Targeted depletion of non-target library molecules using toxic primers in the target capture process of next generation sequencing libraries

Abstract

The present disclosure relates to methods, compositions, and kits for target enrichment by one-way primer extension, wherein the methods, compositions, and kits utilize both a toxic primer and a target capture primer.

Description

BACKGROUND

[0001] For many nucleic acid enrichment techniques, it is useful to first provide a "shotgun library" of nucleic acid molecules, where the longer nucleic acid sequences derived from the sample are subdivided into smaller fragments (i.e., about 50 to 500 nucleotides) compatible with short read sequencing technologies. To prepare a shotgun library, high molecular weight nucleic acid strands (typically cDNA or genomic DNA) are sheared into random fragments, optionally modified by ligation of common end sequences (i.e., adaptors), and selected for size for downstream processing and analysis. For example, it can be useful to selectively capture a portion of the nucleic acids in a shotgun library.

[0002] Currently, there are two major categories of capture techniques: hybridization-based capture and amplification-based capture. Hybridization-based capture methods have the advantage of being able to recover the entirety of the original shotgun library fragments, rather than just replicating and recovering a subset of the original library fragments. However, hybridization-based capture is often associated with lower on-target rates compared to amplification-based methods. Notably, the lower on-target rates result in wasted sequencing capacity due to the need to sequence off-target capture products. In addition, the workflow associated with hybridization-based capture methods can be more complex and have longer turnaround times compared to amplification-based methods. In contrast, amplification-based methods, such as anchored multiplex PCR methods, have the advantages of simpler workflow, faster turnaround times, and higher on-target rates compared to hybridization-based methods, but also have some disadvantages. For example, the incorporation of target capture primer sequences into the library fragments after amplification results in wasted sequencing capacity. In addition, the library fragments do not necessarily represent the original shotgun library, as the templates must be truncated at the target-specific primer binding site.

[0003] In some embodiments, shotgun target capture techniques involve hybridization and extension of biotinylated primers. In some embodiments, target enrichment is subsequently performed using streptavidin-bound magnetic beads. In some embodiments, bound target library fragments can be released from streptavidin using non-biotinylated target release primers, or amplified directly from the streptavidin beads (see, e.g., U.S. Patent Publication Nos. 2020 / 0032244 and 2018 / 0016630, the disclosures of which are incorporated by reference herein in their entireties). However, in some embodiments, the target capture primers can misprime, capturing and enriching unwanted library molecules that can be amplified and sequenced downstream. It is believed that problems can exist in the following cases: (i) the abundance of unwanted molecules far exceeds the abundance of target molecules (e.g., due to the high abundance of ribosomal RNA (rRNA)-derived molecules in cDNA libraries, which typically include about 95% rRNA and about 5% mRNA-derived molecules); and / or (ii) the low abundance of certain fusion transcripts compared to wild-type transcripts makes detection challenging.

[0004] Depletion of unwanted library molecules is a recurring theme in the preparation and analysis of next generation sequencing libraries. For example, rRNA comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Transcriptome profiling using massively parallel sequencing technologies requires enrichment of mature polyadenylated transcripts or targeted depletion of rRNA fragments. Ribosome depletion is a critical step in transcriptomics, allowing efficient detection of functionally relevant coding and non-coding transcripts by removing high abundance rRNA species. It is believed that ribosome depletion, such as occurs prior to cDNA synthesis, is time consuming and / or expensive. SUMMARY

[0005] It is desirable to reduce erroneous priming events during the hybridization and extension process of shotgun capture. It is also desirable to avoid the use of time consuming and costly rRNA depletion methods. In some embodiments, the present disclosure provides an improved method of shotgun target capture in which a toxic primer is included in the target capture primer during any hybridization and extension step. It is believed that inclusion of a toxic primer (not including the capture portion) compared to a target capture primer can prevent or reduce erroneous priming and off-target effects often observed during target enrichment. It is further believed that the presently disclosed method of incorporating a toxic primer in conjunction with a target capture primer does not require time consuming rRNA removal methods, such as ribosome depletion, while still promoting rRNA depletion during the capture and enrichment steps. Additionally, it is believed that the presently disclosed method can reduce the sequencing depth required to detect the desired target nucleic acid sequences.

[0006] One aspect of the present disclosure is a method of enriching at least one target nucleic acid molecule in a library of nucleic acid molecules, the method comprising: hybridizing a first target capture primer to a first target nucleic acid molecule in the library of nucleic acid molecules, wherein the first target capture primer comprises a capture portion; hybridizing a first toxic primer to a first non-target nucleic acid molecule in the library of nucleic acid molecules, wherein the first toxic primer does not comprise any capture portion; extending the first hybridized target capture primer and the first hybridized toxic primer, wherein extension of the first hybridized target capture primer provides a first target capture primer extension complex comprising the first target nucleic acid molecule and the extended first target capture primer; and enriching the first target nucleic acid molecule in the library of nucleic acid molecules relative to the library of nucleic acid molecules. In some embodiments, the first target capture primer and the first toxic primer are added to the library as a primer pool. In some embodiments, each of the nucleic acid molecules in the library of nucleic acid molecules comprises a first end comprising a first adapter and a second end comprising a second adapter. In some embodiments, the method does not comprise performing any ribosome depletion step.

[0007] In some embodiments, the extension of the first hybridized toxic primer prevents or mitigates hybridization of the first target capture primer to the first non-target nucleic acid molecule, i.e., the extension of the first hybridized toxic primer prevents or mitigates mispriming. In other embodiments, the extension of the first hybridized toxic primer displaces the first target primer hybridized to the first non-target nucleic acid molecule, i.e., the extension of the first hybridized toxic primer displaces the first target primer misprimed to the first non-target nucleic acid molecule.

[0008] In some embodiments, the method further comprises amplifying the first target nucleic acid molecule with a first amplification primer and a second amplification primer, wherein the first amplification primer comprises a 3' end that is complementary to the first adaptor, and wherein the second amplification primer comprises a 3' end that is complementary to the second adaptor. In some embodiments, the method further comprises sequencing the amplified target nucleic acid molecule.

[0009] In some embodiments, the enrichment of the first target nucleic acid molecule relative to the library of nucleic acid molecules comprises (i) capture of the first target capture primer extension complex; (i) removal of non-target nucleic acid molecules that are not captured; and (ii) release of the first target nucleic acid molecule from the captured first target capture primer extension complex. In some embodiments, the capture of the first target capture primer extension complex comprises contacting the first target capture primer extension complex with a functionalized substrate.

[0010] In some embodiments, the capture moiety of the first target capture primer comprises a first member of a specific binding entity pair, and wherein the functionalized substrate comprises a second member of the specific binding entity pair. In some embodiments, the first member of the specific binding entity pair is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a sulfhydrylated molecule, and an amino-terminated molecule. In some embodiments, the second member of the specific binding entity pair is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold particle, and an NHS activated moiety. In some embodiments, the capture moiety of the first target capture primer comprises biotin. In some embodiments, the functionalized surface comprises streptavidin. In some embodiments, the functionalized substrate comprises a bead having a surface functionalized with the second member of the specific binding entity pair. In some embodiments, the functionalized substrate comprises a bead having a surface comprising a plurality of streptavidin molecules.

[0011] In some embodiments, the first target capture primer is coupled to a substrate via a capture moiety, and then the first target capture primer hybridizes to a first target nucleic acid molecule in the library of nucleic acid molecules. In some embodiments, the first target capture primer hybridizes to the first target nucleic acid molecule and / or the first hybridized target capture primer is extended, thereby capturing the first target nucleic acid molecule to the substrate.

[0012] In some embodiments, the capturing of the first target capture primer extension complex comprises: (i) hybridizing a universal capture oligonucleotide to the capture portion of the first target capture primer extension complex to form a universal capture oligonucleotide complex, wherein the universal capture oligonucleotide comprises (a) a first member of a specific binding entity pair, and (b) a nucleotide sequence complementary to at least a portion of the capture sequence of the capture portion; (ii) contacting the universal capture complex with a functionalized substrate, wherein the functionalized substrate comprises a second member of the specific binding entity pair.

[0013] In some embodiments, the removing of the non-captured nucleic acid molecules comprises washing away the non-captured non-target nucleic acid molecules. In some embodiments, the releasing of the captured first target capture primer extension complex comprises: (i) hybridizing a release primer to the first target nucleic acid molecule; and (b) extending the hybridized release primer. In some embodiments, the hybridized first target capture primer is extended with a first polymerase; and wherein the hybridized release primer is extended with a second polymerase. In some embodiments, the first polymerase and the second polymerase are different. In some embodiments, the first target capture primer has a melting temperature greater than a melting temperature of the first toxic primer.

[0014] In some embodiments, the library of nucleic acid molecules comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules, wherein the plurality of target nucleic acid molecules is at a lower abundance compared to the plurality of non-target nucleic acid molecules. In some embodiments, the plurality of target nucleic acid molecules comprises less than 10% of the nucleic acid molecules in the library of nucleic acid molecules. In some embodiments, the plurality of target nucleic acid molecules comprises less than 5% of the nucleic acid molecules in the library of nucleic acid molecules. In some embodiments, the plurality of target nucleic acid molecules comprises mRNA-derived nucleic acid molecules. In some embodiments, the plurality of target nucleic acid molecules comprises fusion transcripts.

[0015] Another aspect of the present disclosure is a kit comprising: a first target capture primer complementary to a target nucleic acid molecule in a library of nucleic acid molecules, and wherein the first target capture primer comprises a capture moiety; and a first toxic primer complementary to a non-target nucleic acid molecule in the library of nucleic acid molecules, and wherein the first toxic primer does not comprise a capture moiety. In some embodiments, the first target capture primer has a Tm in a range between about 55 °C to about 65 °C. In some embodiments, the first toxic primer has a Tm in a range between about 65 °C to about 68 °C. In some embodiments, the kit comprises at least one polymerase. In some embodiments, the kit comprises at least two different polymerases. In some embodiments, the kit further comprises a plurality of nucleotides. In some embodiments, the kit further comprises one or more buffer solutions and / or wash solutions. In some embodiments, the kit further comprises a bead having a functionalized surface. In some embodiments, the kit further comprises one or more release primers. In some embodiments, the kit further comprises a first amplification primer and a second amplification primer.

[0016] Another aspect of the present disclosure is a composition comprising: a library of nucleic acid molecules, wherein the library of nucleic acid molecules comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules, wherein the plurality of target nucleic acid molecules is in low abundance compared to the plurality of non-target nucleic acid molecules; a first target capture primer complementary to a first target nucleic acid molecule in the library of nucleic acid molecules, and wherein the first target capture primer comprises a capture moiety; a first toxic primer complementary to a first non-target nucleic acid molecule in the library of nucleic acid molecules, and wherein the first toxic primer does not comprise a capture moiety; and a first polymerase. In some embodiments, the composition further comprises a second polymerase different from the first polymerase. In some embodiments, the composition further comprises a functionalized substrate. In some embodiments, the composition further comprises one or more adaptor molecules. In some embodiments, the composition further comprises one or more release primers. In some embodiments, the composition further comprises a first amplification primer and a second amplification primer.

[0017] Another aspect of the present disclosure is a composition comprising: a library of nucleic acid molecules, wherein the library of nucleic acid molecules comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules; a first target capture primer hybridized to a portion of a first target nucleic acid molecule in the library of nucleic acid molecules, and wherein the first target capture primer comprises a capture moiety that binds to a functionalized substrate; and a first toxic primer hybridized to a portion of a first non-target nucleic acid molecule in the library of nucleic acid molecules, and wherein the first toxic primer does not comprise a capture moiety. In some embodiments, the plurality of target nucleic acid molecules is in low abundance compared to the plurality of non-target nucleic acid molecules.

