have a complexity of at least 4, at least 8, at least 16, at least 100, at least 1,000, at least 10,000 or at least 100,000 or more, depending on the desired result.

[0103] The term “nucleotide” as used herein refers to a phosphate ester of a nucleoside, wherein the esterification site typically corresponds to the hydroxyl group attached to the C-5 position of the pentose sugar. In some cases nucleotides comprise nucleoside polyphosphates. However, the terms “added nucleotide,” “incorporated nucleotide,” “nucleotide added” and20230183-02 / 027644.8509 “nucleotide after incorporation” all refer to a nucleotide residue that is part of an oligonucleotide or polynucleotide chain.

[0008] The term “nucleotide” refers to naturally-occurring nucleotides including guanine,cytosine, adenine, thymine, uracil (G, C, A, T and U respectively), as well as modified pyrimidine and purine derivatives and other non-naturally occurring moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, are functionalized as ethers, amines, or the likes.

[0009] The term “nucleic acid” and “polynucleotide” are used interchangeably herein todescribe a nucleotide-containing polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, and may be produced naturally, chemically, enzymatically or synthetically. The term includes polymers having PNA, LNA or UNA. DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2'20230183-02 / 027644.8509 oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid”, or “UNA”, is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G’ residue and a C’ residue, where these residues correspond to non-naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively.

[0104] The terms “nucleoside”, “nucleotide”, “deoxynucleoside”, and “deoxynucleotide” are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the “nucleoside”, “nucleotide”, “deoxynucleoside”, and “deoxynucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides, nucleotides, deoxynucleosides or deoxynucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

[0105] Natural nucleotides or nucleosides are defined herein as adenine (A), thymine (T), guanine (G), and cytosine (C). It is recognized that certain modifications of these nucleotides or nucleosides occur in nature. However, modifications of A, T, G, and C that occur in nature that affect hydrogen bonded base pairing are considered to be non-naturally occurring. For example, 2-aminoadenosine is found in nature, but is not a “naturally occurring” nucleotide or nucleoside20230183-02 / 027644.8509 as that term is used herein. Other non-limiting examples of modified nucleotides or nucleosides that occur in nature that do not affect base pairing and are considered to be naturally occurring are 5-methylcytosine, 3-methyladenine, O(6)-methylguanine, and 8-oxoguanine, etc. Nucleotides include any nucleotide or nucleotide analog, whether naturally-occurring or synthetic. Exemplary nucleotides include phosphate esters of deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, and uridine. Other nucleotides include an adenine, cytosine, guanine, thymine base, a xanthine or hypoxanthine, 5-bromouracil, 2- aminopurine, deoxyinosine, or methylated cytosine, such as 5-methylcytosine, and N4- methoxydeoxycytosine. Also included are bases of polynucleotide mimetics, such as methylated nucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleic acids, locked nucleic acids and any other structural moiety that can act substantially like a nucleotide or base, for example, by exhibiting base-complementarity with one or more bases that occur in DNA or RNA and / or by being capable of base-complementary incorporation, and includes chain- terminating analogs. A nucleotide corresponds to a specific nucleotide species if they share base- complementarity with respect to at least one base.

[0106] In addition to purines and pyrimidines, modified nucleotides or analogs, as those terms are used herein, include any compound that can form a hydrogen bond with one or more naturally occurring nucleotides or with another nucleotide analog. Any compound that forms at least two hydrogen bonds with T or with a derivative of T is considered to be an analog of A or a modified A. Similarly, any compound that forms at least two hydrogen bonds with A or with a derivative of A is considered to be an analog of T or a modified T. Similarly, any compound that forms at least two hydrogen bonds with G or with a derivative of G is considered to be an analog of C or a modified C. Similarly, any compound that forms at least two hydrogen bonds with C or20230183-02 / 027644.8509 with a derivative of C is considered to be an analog of G or a modified G. It is recognized that under this scheme, some compounds will be considered for example to be both A analogs and G analogs (purine analogs) or both T analogs and C analogs (pyrimidine analogs).

[0107] As used herein, the term “nucleic acid construct” refers to a nucleic acid that is ligated or otherwise attached to another nucleic acid, such as an adaptor. For example, a nucleic acid construct may contain a nucleic acid molecule to be sequenced, a capture site for flowcell attachment, one or more identifier sequences such as SBC and UMI, and primer binding sites for a first and second primer.

[0108] As used herein, the term “capture site” refers to a nucleic acid sequence configured for attachment of a nucleic acid construct to a flowcell or other surface, for NGS sequencing or other analysis processing.

[0109] As used herein, the term “identifier” refers to a nucleic acid sequence that can be used to identify a particular nucleic acid construct. An “identifier” may be a “sample barcode” or “SBC” sequence for identifying a particular biological sample. An “identifier” may also refer to a “molecular barcode” for identification of unique molecules present in the sample. Also, an “identifier” may contain both an SBC and an UMI.