[0018] In some embodiments, the capture moiety comprises a first member of a specific binding entity pair. In some embodiments, the functionalized substrate is a bead having a surface functionalized with a second member of a specific binding entity pair. In some embodiments, the first member of the specific binding entity pair is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a sulfhydrylated molecule, and an amino-terminated molecule. In some embodiments, the second member of the specific binding entity pair is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold particle, and an NHS activated moiety. In some embodiments, the capture moiety of the first target capture primer comprises biotin. In some embodiments, the functionalized surface comprises streptavidin.

[0019] Another aspect of the present disclosure is a composition enriched for a target nucleic acid molecule, the composition prepared by: (i) hybridizing a first target capture primer to a first target nucleic acid molecule in a library of nucleic acid molecules, wherein the library of nucleic acid molecules comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules, wherein the plurality of target nucleic acid molecules is in low abundance compared to the plurality of non-target nucleic acid molecules, and wherein the first target capture primer comprises a capture moiety; (ii) hybridizing a first toxic primer to a first non-target nucleic acid molecule in the library of nucleic acid molecules, wherein the first toxic primer does not comprise any capture moiety; (iii) extending the first hybridized target capture primer and the first hybridized toxic primer, wherein extension of the first hybridized target capture primer provides a first target capture primer extension complex comprising the first target nucleic acid molecule and the extended first target capture primer; and (iv) enriching the first target nucleic acid molecule in the library of nucleic acid molecules relative to the library of nucleic acid molecules.

[0020] In some embodiments, the composition is enriched for a low abundance fusion transcript. In some embodiments, the composition is enriched for mRNA derived molecules. In some embodiments, the composition enriched for target nucleic acid molecules is prepared without performing any ribosome depletion step.

[0021] In some embodiments, the first target capture primer and the first toxic primer are added to the library as a primer pool. In some embodiments, each of the nucleic acid molecules in the library of nucleic acid molecules comprises a first end comprising a first adapter and a second end comprising a second adapter.

[0022] In some embodiments, the method further comprises amplifying the first target nucleic acid molecule with a first amplification primer and a second amplification primer, wherein the first amplification primer comprises a 3' end complementary to the first adapter, and wherein the second amplification primer comprises a 3' end complementary to the second adapter. In some embodiments, the method further comprises sequencing the amplified target nucleic acid molecule.

[0023] In some embodiments, the enrichment of the first target nucleic acid molecule relative to the library of nucleic acid molecules comprises (i) capture of the first target capture primer extension complex; (ii) removal of non-target nucleic acid molecules that are not captured; and (iii) release of the first target nucleic acid molecule from the captured first target capture primer extension complex. In some embodiments, the capture of the first target capture primer extension complex comprises contacting the first target capture primer extension complex with a functionalized substrate.

[0024] In some embodiments, the capture moiety of the first target capture primer comprises a first member of a specific binding entity pair, and wherein the functionalized substrate comprises a second member of the specific binding entity pair. In some embodiments, the first member of the specific binding entity pair is selected from the group consisting of biotin, an antigenic molecule, an enzyme substrate, a receptor ligand, a polysaccharide, a sulfhydrylated molecule, and an amino-terminated molecule. In some embodiments, the second member of the specific binding entity pair is selected from the group consisting of streptavidin, an antibody, an enzyme, a receptor, a lectin, a gold particle, and an NHS activated moiety. In some embodiments, the capture moiety of the first target capture primer comprises biotin. In some embodiments, the functionalized surface comprises streptavidin. In some embodiments, the functionalized substrate comprises a bead having a surface functionalized with the second member of the specific binding entity pair. In some embodiments, the functionalized substrate comprises a bead having a surface comprising a plurality of streptavidin molecules.

[0025] In some embodiments, the first target capture primer is coupled to the substrate via the capture moiety, and then the first target capture primer hybridizes to the first target nucleic acid molecule in the library of nucleic acid molecules. In some embodiments, the first target capture primer hybridizes to the first target nucleic acid molecule and / or the first hybridized target capture primer is extended, thereby capturing the first target nucleic acid molecule to the substrate.

[0026] In some embodiments, the capture of the first target capture primer extension complex comprises: (i) hybridizing a universal capture oligonucleotide to the capture moiety of the first target capture primer extension complex to form a universal capture oligonucleotide complex, wherein the universal capture oligonucleotide comprises (a) a first member of a specific binding entity pair, and (b) a nucleotide sequence that is complementary to at least a portion of the capture sequence of the capture moiety; (ii) contacting the universal capture complex with a functionalized substrate, wherein the functionalized substrate comprises a second member of the specific binding entity pair.

[0027] In some embodiments, the removal of unbound nucleic acid molecules comprises washing away unbound non-target nucleic acid molecules. In some embodiments, the release of the captured first target capture primer extension complex comprises: (i) hybridizing a release primer to the first target nucleic acid molecule; and (b) extending the hybridized release primer. In some embodiments, the hybridized first target capture primer is extended with a first polymerase; and wherein the hybridized release primer is extended with a second polymerase. BRIEF DESCRIPTION OF DRAWINGS

[0028] For a general understanding of the features of the present disclosure, reference is made to the drawings. In the drawings, like reference numbers are used to identify like elements throughout the several views. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] Figure 1A An overview is provided for using a toxic primer in conjunction with a target capture primer to achieve targeted depletion of non-target nucleic acid molecules from a nucleic acid library. In some embodiments, the non-target nucleic acid molecules are rRNA derived cDNA molecules.

[0030] Figure 1B An overview is provided for using a toxic primer in conjunction with a target capture primer to achieve targeted depletion of non-target nucleic acid molecules from a nucleic acid library. In some embodiments, the non-target nucleic acid molecules are rRNA derived cDNA molecules.

[0031] Figure 2A An overview is shown for a method of enriching target nucleic acid molecules in a prepared nucleic acid library according to one embodiment of the present disclosure.

[0032] Figure 2B An overview is shown for a method of enriching target nucleic acid molecules in a prepared nucleic acid library according to one embodiment of the present disclosure.

[0033] Figure 2C An overview is shown for a method of releasing a target nucleic acid molecule from a formed complex comprising at least a portion of the target nucleic acid molecule and an extended hybridized target capture primer according to one embodiment of the present disclosure.

[0034] Figure 3A An overview is shown for a method of primer extension target enrichment according to one embodiment of the present disclosure, wherein hybridization of a toxic primer and extension of the toxic primer along a non-target nucleic acid molecule prevents or reduces the incidence of target capture primer mispriming to the non-target nucleic acid.

[0035] Figure 3B An overview is shown for an alternative method of capturing target nucleic acid molecules using a universal capture oligonucleotide.

[0036] Figure 4A method of primer extension target enrichment is shown, wherein hybridization of a toxic primer and extension of the toxic primer along a non-target nucleic acid molecule cleaves a target capture moiety from an error primed target capture primer, according to one embodiment of the present disclosure.

[0037] Figure 5 An alternative embodiment of the method depicted in Figure 3A An alternative embodiment of the method depicted in

[0038] Figure 6 An alternative embodiment of the method depicted in Figure 4 An alternative embodiment of the method depicted in Figure 6 It is further shown that a target capture primer coupled to a substrate through a capture moiety can be error primed to a non-target nucleic acid molecule, and wherein the capture moiety coupled to the substrate can be released by extension of a hybridized toxic primer.

[0039] Figure 7 A method of releasing an extended hybridized target capture primer from a target nucleic acid molecule prior to amplification and / or sequencing of the target nucleic acid molecule is set forth, according to one embodiment of the present disclosure.

[0040] Figure 8 Results of an experiment are depicted, wherein sgPETE showed an average of about 45% rRNA reads in sequencing reads using 10 ng UHR RNA as input for library preparation and capture. When 1 mM toxic primer was included in the sgPETE capture reaction, the average number of rRNA reads was reduced by about 25%. In ribosome depleted samples, including toxic primer did not result in a significant increase in off-target reads.

[0041] Figure 9A Sequencing read breakdown to 3.5 million is depicted. 2.33 million (± 0.17 million) fusion target reads were included in the toxic primer, and 1.05 million (± 0.08 million) fusion reads were included in the control sample.

[0042] Figure 9B Theoretical sequencing depth and breakdown of reads required for the control sample to detect a similar number of fusion target reads is shown. DETAILED DESCRIPTION

[0043] It should also be understood that, unless specifically stated otherwise, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts are recited.

[0044] Definitions It should also be understood that, unless specifically stated otherwise, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts are recited.

[0045] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to mean "and / or" unless the context clearly dictates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0046] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when used in the context of listing items

[0047] The terms "comprising," "including," "containing," etc. are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Similarly, the terms "comprises", "comprised of", "comprising" and the like can be used synonymously with "including" and "including", respectively, and are used to specify the presence of stated features, integers, steps and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when used in the context of listing items

[0048] As used herein in the specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. Additionally, the term "comprising" is used throughout the specification and claims, and it is understood that the meaning of "comprising" also covers the meanings of "consisting of" and "consisting essentially of."

[0049] References throughout this specification to "one embodiment," "an embodiment," "exemplary embodiment," etc., indicate that the particular feature, structure, or characteristic being described is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in an exemplary embodiment" at

[0050] As used herein, the term "adapter" refers to a nucleotide sequence that can be added to another sequence to impart additional properties to the other sequence. An adapter can be single-stranded or double-stranded, or can have both single-stranded and double-stranded portions.

[0051] As used herein, “amplification” refers to a process of increasing copy number. Amplification can be a process of repeated replication over time to form multiple copies of a template. Amplification can result in an exponential or linear increase in copy number as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), cascade RCA, nucleic acid sequence-based amplification (NASBA), and the like. Further, amplification can apply linear or circular templates. Amplification can be performed under any suitable temperature conditions, such as thermal cycling or isothermal. Further, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature set points, such as a higher denaturation temperature and a lower annealing / extension temperature, or three or more temperature set points, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, and the like. PCR can be performed using a heat-stable polymerase, such as Taq DNA polymerase. PCR generally produces an exponential increase in the amount of product amplicon in successive cycles.

[0052] As used herein, the term “complementary” generally refers to the ability of two nucleotides to pair precisely. The term “complementary” refers to the ability to form a thermodynamically favorable, stable, and specific pairing between the bases of two nucleotides under appropriate temperature and ionic buffer conditions. Complementarity is achieved through different interactions between the nucleobases adenine, thymine (uracil in RNA), guanine, and cytosine, wherein adenine pairs with thymine or uracil, and guanine pairs with cytosine. For example, two nucleic acids are considered to be complementary to each other at a given position if the nucleotide at that position of one nucleic acid is capable of hydrogen bonding with the nucleotide of the other nucleic acid. Complementarity between two single-stranded nucleic acid molecules can be “partial,” in which only some of the nucleotides bind, or it can be complete when total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the “complement” of a second nucleotide sequence if the first nucleotide sequence is complementary to the reverse (i.e., the order of the nucleotides is reversed) of the second sequence. A first nucleotide sequence can be said to be the “reverse complement” of a second sequence if the first nucleotide sequence is complementary to the reverse of the second sequence.

[0053] As used herein, the term “complementary DNA” or “cDNA” refers to DNA copied from RNA. In some embodiments, cDNA is DNA produced on an RNA template by the action of a reverse transcriptase (RNA-dependent DNA polymerase). cDNA copied from mRNA does not include various non-coding sequences that are unique to genomic DNA.

[0054] As used herein, the term "cDNA library" refers to a collection of cDNAs of messenger RNAs expressed in a cell or tissue type, as used herein.

[0055] As used herein, the term "conjugate" refers to two or more molecules (and / or materials such as nanoparticles) covalently linked to a larger construct. In some embodiments, a conjugate includes one or more biological molecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules, such as one or more other biological molecules.

[0056] As used herein, the term "enrichment" refers to a process that increases the relative abundance of a population of molecules (e.g., nucleic acid molecules) in a sample relative to the total amount of the molecules originally present in the sample prior to the treatment. Thus, an enrichment step provides a percentage or fractional increase, rather than a direct increase in the copy number of, for example, a target nucleic acid sequence as an amplification method, such as polymerase chain reaction.

[0057] As used herein, the term "fluid" refers to any liquid or liquid composition, including water, solvents, buffers, solutions (e.g., polar solvents, non-polar solvents), washes or wash solutions, and / or mixtures. A fluid can be aqueous or non-aqueous. In some embodiments, a wash solution includes a surfactant to facilitate spreading of the wash liquid on the sample-bearing surface of a slide. In some embodiments, an acid solution includes deionized water, an acid (e.g., acetic acid), and a solvent. In some embodiments, a basic solution includes deionized water, a base, and a solvent. In some embodiments, a transfer solution includes one or more glycol ethers, such as one or more propylene glycol ethers (e.g., propylene glycol ether, di(propylene glycol) ether, and tri(propylene glycol) ether, ethylene glycol-based glycol ethers (e.g., ethylene glycol ether, di(ethylene glycol) ether, and tri(ethylene glycol) ether), and functional analogs thereof.

[0058] Non-limiting examples of buffering agents include citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2- ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-l-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), and combinations thereof. In other embodiments, the buffer solution can be comprised of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-l-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or combinations thereof. Additional wash, transfer, acid, and base solutions are described in U.S. Patent Application Publication No. 2016 / 0282374, the disclosure of which is incorporated by reference herein in its entirety.

[0059] As used herein, the term "hybridization" refers to base pairing between different nucleic acid molecules in accordance with their nucleotide sequences.

[0060] As used herein, the phrase "next generation sequencing (NGS)" refers to sequencing technologies with high-throughput sequencing compared to traditional Sanger and capillary electrophoresis-based methods, where the sequencing process is performed in parallel, e.g., generating thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing technologies include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (from about 25 to about 500 bp), but produce hundreds of thousands or millions of reads in a relatively short period of time. The term "next generation sequencing" refers to the so-called parallel sequencing by synthesis or sequencing by ligation platforms currently employed by Illumina, Life Technologies, and Helicos Biosciences. Next generation sequencing methods can also include nanopore sequencing methods with electronic detection (Oxford Nanopore and Roche Diagnostics).

[0061] As used herein, the term "nucleic acid" or "polynucleotide" (used interchangeably herein) refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function for which it has been engineered to serve. The term encompasses nucleic acids or polynucleotides comprising known analogues of natural nucleotides that have similar binding properties to the reference nucleotide and are metabolized in a manner similar to naturally occurring nucleotides. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified, such as by conjugation with a labeling component. The disclosure also envisions the use of one or more polynucleotide sequences or fragments as diagnostic or therapeutic agents. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0062] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotides or nucleoside monomer units, wherein the oligomer optionally includes non-nucleotide monomer units and / or other chemical groups attached to internal and / or external positions of the oligomer. The oligomer can be natural or synthetic, and can include naturally occurring oligonucleotides, or oligomers including nucleosides having non-naturally occurring (or modified) base, sugar moieties, phosphodiester-analogous linkages, and / or alternative monomer unit chirality and isomeric structures (e.g., 5'-linkages to 2'-linkages, L-nucleosides, alpha-anomeric nucleosides, beta-anomeric nucleosides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs)).

[0063] As used herein, a“reaction” between any two different reactive groups, such as any two reactive groups of a reagent and a particle, can mean the formation of a covalent bond between the two reactive groups (or two reactive functional groups); or can mean that the two reactive groups (or two reactive functional groups) associate with each other, interact with each other, hybridize with each other, hydrogen bond with each other, etc. In some embodiments, a“reaction” includes a binding event, such as a binding event between reactive functional groups or a binding event between a first member and a second member of a specific binding entity pair.

[0064] As used herein, the term“polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides. DNA polymerases can only add free nucleotides at the 3’ end of the newly formed strand. This results in the elongation of the newly formed strand in the 5’-3’ direction. No known DNA polymerase is able to start a new strand (de novo). DNA polymerases can only add nucleotides on a pre-existing 3’-OH group, so a primer is required where it can add the first nucleotide. Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerases, archaeal DNA polymerases, telomerase, reverse transcriptases, and RNA polymerases. Reverse transcriptases are RNA-dependent DNA polymerases that can synthesize DNA from an RNA template. The reverse transcriptase family contains DNA polymerase function and RNase H function, which can degrade paired RNA bases to DNA. RNA polymerases are enzymes that synthesize RNA using DNA as a template during gene transcription. RNA polymerases polymerize ribonucleotides at the 3’ end of the RNA transcript.

[0065] In some embodiments, a polymerase from an archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus strain KOD (Pfx, GenBank: AAE68738), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degrees Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus GE8 (GenBank: CAC12850), Thermococcus JDF-3 (GenBank: AX135456, WO0132887), Thermococcus TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849), Pyrococcus horikoshii (Pyrococcus horikoshii), Pyrococcus sp. AM4 (GenBank: AY028593), Pyrococcus sp. ST04 (GenBank: AY028592), Pyrococcus sp. K1 (GenBank: AY028591), Pyrococcus sp. K2 (GenBank: AY028590), Pyrococcus sp. K3 (GenBank: AY028589), Pyrococcus sp. K4 (GenBank: AY028588), Pyrococcus sp. K5 (GenBank: AY028587), Pyrococcus sp. K6 (GenBank: AY028586), Pyrococcus sp. K7 (GenBank: AY028585), Pyrococcus sp. K8 (GenBank: AY028584), Pyrococcus sp. K9 (GenBank: AY028583), Pyrococcus sp. K10 (GenBank: AY028582), Pyrococcus sp. K11 (GenBank: AY028581), Pyrococcus sp. K12 (GenBank: AY028580), Pyrococcus sp. K13 (GenBank: AY028579), Pyrococcus sp. K14 (GenBank: AY028578), Pyrococcus sp. K15 (GenBank: AY028577), Pyrococcus sp. K16 (GenBank: AY028576), Pyrococcus sp. K17 (GenBank: AY028575), Pyrococcus sp. K18 (GenBank: AY028574), Pyrococcus sp. K19 (GenBank: AY028573), Pyrococcus sp. K20 (GenBank: AY028572), Pyrococcus sp. K21 (GenBank: AY028571), Pyrococcus sp. K22 (GenBank: AY028570), Pyrococcus sp. K23 (GenBank: AY028569), Pyrococcus sp. K24 (GenBank: AY028568), Pyrococcus sp. K25 (GenBank: AY028567), Pyrococcus sp. K26 (GenBank: AY028566), Pyrococcus sp. K27 (GenBank: AY028565), Pyrococcus sp. K28 (GenBank: AY028564), Pyrococcus sp. K29 (GenBank: AY028563), Pyrococcus sp. K30 (GenBank: AY028562), Pyrococcus sp. K31 (GenBank: AY028561), Pyrococcus sp. K32 (GenBank: AY028560), Pyrococcus sp. K33 (GenBank: AY028559), Pyrococcus sp. K34 (GenBank: AY028558), Pyrococcus sp. K35 (GenBank: AY028557), Pyrococcus sp. K36 (GenBank: AY028556), Pyrococcus sp. K37 (GenBank: AY028555), Pyrococcus sp. K38 (GenBank: AY028554), Pyrococcus sp. K39 (Gen*Thermococcus horikoshii* (GenBank: NP143776), *Thermococcus* GE23 (GenBank: CAA90887), *Thermococcus* ST700 (GenBank: CAC 12847), *Thermococcus pacificus* (GenBank: AX411312.1), *Thermococcus zilligii* (GenBank: DQ3366890), *Thermococcus aggtegans*, *Thermococcus barossii*, *Thermococcus celer* (GenBank: DD259850.1), *Thermococcus cephalopoda* Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), species of the genus *Desulfococcus* TOK, *Desulfurococcus*, *Pyrolobus*, *Pyrodictium*, *Staphylothermus*, *Vulcanisaetta*, Methanococcus (GenBank: P52025), and other archaea B polymerases, such as GenBank AAC62712, P956901, BAAA07579), thermophilic bacteria of the genus *Thermus* (e.g., *Thermus flavus*, *Thermus ruber*, *Thermus thermophilus*, *Thermus mollusc*). *Lacteus*, *Thermus rubens*, *Thermus aquaticus*Aquifex aeolicus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, Thermococcus GT, Pyrococcus GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis, and Thermococcus sp. 9N-7 polymerases.

[0066] As used herein, the term "thermostable polymerase" refers to an enzyme that is thermostable, i.e., resistant to heat, that retains sufficient activity to effect a subsequent polynucleotide extension reaction after being subjected to the heating conditions required for nucleic acid denaturation for the time required at high temperatures, and does not irreversibly denature (become inactive). The heating conditions required for nucleic acid denaturation are well known in the art and are exemplified in, e.g., U.S. Patent Nos. 4,683,202, 4,683,195, and 4,965,188, which are incorporated herein by reference. As used herein, a thermostable polymerase is suitable for use in temperature cycling reactions, such as polymerase chain reaction ("PCR"), primer extension reactions, or end modification (e.g., terminal transferase, degradation, or blunting) reactions. Irreversible denaturation for purposes herein refers to a permanent and complete loss of enzyme activity. For a thermostable polymerase, enzyme activity refers to the catalysis of the combination of nucleotides in the proper manner to form a polynucleotide extension product that is complementary to a template nucleic acid strand. Thermostable DNA polymerases from thermophilic bacteria include, e.g., DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus sp spl7, Thermus sp Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosipho afficanus, and other thermostable DNA polymerases disclosed above.

[0067] In some cases, the nucleic acid (e.g., DNA or RNA) polymerase can be a modified naturally occurring Type A polymerase. Further embodiments of the present invention generally relate to a method wherein the modified Type A polymerase can be selected from any species of any genus of Meiothermus, Thermotoga, or Thermomicrobium, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or fill-in), or amplification reactions. Another embodiment of the present invention generally depends from a method wherein the polymerase can be isolated from any one of Thermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, or Thermus filiformis, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or fill-in), or amplification reactions. Further embodiments of the present invention generally encompass a method wherein the modified Type A polymerase can be isolated from Bacillus stearothermophilus, Sphaerobacter thermophilus, Dictoglomus thermophilum, or Escherichia coli, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or fill-in), or amplification reactions. In another embodiment, the present invention generally relates to a method wherein the modified Type A polymerase can be a mutant Taq-E507K polymerase, for example, in primer extension, end modification (e.g., terminal transferase, degradation, or fill-in), or amplification reactions. Another embodiment of the present invention generally depends from a method wherein a thermostable polymerase can be used to cause amplification of a target nucleic acid.

[0068] As used herein, the term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction (extending from the 3' end of the primer) and is complementary to the sequence of the template molecule. PCR amplification primers may be referred to as "forward" and "reverse" primers, one complementary to the nucleic acid strand and the other to the complementary sequence of that strand. Typically, primers comprise fewer than about 100 nucleotides, and preferably fewer than about 30 nucleotides. Exemplary primers range from about 5 to about 25 nucleotides. Primers may contain, for example, RNA and / or DNA bases, as well as non-naturally occurring bases. The directionality of the newly formed strand (daughter strand) is opposite to the direction of DNA polymerase movement along the template strand. In some cases, target capture primers specifically hybridize to the target polynucleotide under hybridization conditions. Such hybridization conditions may include, but are not limited to, isothermal amplification buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4), 50 mM KCl, 2 mM MgSO4, 0.1% Hybridization was carried out at temperatures of approximately 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C (pH 8.8 at 20°C and 25°C).