[0110] The term “antibody” is well understood by those in the field and is used interchangeably herein with “immunoglobulin” Those terms refer to a protein consisting of one or more polypeptides that specifically binds an antigen. One example of an antibody is the naturally occurring structural unit found in humans and other mammals which comprises a tetramer of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector20230183-02 / 027644.8509 functions. The term antibody encompasses monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, murine antibodies, rabbit antibodies, camelid antibodies, and antibodies from other mammalian and non-mammalian species. The term antibody also encompasses single-chain antibodies, bi-specific hybrid antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non- antibody protein. The term antibody also encompasses includes antigen-binding fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments.

[0111] The term “binding pair” as used herein refers to a pair of binding partners that exhibit specific binding between them. In some embodiments, a binding pair can selectively interact through covalent or non-covalent binding. In some embodiments, a binding pair can selectively interact by hybridization, ionic bonding, hydrogen bonding, van der Waals interactions, or any combination of these forces. In some embodiments, a binding partner can comprise, for example, biotin, avidin, streptavidin, digoxigenin, inosine, avidin, GST sequences, modified GST sequences, biotin ligase recognition (BiTag) sequences, S tags, SNAP-tags, enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, or combinations thereof. Examples of binding pairs include biotin:avidin, biotin:streptavidin, antibody:antigen, complementary nucleic acids, hapten / antibody, lectin / carbohydrate, apoprotein / cofactor and biotin / streptavidin.

[0112] The term “specific binding” refers to the ability of a binding partner to preferentially bind to its reciprocal binding partner that is present in a homogeneous mixture of different molecules. In some embodiments, specific binding discriminates between a reciprocal binding partner and other molecules by at least 100-fold, 1000-fold, 10,000-fold, 100,000-fold,20230183-02 / 027644.8509 or more. In some embodiments, the affinity between binding partners of a binding pair when they are specifically bound in a complex is characterized by a KD (dissociation constant) of less than 10-6M, less than 10-7M, less than 10-8M, less than 10-9M, less than 10-10M, less than 10-11M, or less than about 10-12M, or less.

[0113] As used herein, a “capture binding partner” refers to a binding partner that is configured to capture (e.g., isolate, purify, immobilize, extract) a nucleic acid tagged with its reciprocal binding partner. For example, streptavidin coated on a bead would be a capture binding partner for an input nucleic acid complex having a tag comprising a biotin moiety. A capture binding partner and its reciprocal binding partner may comprise any suitable binding pair. EXAMPLES Example 1

[0114] In this example, an experiment was conducted to test an embodiment of the present methods. 3ng of cell free DNA was used as input nucleic acid and ligated with Y-adaptors with or without a digoxigenin tag. The tagged molecules were then captured using a PanCancer71 panel (20kb) using a target enrichment procedure with bridge probes and anchor probes as illustrated in FIG.1B. The captured molecules were sequenced on an Illumina NextSeq1000 instrument. Table 1 shows the adaptors conjugated to a digoxigenin tag ligated to the input DNA with efficiency comparable to Y adaptors without a conjugated tag.

[0115] After capture of input molecules having the target sequences, the DIG-tagged input nucleic acid constructs were incubated with anti-DIG antibody conjugated with biotin, thereby forming input nucleic acid complexes comprising a first tag (DIG) and a second tag (biotin).20230183-02 / 027644.8509 The complexes were then bound to streptavidin coated beads (SA beads), since as streptavidin is a reciprocal binding partner of the binding partner of the second tag (biotin). The SA beads were washed, and the composition enriched for target nucleic acids of the enriched were subjected to indexing PCR. The sequencing results showed that binding of the complex with the second tag was efficient, with about 80% recovery (about 800 vs about 1100 copies of molecular recovery).

[0116] The results of this experiment (summarized in Table 1) demonstrate the methods were successful. Table 1 Experiment raw raw median molecular % % Median % 0.2X ise20230183-02 / 027644.8509 Table 2 raw raw dedup % % Median % 0.2X SampleID median median Insert region-Exemplary Embodiments

[0117] Embodiment 1. A method of preparing nucleic acid molecules for sequencing comprising attaching a first tag to an input nucleic acid, wherein the first tag comprises a binding partner of a first binding pair; and attaching a second tag to the first tag to produce an input nucleic acid complex, wherein the second tag comprises a reciprocal binding partner of the first binding pair.

[0118] Embodiment 2. The method of embodiment 1, wherein the binding partner in the first tag is selected from digoxigenin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris-Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine, an aldehyde, an alkyne or an azide.20230183-02 / 027644.8509

[0119] Embodiment 3. The method of embodiment 2, wherein the reciprocal binding partner in the second tag is selected from an anti-DIG antibody, an anti-BrdU antibody, an anti- DNP antibody, poly-Histidine, a tyrosine, a thiol, an amine, an aldehyde, an alkyne, or an azide.

[0120] Embodiment 4. The method of embodiment 1, wherein the binding partner in the first tag is digoxigenin (DIG).

[0121] Embodiment 5. The method of embodiment 4, wherein the second tag comprises anti-DIG antibody as the reciprocal binding partner of the first binding pair.

[0122] Embodiment 6. The method of embodiment 1, wherein the second tag further comprises a binding partner of a second binding pair.