[0069] As used herein, the term "sequence" when referring to nucleic acid molecules refers to the order of nucleotides (or bases) within the molecule. In cases where different types of nucleotides are present in a nucleic acid molecule, the sequence includes the identification of the types (or bases) of nucleotides at various positions within the molecule. A sequence is a characteristic of all or part of a nucleic acid molecule. The term can be similarly used to describe the sequential and positional identity of monomeric units in other polymers (e.g., amino acid monomeric units in protein polymers).

[0070] As used herein, the term "sequencing" refers to determining the sequence and position of bases in a nucleic acid molecule. More specifically, the term "sequencing" refers to a biochemical method used to determine the sequence of nucleotide bases, adenine, guanine, cytosine, and thymine in a DNA oligonucleotide. As used herein, sequencing can include, but is not limited to, parallel sequencing or any other sequencing method known to those skilled in the art, such as chain termination, rapid DNA sequencing, wandering-spot analysis, Maxam-Gilbert sequencing, dye-terminated sequencing, or the use of any other modern automated DNA sequencing instrument.

[0071] As used herein, the term "substrate" refers to any material capable of interacting with a capture moiety. In some embodiments, the substrate is a solid support. In some embodiments, the solid support can encompass any type of solid, porous, or hollow sphere, sphere, bearing, cylinder, capillary, channel, or other similar structure composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) on which a nucleic acid molecule can be immobilized (e.g., covalently or non-covalently). In some embodiments, the solid support can comprise discrete particles, which can be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cube, cuboid, pyramid, cylinder, cone, oval, or disc, etc. In some embodiments, the solid support can include a silicon wafer, microparticle, nanoparticle, plate, array, capillary, flatbed support such as a glass fiber filter sheet, glass surface, metal surface (steel, gold, silver, aluminum, silicon, and copper), glass support, plastic support, silica gel support, chip, filter sheet, membrane, microplate, glass slide, plastic material including a multi-well plate or membrane (e.g., polyethylene, polypropylene, polyamide, polyvinylidene difluoride, etc.), and / or a wafer, comb, pin, or needle (e.g., a pin array suitable for combinatorial synthesis or analysis) or beads in an array of recesses or nanoliter scale wells in a flatbed surface such as a wafer (e.g., a silicon wafer) with or without a filter bottom. In some embodiments, the solid support can be a liquid phase support that can be suspended in a solution (e.g., a glass bead, a magnetic bead, or other similar particle) or a solid phase support (e.g., a silicon wafer, a glass slide, etc.). Non-limiting examples of liquid phase supports include superparamagnetic spherical polymeric particles such as INVITROGEN's DYNABEADS magnetic beads or magnetic glass particles such as those described in U.S. Patent Nos. 6,656,568, 6,274,386, 7,371,830, 6,870,047, 6,255,477, 6,746,874, and 6,258,531, the disclosures of which are incorporated by reference herein in their entireties.

[0072] As used herein, the term "specific binding entity" refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to one another to the substantial exclusion of binding to other molecules (e.g., the binding constant of a specific binding pair can be at least 10 3 M -1 , 10 4 M -1 , or 10 5 M -1 ) greater than the binding constant of either of the two members of a binding pair of other molecules in a biological sample. Examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, streptavidin, and protein A, among others). Specific binding moieties can also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0073] As used herein, the term “substantially” means a qualitative condition of being, either entirely or nearly entirely, within a range or degree of some characteristic or property. In some embodiments, “substantially” means within about 5%. In some embodiments, “substantially” means within about 10%. In some embodiments, “substantially” means within about 15%. In some embodiments, “substantially” means within about 20%.

[0074] As used herein, the term “target” or “target sequence” refers to a target nucleic acid molecule sequence, such as those that hybridize to oligonucleotide probes.

[0075] As used herein, the term “universal primer” refers to a primer that is capable of hybridizing to a target polynucleotide having a common complementary universal primer binding site and supporting amplification. Similarly, the term “universal primer pair” refers to a pair of forward and reverse primers that are capable of hybridizing and supporting PCR amplification of a target polynucleotide having common complementary forward and reverse universal primer binding sites. Such universal primer(s) and universal primer binding site(s) can allow for single- or dual-primer mediated universal amplification (e.g., universal PCR) of a region of a polynucleotide of interest. The headings provided herein are for convenience only and are not intended to limit or otherwise affect the scope or meaning of the embodiments disclosed herein.

[0076] SUMMARY

[0077] The present disclosure relates to methods, compositions, and kits for target enrichment by one-way primer extension, wherein the methods, compositions, and kits utilize both a toxic primer and a target capture primer. One aspect of the present disclosure is a method of enriching a plurality of target nucleic acid molecules, e.g., between about 1 and about 10,000 target nucleic acid molecules, or between about 1 and about 5,000 target nucleic acid molecules, or between about 1 and about 1,000 target nucleic acid molecules, wherein the method applies both a toxic primer and a target capture primer.

[0078] The present disclosure also relates to methods of amplifying a target enrichment sample, such as a target enrichment sample prepared using any of the methods described herein. Additionally, the present disclosure also relates to methods of sequencing using a target enrichment sample, such as a target enrichment sample prepared using any of the methods described herein. The methods of the present disclosure can be used as part of a sequencing protocol, including high-throughput single molecule sequencing protocols. In some embodiments, the methods of the present disclosure generate a library of enriched target nucleic acid molecules to be sequenced. The enriched target nucleic acid molecules in the library can be optionally incorporated with barcodes for molecular and sample identification, such as described in U.S. Pub. No. 2020 / 0032244 and U.S. Pat. Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678, and 8,722,368, the disclosures of which are incorporated by reference herein in their entireties.

[0079] In some embodiments, the disclosure describes a universal method of enrichment based on one-way dual-probe primer extension that applies both a toxic primer and a target capture primer. In some embodiments, the method comprises: hybridizing a first target capture primer to a first target nucleic acid molecule in a library of nucleic acid molecules, wherein the first target capture primer comprises a capture moiety; hybridizing a first toxic primer to a first non-target nucleic acid molecule in the library of nucleic acid molecules, wherein the first toxic primer does not comprise any capture moiety; extending the first hybridized target capture primer and the first hybridized toxic primer, wherein extension of the first hybridized target capture primer provides a first target capture primer extension complex comprising the first target nucleic acid molecule and the extended first target capture primer; and enriching the first target nucleic acid molecule in the library of nucleic acid molecules relative to the library of nucleic acid molecules. In some embodiments, the introduced toxic primer hybridizes and extends on the non-target nucleic acid molecule (non-target nucleic acid molecule) and prevents or reduces mispriming of the target capture primer to the non-target nucleic acid molecule. In other embodiments, the toxic primer hybridizes upstream of the target capture primer that is misprimed and hybridized to the non-target nucleic acid molecule and then extends from the misprimed and hybridized target capture primer and displaces the capture moiety. Both of these mechanisms of action are believed to prevent or reduce downstream enrichment of misprimed non-target nucleic acid molecules.

[0080] Advantageously, the compositions, kits, and methods of the disclosure allow for enrichment of low-abundance target nucleic acid molecules in a library of nucleic acids that includes low-abundance target nucleic acid molecules (e.g., mRNA; fusion transcripts) and high-abundance non-target nucleic acid molecules (e.g., rRNA; wild-type transcripts). In some embodiments, a toxic primer is applied to achieve targeted depletion of undesirable molecules, i.e., non-target nucleic acid molecules, from next-generation sequencing libraries. This can include rRNA-derived cDNA molecules, as well as other non-target molecules that are undesirably captured and subsequently amplified downstream (see, e.g., Figure 1A ). Additionally, in the context of cDNA libraries, the compositions, kits, and methods of the disclosure facilitate enrichment of low-abundance target molecules without the need for costly and time-consuming ribosome depletion steps. In other embodiments, a toxic primer is applied to achieve enrichment of desired molecules from next-generation sequencing capture libraries by depleting background molecule libraries. For example, in the search for new fusion transcripts, this embodiment would include depletion of wild-type transcripts (see, e.g., Figure 1B ).

[0081] Referring to Figure 2AIn some embodiments, the methods disclosed herein include obtaining a nucleic acid library comprising a plurality of nucleic acid molecules (step 101). In some embodiments, the nucleic acid molecules in the obtained nucleic acid library are selected from DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or a combination thereof. In some embodiments, the plurality of nucleic acid molecules comprises single-stranded polynucleotides. In some embodiments, the plurality of nucleic acid molecules is derived from a tissue sample, such as a tissue sample derived from a mammalian subject. In other embodiments, the plurality of nucleic acid molecules is derived from a cytological sample, such as a cytological sample derived from a mammalian subject.

[0082] In some embodiments, the obtained nucleic acid library includes a plurality of target nucleic acid molecules and / or a plurality of non-target nucleic acid molecules. In some embodiments, the non-target nucleic acid molecules are in high abundance compared to the target nucleic acid molecules in the nucleic acid library. In some embodiments, the non-target nucleic acid molecules comprise at least about 70% of the nucleic acid molecules in the obtained nucleic acid library. In other embodiments, the non-target nucleic acid molecules comprise at least about 75% of the nucleic acid molecules in the obtained nucleic acid library. In yet other embodiments, the non-target nucleic acid molecules comprise at least about 80% of the nucleic acid molecules in the obtained nucleic acid library. In further embodiments, the non-target nucleic acid molecules comprise at least about 85% of the nucleic acid molecules in the obtained nucleic acid library. In still further embodiments, the non-target nucleic acid molecules comprise at least about 90% of the nucleic acid molecules in the obtained nucleic acid library. In yet further embodiments, the non-target nucleic acid molecules comprise at least about 95% of the nucleic acid molecules in the obtained nucleic acid library. In some embodiments, the non-target nucleic acid molecules comprise at least about 96% of the nucleic acid molecules in the obtained nucleic acid library. In some embodiments, the non-target nucleic acid molecules comprise at least about 97% of the nucleic acid molecules in the obtained nucleic acid library. In some embodiments, the non-target nucleic acid molecules comprise at least about 98% of the nucleic acid molecules in the obtained nucleic acid library. In some embodiments, the non-target nucleic acid molecules comprise at least about 99% of the nucleic acid molecules in the obtained nucleic acid library.

[0083] In some embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 90% rRNA. In some embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 92% rRNA. In other embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 94% rRNA. In yet other embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 95% rRNA. In further embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 96% rRNA. In further embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 97% rRNA. In further embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 98% rRNA. In further embodiments, the obtained nucleic acid library is a cDNA library comprising mRNA-derived molecules and at least about 99% rRNA. In some embodiments, the cDNA library comprises rRNA and mRNA-derived molecules, and wherein the cDNA library is derived from a biological sample that has not been previously treated with a ribosome depletion method. In some embodiments, it is desirable to enrich for mRNA-derived molecules without the need to perform a ribosome depletion step.

[0084] In some embodiments, the obtained nucleic acid library comprises fusion transcripts and wild-type transcripts. In some embodiments, the fusion transcripts are in lower abundance compared to the wild-type transcripts. In some embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 :3. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 :4. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 :5. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 : 10. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 :20. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 :50. In other embodiments, the ratio of fusion transcripts to wild-type transcripts in the obtained nucleic acid library is at least about 1 : 100.