[0123] Embodiment 7. The method of embodiment 6, wherein the binding partner of the second binding pair is selected from biotin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris-Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine, an aldehyde, an alkyne or an azide or other groups reacting by click chemistry, and mixtures thereof; with the proviso that the binding partner of the second binding pair is not a binding partner of the first binding pair.

[0124] Embodiment 8. The method of embodiment 7, wherein the reciprocal binding partner of the second pair is selected from an avidin moiety, an anti-DIG antibody, an anti-BrdU antibody, an anti-DNP antibody, poly-Histidine, a tyrosine residue, a thiol, an amine, an aldehyde, an alkyne, or an azide.

[0125] Embodiment 9. The method of any of embodiments 1-8, wherein the first tag is covalently bound to the input nucleic acid.

[0126] Embodiment 10. The method of any of embodiments 1-9, wherein the second tag is non-covalently bound to the first tag.20230183-02 / 027644.8509

[0127] Embodiment 11. The method of any of embodiments 1-10, further comprising attaching the second tag to a reciprocal binding pair of the second binding pair.

[0128] Embodiment 12. The method of embodiment 11, wherein the reciprocal binding pair of the second binding pair is attached to the solid support.

[0129] Embodiment 13. The method of embodiment 12, further comprising loading the input nucleic acid complex onto a flowcell or immobilizing the complex on a substrate.

[0130] Embodiment 14. The method of any of embodiments 1-13, further comprising hybridizing the input nucleic acid construct to a probe.

[0131] Embodiment 15. The method of embodiment 14, wherein the probe is a bridge probe.

[0132] Embodiment 16. The method of embodiment 15, further comprising hybridizing the bridge probe with an anchor probe.

[0133] Embodiment 17. The method of embodiment 16, the anchor probe is attached to an enrichment tag such as a biotin moiety.

[0134] In view of this disclosure it is noted that the methods and kits can be implemented in keeping with the present teachings. Further, the various components, materials, structures and parameters are included by way of illustration and example only and not in any limiting sense. In view of this disclosure, the present teachings can be implemented in other applications and components, materials, structures and equipment to implement these applications can be determined, while remaining within the scope of the appended claims.

Claims

20230183-02 / 027644.8509 CLAIMS We claim:

1. A method of preparing nucleic acid molecules for sequencing comprising: attaching a first tag to an input nucleic acid, wherein the first tag comprises a binding partner of a first binding pair; and attaching a second tag to the first tag to produce an input nucleic acid complex, wherein the second tag comprises a reciprocal binding partner of the first binding pair.

2. The method of claim 1, wherein the binding partner in the first tag is selected from digoxigenin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris-Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine, an aldehyde, an alkyne or an azide.

3. The method of claim 2, wherein the reciprocal binding partner in the second tag is selected from an anti-DIG antibody, an anti-BrdU antibody, an anti-DNP antibody, poly- Histidine, a tyrosine, a thiol, an amine, an aldehyde, an alkyne, or an azide.

4. The method of claim 1, wherein the binding partner in the first tag is digoxigenin (DIG).

5. The method of claim 4, wherein the second tag comprises anti-DIG antibody as the reciprocal binding partner of the first binding pair.20230183-02 / 027644.8509 6. The method of claim 1, wherein the second tag further comprises a binding partner of a second binding pair.

7. The method of claim 6, wherein the binding partner of the second binding pair is selected from biotin, 5-bromo-2’-deoxyuridine (BrdU), 2,4-dinitrophenyl (DNP), nitrilotriacetic acid or a nitrilotriacetate (NTA) such as nickel nitrilotriacetate (Ni-NTA), tris-Nitrilotriacetate (tris-NTA), a tyramine, a thiol, an amine, an aldehyde, an alkyne or an azide or other groups reacting by click chemistry, and mixtures thereof; with the proviso that the binding partner of the second binding pair is not a binding partner of the first binding pair.

8. The method of claim 7, wherein the reciprocal binding partner of the second pair is selected from an avidin moiety, an anti-DIG antibody, an anti-BrdU antibody, an anti-DNP antibody, poly-Histidine, a tyrosine residue, a thiol, an amine, an aldehyde, an alkyne, or an azide.

9. The method of any of claims 1-8, wherein the first tag is covalently bound to the input nucleic acid.

10. The method of claim 9, wherein the second tag is non-covalently bound to the first tag.20230183-02 / 027644.8509 11. The method of claim 10, further comprising attaching the second tag to a reciprocal binding pair of the second binding pair.

12. The method of claim 11, wherein the reciprocal binding pair of the second binding pair is attached to the solid support.

13. The method of claim 12, further comprising loading the input nucleic acid complex onto a flowcell or immobilizing the complex on a substrate.

14. The method of claim 1, further comprising hybridizing the input nucleic acid construct to a probe.

15. The method of claim 14, wherein the probe is a bridge probe.

16. The method of claim 15, further comprising hybridizing the bridge probe with an anchor probe.

17. The method of claim 16, the anchor probe is attached to an enrichment tag such as a biotin moiety.

Images