[0085] In some embodiments, the obtained nucleic acid library is derived from one or more tumor samples, and the target nucleic acid molecules include low abundance fusion transcripts. Non-limiting examples of fusion transcripts for detection include EML4-ALK, ETV6-NTRK3, and ALK-RET fusion oncogenes. In other embodiments, the obtained nucleic acid library includes expressed housekeeping genes, wherein the expressed housekeeping genes are of low abundance relative to non-target nucleic acid molecules in the obtained nucleic acid library. Non-limiting examples of housekeeping genes include VCP, RAB7A, and CHMP2A.

[0086] In some embodiments, nucleic acid fragments are first prepared from a biological sample, such as a tissue sample and / or a cytological sample. A DNA sequencing library can be constructed from genomic DNA for genomic analysis, or from cDNA prepared from RNA or mRNA for transcriptomic analysis, and it can be constructed from DNA or cDNA of any organism species from which these nucleic acids can be extracted. In some embodiments, the obtained sample is sheared to provide a population of nucleic acid fragments. In some embodiments, shearing of the obtained genomic sample is achieved using mechanical (e.g., nebulization or sonication) and / or enzymatic fragmentation (e.g., restriction endonucleases). In some embodiments, the generated nucleic acid fragments are of random size. In some embodiments, the generated nucleic acid fragments have a length of less than about 1000 base pairs. In other embodiments, the generated nucleic acid fragments include sequence fragments having a sequence size of between about 100 to about 1000 base pairs in length. In yet other embodiments, the generated nucleic acid fragments include sequence fragments having a sequence size of between about 500 to about 750 base pairs in length.

[0087] In some embodiments, adaptors are then added to the population of nucleic acid molecules via a ligation reaction. In some embodiments, the adaptors include one or more barcode sequences. Methods of ligating adaptors to nucleic acid molecules are described in U.S. Patent Publication Nos. 2017 / 0037459, 2018 / 0334709, 2018 / 0016630, and in PCT Publication No. WO2017021449, the disclosures of which are incorporated by reference herein in their entireties.

[0088] Following fragmentation and preparation of the nucleic acid library (step 101), a pool of primers is introduced to the nucleic acid library or reaction mixture including the nucleic acid library and other components to effect hybridization (e.g., a buffer or the like). Following introduction, one or more toxic primers and one or more target capture primers of the pool of primers at least partially hybridize to one or more non-target and / or target nucleic acid molecules, respectively (steps 102, 103).

[0089] In some embodiments, the primer pool comprises one or more toxic primers and one or more target capture primers. In some embodiments, the primer pool comprises between about 1 and about 1000 different toxic primers. In some embodiments, the primer pool comprises between about 50 and about 750 different toxic primers. In other embodiments, the primer pool comprises between about 100 and about 500 different toxic primers. In some embodiments, the primer pool comprises between about 50 and about 2500 different target capture primers. In other embodiments, the primer pool comprises between about 150 and about 1000 different target capture primers.

[0090] In some embodiments, one or more of the target capture primers are target specific, and thus, are designed to hybridize to a subset of complementary nucleic acid molecules in a nucleic acid library comprising desired target genes, exons, and / or other genomic regions of interest (hereinafter “target nucleic acid molecules”). In some embodiments, the target capture primers comprise the Roche SeqCap EZ Probes pool (available from Roche Sequencing and Life Sciences, Indianapolis, IND). In some embodiments, the target capture primers comprise a pool of Ampliseq primers commercially available from ThermoFisher.

[0091] In some embodiments, the target capture primers can be composed of ribonucleic acids, deoxyribonucleic acids, or other nucleic acid analogs known in the art. In some embodiments, the target capture primers can comprise one or more unnatural nucleotides, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), gamma-PNAs, glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). In some embodiments, the target capture primers range in length from about 20 nucleotides to about 100 nucleotides. In other embodiments, the target capture primers range in length from about 40 nucleotides to about 80 nucleotides. In yet other embodiments, the target capture primers range in length from about 50 nucleotides to about 75 nucleotides.

[0092] In some embodiments, each of the one or more target capture primers is a conjugate comprising (i) a capture moiety; and (ii) a sequence that is substantially complementary to a sequence of one or more target nucleic acid molecules in the nucleic acid library. In some embodiments, each of the one or more target capture primers is capable of binding to a functionalized substrate through the capture moiety by hybridization and / or extension, and as described herein (see, e.g., Figure 3A and 4 ). In other embodiments, the target capture primers bind to a functionalized substrate through the capture moiety prior to hybridization, as described herein (see, e.g. Figure 5 and 6 ).

[0093] In some embodiments, the capture portion of each of the one or more target capture primers comprises a first moiety (e.g., a first reactive functional group) that is reactive with a second moiety (e.g., a second reactive functional group) of another entity (e.g., a second moiety conjugated to a functionalized substrate). In some embodiments, the first moiety is a first member of a particular binding entity pair; and the second moiety is a second member of the same particular binding entity pair. In some embodiments, “reacting” between the first moiety and the second moiety can mean that a covalent bond is formed between the two reactive groups or two reactive functional groups of the two moieties; or can mean that the two reactive groups or two reactive functional groups of the two moieties associate with each other, interact with each other, hybridize to each other, hydrogen bond to each other, etc. Thus, in some embodiments, “reacting” includes a binding event, such as the binding of a hapten to an anti-hapten antibody or the binding of biotin to streptavidin. In some embodiments, each of the target capture primers comprises the same capture moiety, e.g., biotin. In other embodiments, different subsets of the target capture primers comprise different capture moieties.

[0094] In some embodiments, the capture moiety can comprise biotin to bind to a functionalized substrate comprising avidin or streptavidin. In other embodiments, the capture moiety can comprise a thiolated molecule to bind to a functionalized substrate comprising gold particles. In yet other embodiments, the capture moiety can comprise an amine-terminated molecule to bind to an NHS-activated substrate.

[0095] In some embodiments, the capture moiety comprises an immobilized antibody that can be used to bind to a molecule comprising or conjugated to a particular antigen molecule, such as an antigen molecule that binds to a functionalized substrate. In other embodiments, the capture moiety comprises an antigen molecule that can be used to bind to an immobilized antibody, such as an antibody that binds to a functionalized substrate.

[0096] In some embodiments, the capture moiety comprises an enzyme that can be used to bind to a molecule comprising or conjugated to a particular enzyme substrate. In other embodiments, the capture moiety comprises a substrate for an enzyme that can be used to bind to an enzyme, such as an enzyme coupled to a functionalized substrate.

[0097] In some embodiments, the capture moiety comprises a receptor that can be used to bind to a molecule comprising or conjugated to a particular receptor ligand, such as a receptor ligand that binds to a functionalized substrate. In other embodiments, the capture moiety comprises one or more receptor ligands that can be used to bind to a molecule comprising a receptor, such as a receptor that binds to a functionalized substrate.

[0098] In some embodiments, the capturing portion includes a lectin that can be used to bind molecules comprising or conjugated to a specific glycan (such as a specific glycan that binds to a functionalized substrate). In other embodiments, the capturing portion includes one or more glycans that can be used to bind molecules comprising or conjugated to one or more lectins (such as one or more lectins that bind to a functionalized substrate).

[0099] In a further embodiment, the capture portion includes one or more nucleic acid sequences that can be used to bind molecules including or conjugated to complementary base sequences. In other embodiments, the capture portion may include a bound DNA / RNA aptamer capable of specifically binding to target analytes such as small molecules, peptides, proteins, or cells.

[0100] Each of one or more toxic primers is designed to hybridize with a second subset of complementary nucleic acid molecules in a nucleic acid library that does not include the desired gene, exons, and / or other genomic regions of interest (hereinafter referred to as "non-target nucleic acid molecules"). Unlike target capture primers, the toxic primers of this disclosure do not conjugate to any capture portion and therefore cannot react with any functionalized substrates.

[0101] In some embodiments, the toxic primer may consist of ribonucleic acid, deoxyribonucleic acid, or other nucleic acid analogs known in the art. In some embodiments, the toxic primer may include one or more non-natural nucleotides, such as locked nucleic acid (LNA), peptide nucleic acid (PNA), γ-PNA, glycol nucleic acid (GNA), and threonic acid (TNA). In some embodiments, the length of the toxic primer ranges from about 20 nucleotides to about 100 nucleotides. In other embodiments, the length of the toxic primer ranges from about 40 nucleotides to about 80 nucleotides. In still other embodiments, the length of the toxic primer ranges from about 50 nucleotides to about 75 nucleotides.

[0102] In some embodiments, the toxic primers have a lower melting temperature (T0) compared to the target capture primers. m In some embodiments, the T of the toxic primer m T of the target-capture primer m At least about 2% lower. In other embodiments, the T of the toxic primer... m T of the target-capture primer m The level is at least about 5%. In some other embodiments, the T of the toxic primer... m T of the target-capture primer m The level is at least about 10% lower. In a further embodiment, the T of the toxic primer... m T of the target-capture primer m The level is at least about 15% lower. In a further embodiment, the T of the toxic primer... m T of the target-capture primerm At least 20% lower.

[0103] In some embodiments, the melting temperature of the first target-capturing primer is at least about 1°C higher than the melting temperature of the first toxic primer. In other embodiments, the melting temperature of the first target-capturing primer is at least about 2°C lower than the melting temperature of the first toxic primer. In still some embodiments, the melting temperature of the first target-capturing primer is at least about 3°C ​​higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 4°C higher than the melting temperature of the first toxic primer. In a still further embodiment, the melting temperature of the first target-capturing primer is at least about 5°C higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 6°C higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 7°C higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 8°C higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 9°C higher than the melting temperature of the first toxic primer. In a further embodiment, the melting temperature of the first target-capturing primer is at least about 10°C higher than that of the first toxic primer.

[0104] In some embodiments, the T of the toxic primer m At least about 50°C. In some embodiments, the T of the toxic primer m At least about 55°C. In some embodiments, the T of the toxic primer m At least about 60°C. In some embodiments, the T of the toxic primer m At least about 65°C. In some embodiments, the T of the toxic primer m At least about 70°C. In some embodiments, the T of the toxic primer m At least about 75°C. In some embodiments, the T of the toxic primer m At least approximately 80°C.

[0105] In some embodiments, the concentration of the target capture primers and the concentration of the toxic primers are approximately the same. In other embodiments, the concentration of the target capture primers and the concentration of the toxic primers are different. In some embodiments, the concentration of the toxic primers is at least two-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least five-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least ten-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least twenty-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least fifty-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least one hundred-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least two hundred-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least five hundred-fold higher than the concentration of the target capture primers. In some embodiments, one or more of the target capture primers can misprime, i.e., hybridize to a non-target nucleic acid molecule rather than a target nucleic acid molecule. Mispriming events are illustrated herein Figure 3A and 4 In some embodiments, the concentration of the target capture primers and the concentration of the toxic primers are approximately the same. In other embodiments, the concentration of the target capture primers and the concentration of the toxic primers are different. In some embodiments, the concentration of the toxic primers is at least two-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least five-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least ten-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least twenty-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least fifty-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least one hundred-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least two hundred-fold higher than the concentration of the target capture primers. In other embodiments, the concentration of the toxic primers is at least five hundred-fold higher than the concentration of the target capture primers. In some embodiments, one or more of the target capture primers can misprime, i.e., hybridize to a non-target nucleic acid molecule rather than a target nucleic acid molecule. Mispriming events are illustrated herein

[0106] After one or more target capture primers hybridize to one or more target nucleic acid molecules (step 102) and one or more toxic primers hybridize to one or more non-target nucleic acid molecules (step 103), each of the one or more hybridized target capture primers and the one or more hybridized toxic primers are extended (step 104) to form one or more extended target capture primer complexes and / or one or more extended toxic primer complexes. In those instances where a target capture primer is misprimed, the misprimed and hybridized target capture primer will extend along the non-target nucleic acid molecule, as further illustrated herein (see, e.g. Figure 3A and Figures 4-6 ).

[0107] In some embodiments, the one or more hybridized target capture primers are extended by using a first polymerase, thereby forming one or more double-stranded products, each double-stranded product comprising a target nucleic acid molecule hybridized to the extended target capture primer. In some embodiments, each of the one or more extended target capture primer complexes comprises an extended target capture primer (which itself comprises a hybridized target capture primer, which comprises a capture moiety) and a target nucleic acid sequence hybridized to the target capture primer. In some embodiments, the extended target capture primer comprises a reverse complement of at least a portion of the target nucleic acid molecule hybridized to the target capture primer.

[0108] In the event that one or more of the target capture primers are misprimed, those misprimed target capture primers can also be extended using the first polymerase. In some embodiments, one or more misprimed hybridized target capture primers (hybridized to non-target nucleic acid molecules) are extended by using the first polymerase, thereby forming one or more double-stranded products, each comprising a non-target nucleic acid molecule hybridized to the extended target capture primer.

[0109] Likewise, one or more hybridized toxic primers are extended by using the second polymerase, thereby forming one or more double-stranded products, each comprising a non-target nucleic acid molecule hybridized to the extended toxic primer. In some embodiments, each of the one or more extended toxic primer complexes comprises an extended target capture primer and a non-target nucleic acid sequence hybridized to the toxic primer. In some embodiments, the extended toxic primer comprises at least a portion of the reverse complement of the non-target nucleic acid molecule hybridized to the toxic primer.

[0110] In some embodiments, the first polymerase used to extend one or more hybridized target capture primers is the same as the second polymerase used to extend one or more hybridized toxic primers. In other embodiments, the first polymerase used to extend one or more hybridized target capture primers is different than the second polymerase used to extend one or more hybridized toxic primers. Depending on the type of nucleic acid molecules being analyzed, the polymerase can be a DNA-dependent DNA polymerase (“DNA polymerase”) or an RNA-dependent DNA polymerase (“reverse transcriptase”). Suitable polymerases are selected from Taq or Taq- derived polymerases (e.g., KAPA 2G polymerase from KAPA BIOSYSTEMS); or B- family DNA polymerases (e.g., KAPA HIFI polymerase from KAPA BIOSYSTEMS).

[0111] In some embodiments, the hybridization and extension processes (steps 102, 103, and 104) are performed simultaneously. In other embodiments, the hybridization and extension processes (steps 102, 103, and 104) are performed sequentially. In some embodiments, the length of extension of one or more hybridized target capture primers and one or more hybridized toxic primers can be actively controlled, such as by inactivating the added first and / or second polymerases, or passively allowed to complete, such as by depleting limiting reagents. Such methods are further described in U.S. Patent Publication No. 2020 / 0032244, the disclosure of which is incorporated by reference herein in its entirety.

[0112] Referring again to Figure 2AFollowing extension of both the hybridized target capture primers and the hybridized toxic primers (step 104), the nucleic acid library is enriched for the presence of the one or more target nucleic acid molecules. In some embodiments, enrichment involves increasing the concentration of the one or more target nucleic acid molecules by depleting (i.e., removing) other members of the nucleic acid molecule library. In some embodiments, enrichment includes the step of forming one or more released primer extension complexes.

[0113] One method of target nucleic acid molecule enrichment is shown in FIG. 2. For example, one or more target capture primer extension complexes are captured in step 201. Capture of the one or more target capture primer extension complexes can be achieved in a variety of ways as described herein and can be achieved prior to, concurrently with, or after the hybridization and / or extension steps described above. Figure 2B

[0114] In some embodiments, capture includes contacting the target capture primer extension complexes with an appropriately functionalized substrate (e.g., a bead) following the hybridization and / or extension steps, allowing the capture portion of each target capture primer extension complex to react with a corresponding portion of the functionalized substrate, thereby binding the one or more target capture primer extension complexes to the functionalized substrate. For example, the target capture primer extension complexes can include a capture portion comprising biotin, which can bind to a functionalized substrate comprising streptavidin. In these embodiments, the capture portion is coupled to the functionalized substrate (e.g., a bead) prior to the hybridization step (i.e., prior to step 102), allowing capture to occur by hybridization of each target capture primer to its corresponding target nucleic acid. Other methods of capturing target capture primer extension complexes are further described herein and are also described in U.S. Pub. No. 2020 / 0032244, the disclosure of which is incorporated by reference herein in its entirety.

[0115] In some embodiments, the functionalized substrate comprises a bead having an appropriately functionalized surface (where the substrate can be functionalized with any of the moieties described above). In some embodiments, the bead is included in a column or a microfluidic device. In some embodiments, the bead is a magnetic bead. In other embodiments, the bead is a non-magnetic bead.

[0116] ​After capturing the one or more target capture primer extension complexes, one or more purification processes are performed to remove non-target nucleic acid molecules as well as any other unused reaction components (e.g., nucleotides, primer molecules, enzymes, buffers, etc.) (step 202). For example, the non-target nucleic acid molecules can be removed by flowing a wash solution and / or buffer through a column comprising a functionalized substrate (e.g., a column comprising a plurality of beads having a suitably functionalized surface). In some embodiments, the wash is performed at least once. In other embodiments, the wash is performed at least twice. In yet other embodiments, the wash is performed at least three times. The skilled artisan will recognize that the one or more target capture primer extension complexes bound to the functionalized substrate (e.g., beads) will remain bound to the functionalized substrate as the wash solution and / or buffer is flowed through the column packed with the functionalized substrate, while those unbound non-target nucleic acid molecules will be washed away, thereby producing a reaction mixture enriched with the one or more target nucleic acid molecules.

[0117] In some embodiments, the target nucleic acid molecules can be optionally amplified after enrichment. In some embodiments, the captured target nucleic acid molecules are directly amplified while coupled to the functionalized surface. Such methods are described in U.S. Patent Nos. 10,240,192, 10,160995, and 7,842,457, the disclosures of which are incorporated by reference herein in their entireties. In other embodiments, the one or more target nucleic acid molecules are released from the captured target capture primer extension complex prior to an amplification step (step 203).

[0118] In some embodiments, the release of the one or more target nucleic acid molecules follows Figure 2C the workflow described. For example, in some embodiments, one or more release primers hybridize to the one or more target nucleic acid molecules (step 211). In some embodiments, the one or more release primers are designed to hybridize to a portion of the one or more target nucleic acid molecules and between the first and second adaptors. In other embodiments, the one or more release primers are designed to hybridize to a portion of the target nucleic acid molecule upstream relative to the one or more target capture primer extension complexes. In some embodiments, primer design algorithms are designed to select primers as close to the capture primers as possible. In some instances, there is no gap between the capture and release primers. In some embodiments, the release primers will not have a capture portion, similar to a toxic primer. However, it will have a similar Tm to the capture primers.

[0119] Next, the one or more hybridized release primers are extended using a polymerase, thereby forming one or more extended hybridized release primers (step 212). In some embodiments, the extension of the one or more hybridized release primers with the polymerase releases the one or more target-extended target capture primers from the one or more target capture primer extension complexes. In some cases, the polymerase exhibits strand displacement activity. In some cases, the polymerase exhibits 5'-3' exonuclease activity (which digests single strands of double-stranded nucleic acid molecules) (referred to herein as 5'-3' double-stranded exonuclease activity) or double-stranded exonuclease activity. In some cases, the polymerase exhibits both strand displacement and 5'-3' double-stranded exonuclease activity. In some cases, the strand displacement, 5'-3' double-stranded exonuclease activity, or a combination thereof, can transfer the target nucleic acid molecules (e.g., the original target nucleic acid molecules from the provided sample) into solution.

[0120] For example, in some embodiments, the first double-stranded product containing the target nucleic acid molecule hybridized to the extended target capture primer is immobilized on a functionalized substrate, e.g., by affinity capture through a ligand (e.g., biotin or a derivative thereof) of the capture moiety of the extended target capture primer. In such embodiments, the strand displacement activity of the polymerase extending the hybridized release primer transfers the target nucleic acid molecule from the sample into solution. Alternatively, the 5'-3' exonuclease activity can degrade the extended target capture primer immobilized by affinity capture and hybridized to the target nucleic acid molecule, wherein the 5'-3' exonuclease activity thereby releases the target nucleotide into solution. Other methods of releasing the target nucleic acid molecule from the formed target capture primer extension complex are described in U.S. Patent Publication No. 2018 / 0016630, the disclosure of which is incorporated by reference herein in its entirety.

[0121] The released one or more target nucleic acid molecules can then be used in one or more downstream processes, e.g., sequencing, amplification, further coupling, etc. Referring again to Figure 2AIn some embodiments, a nucleic acid library enriched with one or more target nucleic acid molecules is optionally amplified (step 106). In some embodiments, the optional amplification step (106) includes either linear or exponential amplification (e.g., polymerase chain reaction (PCR)). As used herein, “PCR” refers to the in vitro amplification of a specific DNA sequence by simultaneous primer extension of the DNA complementary strand. As used herein, PCR encompasses derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, droplet digital PCR, and assembly PCR. Amplification of the enriched target nucleic acid molecules can include non-PCR-based methods. Non-limiting examples of non-PCR methods include nucleic acid sequence-dependent amplification (NASBA), transcription-mediated amplification (TMA), whole transcriptome amplification (WTA), whole genome amplification (WGA), multiple substitution amplification (MDA), strand substitution amplification (SDA), real-time SDA, rolling circle amplification, and / or loop-to-loop amplification.

[0122] Generally, amplification (step 106) involves amplifying a target nucleic acid molecule using a polymerase, a first amplification primer, and a second amplification primer. In some embodiments, the first and second amplification primers are designed to be complementary to an adaptor sequence incorporated into one or more target nucleic acid molecules in the nucleic acid molecule library of step 101. For example, the first amplification primer may have a 3′ end complementary to a first adaptor, and the second amplification primer may have a 3′ end complementary to a second adaptor. In some embodiments, the amplification primers may include any sequence present in the target nucleic acid molecule being amplified (e.g., gene / target-specific primers, universal primers, etc.) and may support the synthesis of one or both strands (i.e., both the upper and lower strands of the double-stranded nucleic acid molecule corresponding to the template for the amplification reaction). In some embodiments, the first and second amplification primers are universal primers. Other amplification methods are described in U.S. Publication Nos. 2020 / 0032244 and 2018 / 0016630, the disclosures of which are incorporated herein by reference in their entirety.

[0123] In some embodiments, sequencing may be optionally performed according to any method known to those skilled in the art (see [link to documentation]). Figure 2Aof step 107). In some embodiments, sequencing methods include Sanger sequencing and dye terminator sequencing, as well as next generation sequencing technologies such as pyrosequencing, nanopore sequencing, micro-well based sequencing, nanoball sequencing, MPSS, SOLiD, Illumina, Ion Torrent, Starlite, SMRT, tSMS, sequencing by synthesis, sequencing by ligation, mass spectrometry sequencing, polymerase sequencing, RNA polymerase (RNAP) sequencing, microscopy-based sequencing, microfluidic Sanger sequencing, microscopy-based sequencing, RNAP sequencing, and the like. For example, instrumentation and methods for sequencing are disclosed in PCT Publication Nos. WO2014144478, WO2015058093, WO2014106076, and WO2013068528, the disclosures of which are incorporated by reference herein in their entireties.

[0124] In some embodiments, sequencing (step 107) can be performed by a number of different methods, such as by employing a sequencing by synthesis technique. Sequencing by synthesis, according to the art, is defined as any sequencing method that monitors the production of a by-product following the incorporation of a particular deoxy-nucleotide triphosphate during the sequencing reaction (Hyman, 1988, Anal. Biochem. 174:423-436; Ronaghi et al., 1998, Science 281 :363-365). One prominent embodiment of sequencing by synthesis is the pyrosequencing method. In this case, the production of pyrophosphate during nucleotide incorporation is monitored by an enzymatic cascade that results in the production of a chemiluminescent signal. The 454 Genome Sequencer system (Roche Applied Science Cat. No. 04 760 085 001) is an example of sequencing by synthesis, which is based on the pyrosequencing technology. As described in the product literature, for sequencing on the 454 GS20 or 454 FLX instruments, the average genomic DNA fragment size is in the range of 200 or 600 bp, respectively.

[0125] In some embodiments, sequencing by synthesis can alternatively be based on the termination dye type of the sequencing reaction. In this case, the incorporated dye deoxy-nucleotide triphosphate (ddNTP) building block contains a detectable label, which is preferably a fluorescent label that prevents further extension of the nascent DNA strand. The label is then removed and detected after incorporation of the ddNTP building block into the template / primer extension hybrid, for example, by using a DNA polymerase that contains a 3'-5' exonuclease or proofreading activity.

[0126] In some embodiments, and in the case of the Genome Sequencer System workflow (Roche Applied Science Cat. No. 04 896 548 001), in the first step, amplification (cloning) is performed by emulsion PCR. Thus, the step of amplification is performed by the emulsion PCR method, which is also within the scope of the present disclosure. The beads carrying the clonally amplified target nucleic acid molecules can then be arbitrarily transferred to a picotiter plate and subjected to pyrosequencing reactions for sequence determination according to the manufacturer's protocol.

[0127] In some embodiments, sequencing is performed using next-generation sequencing methods, such as those provided by Illumina, Inc. ("Illumina sequencing methods"). Without wishing to be bound by any particular theory, Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid, accurate sequencing. The process identifies DNA bases simultaneously as they are incorporated into a growing strand of nucleic acid. Each base emits a unique fluorescent signal as it is added to the growing chain, which is used to determine the order of the DNA sequence.

[0128] As noted above, it is desirable to prevent target capture primers from inadvertently "mispriming" (i.e., binding to non-target sequences). In the first non-limiting embodiment in Figure 3A The ability of a toxic primer to prevent or reduce mispriming from scratch is illustrated in the first non-limiting embodiment in Figure 4 The ability of a toxic primer to prevent or reduce capture of misprimed non-target molecules is illustrated in the second non-limiting embodiment in

[0129] Figure 3A A method of target nucleic acid enrichment is depicted in which the extended toxic primer prevents or reduces the incidence of mispriming of a target capture primer. In this embodiment, the extension of the hybridized toxic primer prevents the target capture primer from hybridizing to a non-target nucleic acid molecule. Turning first to Figure 3AFigure 301, a library of nucleic acid molecules is first prepared, where the library of nucleic acid molecules includes one or more target nucleic acid molecules 323 and one or more non-target nucleic acid molecules 333. In some embodiments, the one or more target nucleic acid molecules 323 are in low abundance compared to the one or more non-target nucleic acid molecules in the library of nucleic acid molecules. For example, in some embodiments, the one or more target nucleic acid molecules can represent less than 10% of the nucleic acid molecules in the library of nucleic acid molecules. In other embodiments, the one or more target nucleic acid molecules can represent less than 5% of the nucleic acid molecules in the library of nucleic acid molecules. In some embodiments, adaptors (321, 322, 331, and 332) are attached to both ends of the one or more target nucleic acid molecules 323 and the one or more non-target nucleic acid molecules 333.

[0130] Figure 301 shows a target nucleic acid molecule 323, where the target nucleic acid molecule includes a first end comprising a first adaptor 301 and a second end comprising a second adaptor 322. Figure 301 further shows a non-target nucleic acid molecule 333, where the non-target nucleic acid molecule includes a first end comprising a first adaptor 331 and a second end comprising a second adaptor 333. The first adaptors 321 and 331 are located at the 5' end of the target and non-target nucleic acid molecules, respectively. The second adaptors 322 and 332 are located at the 3' end of the target and non-target nucleic acid molecules, respectively. In some embodiments, the first adaptors 321 and 331 are identical; similarly, in some embodiments, the second adaptors 331 and 332 are identical.

[0131] After the library of nucleic acid molecules is prepared, one or more target capture primers 324 and one or more poison primers 334 are hybridized to the target and non-target nucleic acid molecules 323 and 333, respectively (see Figure 302). In some embodiments, the one or more target capture primers 324 each include a target capture region 325 that is complementary to at least a portion of the target nucleic acid molecules 323 and a capture moiety 326. The capture moiety can include any of those described above. In some embodiments, the capture moiety is biotin. In some embodiments, the one or more poison primers 334 include a non-target specific region that is complementary to at least a portion of the non-target nucleic acid molecules 333. Notably, the one or more poison primers 334 do not include a capture moiety and, therefore, cannot be captured during any enrichment step.

[0132] As Figure 3AAs shown in inset 303, each of one or more hybridization target capture primers and one or more toxic primers is extended. In some embodiments, one or more hybridization target capture primers 324 are extended with a first polymerase (not shown) to produce one or more target capture primer extension complexes 328. Each of the one or more target capture primer extension complexes 328 comprises a target nucleic acid molecule 323 and an extended target capture primer 327 (wherein the dashed lines indicate the extended portions of the hybridization target capture primer 324). As further shown in inset 303, one or more hybridization toxic primers 334 are extended with a second polymerase (not shown) to produce one or more extended toxic primers 335 (wherein the dashed lines again indicate the extended portions of the hybridization toxic primer 334).

[0133] Skilled technicians will recognize that, in some embodiments, the extension of the toxic primer prevents or mitigates the erroneous priming of the target-capture primer to the non-target strand (in contrast). Figure 3A small image 303 and Figure 4 (See figures 402 and 403). In other words, the formation of double-stranded polynucleotides prevents or mitigates the binding of target capture primers to non-target nucleic acid molecules, thereby preventing or mitigating erroneous priming.

[0134] Next, the target-capture primer extension complex 328 was captured onto the functionalized substrate 329 (see [link]). Figure 3A (See small figure 304). In some embodiments, the functionalized substrate includes a liquid support (e.g., beads or another similar particle) or a solid support (e.g., a silicon wafer, a glass slide, etc.). Examples of magnetic glass particles and devices using magnetic glass particles are described in U.S. Patent Nos. 656,568, 6,274,386, 7,371,830, 6,870,047, 6,255,477, 6,746,874, and 6,258,531, the disclosures of which are incorporated herein by reference in their entirety. In the embodiment shown in small figure 304, the target capture primer extension complex 328 is captured on the functionalized substrate 329 via a capture portion 326. In some embodiments, the capture portion 326 comprises biotin, and the functionalized substrate comprises streptavidin. Alternative methods of target capture are further described herein (see, for example, Figure 5 (Example).

[0135] In some embodiments, and referring to Figure 3B The capture involves hybridizing a universal capture oligonucleotide 351 with a target capture primer 324. In this specific embodiment, the target capture primer 324 includes a portion comprising a capture sequence (i.e., a universal nucleic acid sequence 350) (see inset 310). The introduced universal capture oligonucleotide 351 includes a portion having a nucleic acid sequence complementary to the universal capture sequence 350 of the target capture primer 324; and further includes a capture portion 351. Figure 3AThe embodiment depicted in panel 304 is the same as the embodiment described above with respect to panel 301, with the addition that the capture further comprises the introduction of a functionalized substrate 329, wherein the functionalized substrate 329 comprises a moiety capable of reacting or binding to the capture moiety 351.

[0136] Following capture, the target capture primer extension complex 328 is enriched relative to the nucleic acid molecules present in the nucleic acid library. In some embodiments, the enrichment comprises removal of nucleic acid molecules that are not captured onto the functionalized substrate. In some embodiments, the non-captured nucleic acid molecules can be removed by flowing through one or more washes and / or buffers through a column comprising a liquid phase support. By comparing panels 301 and 305, the skilled artisan will recognize that following capture of one or more target capture primer extension complexes and removal of non-captured nucleic acid molecules, the initial reaction mixture is enriched in one or more captured target nucleic acid molecules. In some embodiments, the captured target nucleic acid molecules are directly amplified while coupled to the functionalized surface. In other embodiments, as described further herein (see Figure 7 ), the target capture primer extension complex 328 can be released from the functionalized substrate 329, such that the target nucleic acid molecule can be used for further downstream process operations (e.g., amplification and / or sequencing).

[0137] Figure 4 Methods of target nucleic acid enrichment are depicted, wherein the extension of a toxic primer prevents capture of non-target nucleic acid molecules that are misprimed by the target capture primer. As described herein, extension of a toxic primer hybridized to a non-target nucleic acid molecule will cleave the capture moiety from the resulting target capture primer extension complex. Turning first to Figure 4 panel 401, a library of nucleic acid molecules is first prepared, wherein the library of nucleic acid molecules comprises one or more target nucleic acid molecules 423 and one or more non-target nucleic acid molecules 433. In some embodiments, adaptors (421, 422, 431, and 432) are attached to both ends of the one or more target nucleic acid molecules 423 and the one or more non-target nucleic acid molecules 433. In some embodiments, the one or more target nucleic acid molecules 423 are present at a low abundance compared to the one or more non-target nucleic acid molecules in the library of nucleic acid molecules. For example, in some embodiments, the one or more target nucleic acid molecules can represent less than 10% of the nucleic acid molecules in the library of nucleic acid molecules. In other embodiments, the one or more target nucleic acid molecules can represent less than 5% of the nucleic acid molecules in the library of nucleic acid molecules. Panel 401 shows a target nucleic acid molecule 423, wherein the target nucleic acid molecule comprises a first end comprising a first adaptor 401 and a second end comprising a second adaptor 422.

[0138] Figure 401 further illustrates a non-target nucleic acid molecule 433, where the non-target nucleic acid molecule includes a first end comprising a first adaptor 431 and a second end comprising a second adaptor 433. The first adaptors 421 and 431 are located at the 5' end of the target and non-target nucleic acid molecules, respectively. The second adaptors 422 and 432 are located at the 3' end of the target and non-target nucleic acid molecules, respectively. In some embodiments, the first adaptors 421 and 431 are identical; similarly, in some embodiments, the second adaptors 431 and 432 are identical.

[0139] After the nucleic acid library is prepared, one or more target capture primers 424 and one or more toxic primers 434 are hybridized to the target and non-target nucleic acid molecules 423 and 433, respectively (see Figure 402). In some embodiments, the one or more target capture primers 424 each include a target capture region 425 that is complementary to at least a portion of the target nucleic acid molecule 423 and a capture moiety 426 (e.g., biotin). Figure 4

[0140] Figure 4 Figure 402 also illustrates the mispriming of the target capture primers 424 to the non-target nucleic acid molecule sequence 433. That is, in some embodiments, the target capture primers 424, which include the capture moiety 426, can hybridize to the non-target nucleic acid molecule 433, i.e., the target capture primers become "misprimed." Thus, the non-target nucleic acid molecule can include both hybridized toxic primers as well as hybridized and misprimed target capture primers.

[0141] In some embodiments, the one or more toxic primers 434 include a non-target specific region that is complementary to at least a portion of the non-target nucleic acid molecule 433. Notably, the one or more toxic primers 434 do not include a capture moiety and, thus, are not captured in downstream processes.

[0142] As Figure 4 ​As shown in inset 403, each of one or more hybridization target capture primers (including those that have been mistriggered) and one or more toxic primers are extended. In some embodiments, one or more target capture primers 424 that hybridize with a target nucleic acid molecule are extended with a first polymerase (not shown) to produce one or more target capture primer extension complexes 428. Each of the one or more target capture primer extension complexes 428 comprises a target nucleic acid molecule 423 and an extended target capture primer 427 (where the dashed line indicates the extended portion of the hybridized target capture primer 424). In some embodiments, target capture primers that hybridize with non-target nucleic acid molecules are also extended with the first polymerase (not shown) to produce one or more mistriggered extension complexes 430. As further shown in inset 403, one or more hybridization toxic primers 434 are extended with a second polymerase (not shown) to produce one or more extended toxic primers 435 (where the dashed line again indicates the extended portion of the hybridized toxic primer 434).

[0143] like Figure 4 As further illustrated in small figure 404, as the toxic primer continues to extend, the extended toxic primer severs the capture portion 426 from the target capture primer, thereby releasing the capture portion from the target capture primer extension complex. Skilled technicians will recognize that the extension of the hybridized toxic primer prevents or reduces the occurrence of capture of non-target nucleic acid molecules.

[0144] Next, the primer extension complex 428 was captured using the functionalized substrate 429 capture target (see [link]). Figure 4 (Figure 405). In some embodiments, the functionalized substrate is a liquid support (e.g., beads or other similar particles) or a solid support (e.g., a silicon wafer, a glass slide, etc.). As described above, any magnetic glass particles and apparatuses using magnetic glass particles described in U.S. Patent Nos. 656,568, 6,274,386, 7,371,830, 6,870,047, 6,255,477, 6,746,874, and 6,258,531 can be applied. In the embodiment shown in Figure 405, the target capture primer extension complex 428 is captured on the functionalized substrate 429 via the capture portion 426. Alternative methods of target capture are described herein (see, for example, Figure 6 (Example).

[0145] After capture, the target capture primer extension complex 428 is enriched relative to the nucleic acid molecules present in the nucleic acid library. In some embodiments, enrichment includes removal of nucleic acid molecules that were not captured onto the functionalized substrate, e.g., uncaptured nucleic acid molecules can be removed by flowing through one or more washes and / or buffers through a column comprising a liquid phase support. By comparing small figures 401 and 406, the skilled artisan will recognize that after capturing one or more target capture primer extension complexes and removing uncaptured nucleic acid molecules, the initial reaction mixture is enriched with one or more captured target nucleic acid molecules. In some embodiments, the captured target nucleic acid molecules are directly amplified while coupled to the functionalized surface. In other embodiments, as described further herein (see Figure 7 ), the target capture primer extension complex 428 can be released from the functionalized substrate 429, thereby making the target nucleic acid molecules available for further downstream process operations (e.g., and / or sequencing).

[0146] As described herein, the capture portion of the target capture primer can be bound to the functionalized substrate prior to hybridization of the target capture primer to the target nucleic acid molecule. Figure 5 An alternative workflow is depicted, in which the target capture primer 324 is bound to the functionalized substrate 329 via a capture portion 326 prior to hybridization to the target nucleic acid molecule 323. In some embodiments, the target capture primer 324 bound to the functionalized substrate 329 is hybridized to the target nucleic acid molecule 323 (see small figure 502). At the same time, the toxic primer 334 is hybridized to the non-target nucleic acid molecule 333 (see small figure 502). As described in the embodiments described above, both the hybridized target capture primer 324 and the hybridized toxic primer 334 are extended. In some embodiments, extension of the toxic primer prevents or reduces mispriming of the target capture primer to the non-target nucleic acid molecule 333. As shown in small figure 503, the target capture primer extension complex 328 is bound to the functionalized substrate 329 via the capture portion 326, and thus does not require a separate capture step (as compared to small figure 304). Figure 3A Figure 3A As described herein, the target capture primer extension complex 328 can be released from the functionalized substrate 329, thereby making the target nucleic acid molecules available for further downstream process operations (e.g., sequencing).

[0147] Figure 6 ​Another alternative workflow is depicted, in which the target capture primer 424 is bound to the functionalized substrate 429 via the capture moiety 426 prior to hybridization with the target nucleic acid molecule 423. In some embodiments, the target capture primer 424 bound to the functionalized substrate 429 is hybridized with the target nucleic acid molecule 423 (see panel 602). At the same time, the toxic primer 434 hybridizes with the non-target nucleic acid molecule 433 (see panel 502). Also shown in panel 602 is the target capture primer 424 (again bound to the functionalized substrate 429 via the capture moiety 426) mispriming to the non-target nucleic acid molecule 433. As with Figure 4 Both the hybridized target capture primer 424 and the hybridized toxic primer 434 are extended (see panel 603), as with the described embodiments. As depicted in panel 604, as the toxic primer 434 continues to extend, the extended toxic primer cleaves the capture moiety 426 from the target capture primer and binds to the functionalized substrate 429, thereby releasing the capture moiety from the target capture primer extension complex 428. Figure 6

[0148] As shown in panel 604, the target capture primer extension complex 428 is bound to the functionalized substrate 429 via the capture moiety 426, and thus does not require a separate capture step (as compared to panel 405). In some embodiments, the enrichment includes removing nucleic acid molecules that are not captured onto the functionalized substrate, e.g., the nucleic acid molecules can be removed by flowing through one or more washes and / or buffers through a column comprising a liquid carrier. As described further herein, the target capture primer extension complex 428 can be released from the functionalized substrate 429, such that the target nucleic acid molecule can be used for further downstream process operations (e.g., sequencing). Figure 4 As previously described, in some embodiments, the target capture primer extension complex can be released from the functionalized substrate, such that the target nucleic acid molecule can be amplified and / or sequenced.

[0149] The coupling of the target capture primer extension complex 728 to the functionalized substrate 729 via the capture moiety 726 is depicted (see panel 701). In some embodiments, a release primer 730 hybridizes with the target nucleic acid molecule 723 (see panel 702). In some embodiments, the release primer 730 is complementary to the target nucleic acid molecule 723 and hybridizes with the target nucleic acid molecule 723 at a 5' position relative to the target capture primer extension complex 728. As shown in panel 702, both the target capture primer extension complex 728 (attached to the functionalized substrate 729) and the release primer 730 hybridize with the target nucleic acid molecule 723. Figure 7 Figure 7 Referring to panel 703, the release primer 730 is extended, which cleaves the capture moiety 726 from the target capture primer extension complex 728 and binds to the functionalized substrate 729, thereby releasing the capture moiety from the target capture primer extension complex 728.

[0150] Referring to panel 703, the release primer 730 is extended, which cleaves the capture moiety 726 from the target capture primer extension complex 728 and binds to the functionalized substrate 729, thereby releasing the capture moiety from the target capture primer extension complex 728. Figure 7 ​​Figure 7B shows a small diagram 703 of the extension of the hybridized release primer 730 with a third polymerase (not shown) to provide an extended release primer 731. In some embodiments, the extension of the hybridized release primer 730 releases the extended target capture primer 727 from the target capture primer extension complex 728. In some embodiments, the extended target capture primer 727 remains attached to the solid support 218.

[0151] Figure 7B shows a small diagram 703 of the extension of the hybridized release primer 730 with a third polymerase (not shown) to provide an extended release primer 731. In some embodiments, the extension of the hybridized release primer 730 releases the extended target capture primer 727 from the target capture primer extension complex 728. In some embodiments, the extended target capture primer 727 remains attached to the solid support 218.

[0152] Examples Principle validation was performed using the universal human reference UHR (Agilent) RNA as test material with an input of 25 ng. Kapa RNA HyperPrep was used to prepare the cDNA library. Kapa Ribo-Erase was used to prepare the rRNA depleted library as a positive control. Using the reference GRCh38, two sets of toxic primers were designed against the two strands of human rRNA derived cDNA. The rRNA toxic primers consisted of 98 primers (SEQ ID NOs: 1-98) designed with a T m Between 54°C and 58°C. The NCBI nucleotide archive (https: / / www.ncbi.nlm.nih.gov / nuccore) was searched for “Homo sapiens” [Organism] AND “ribosomal ma” [All Fields] AND biomol_rrna [PROP]” to identify all human rRNA genes. The nucleotide sequences were aligned using clustalW to investigate sequence similarity. Based on sequence similarity, the list was reduced to representative rRNAs (Table 1). All possible primers of 16-31 bp in length were created against the listed rRNA sequences. The primer sequences were aligned to the human reference genome GRCh38 (https: / / github.com / dib-lab / ged-docs / wiki / BLAT) using blat. The putative primers with good PCR amplification properties were selected based on the number of mapping sites and melting point (> 54°C). Primers were chosen to span each rRNA target on the positive and negative strand with little overlap between primers. The toxic primers were included in the capture reaction with the capture primers. The target capture primers consisted of 152 primers designed to detect known gene fusions with an average T mwas 55°C. Target capture primers were accompanied by 152 primers designed to release the captured fragments from streptavidin in a subsequent release reaction (“release primers”). Input for capture extension included reactions with toxic primers at 1 mM and reactions without toxic primers as a control. Capture extension reactions included: 1 pg cDNA, Kapa 2G Multiplex Mix (5X), 10 pL Blocking Oligo (37.3 mM), 2 pL capture primer pool (1.64 nM / primer), and 2 pL, toxic primer pool (5 nM / primer), and 16 pL ddH2O. Capture extension reactions were performed using the following thermocycling: 95°C for 2 minutes, 80°C for 1 second, 2% down to 60°C for 10 minutes, 65°C for 2 minutes and held at 4°C. Capture extension products were captured by incubation with streptavidin magnetic beads in lx binding buffer (100 mM NaCl, 10 mM Tris-HCl, 2% SDS). Beads were washed using wash buffer. Release primers were hybridized to the bead captured targets for 30 minutes at 55°C. Release primer extension was performed using sgPETE release mix at 50°C for 2 minutes. Final release reactions were amplified using Kapa HiFi ReadyMix for 15 cycles of amplification. Sequencing was performed to assess whether toxic primer depletion was successful by using a 2x150bp read workflow on an Illumina Nextseq 500. The number of demultiplexed reads per sample ranged from 3.98 million to 101 million. For bioinformatics analysis, the number of reads was subsampled to 35 million reads for all samples. De-duplication of quality filtered sequencing reads using fastp was performed using UMI tools using default settings. Sequencing reads mapped to rRNA reads were mapped using BWM MEM. The remaining non-rRNA reads were mapped to RNA reverence Hg38 using STAR alignment software. Figure 8 The UHR control sample had no rRNA depleted cDNA as input and no toxic primers in the capture reaction. The UHR no RiboErase 1 mM sample had rRNA depleted cDNA as input and 1 mM toxic primers in the capture reaction. The sample with toxic primers showed a decrease in off-target rRNA fragment abundance sequenced compared to the control sample (average of 44.83% (± 1.97%)) to an average of 19.47% (± 4.17%). The number of expected targets also increased when toxic primers were included in the capture reaction. Out of the 35 million reads subsampled, 23.3 million (± 1.7 million) fusion target reads were detected with toxic primers compared to 10.5 million (± 0.8 million) sequencing reads detected in the control sample Figure 9A). The inclusion of the toxic primers results in the fusion targets having higher read depth with the same overall sequencing depth. In this example, the control sample would have theoretically required 7.71 million reads to reach the same read depth of the fusion targets as the toxic primer treatment ( Figure 9B ). There was no significant difference in % reads off-target and un-mapped reads between the Ribo-Erase treated samples and the Ribo-Erase treated samples including toxic primers. Thus, for the Ribo-Erase treated samples, the inclusion of toxic primers was observed to have no negative impact on sequencing metrics Figure 8

[0153] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are hereby incorporated by reference, in their entirety. As necessary, modifications can be made to various aspects of the embodiments in order to employ concepts of the various patents, applications and publications, to provide other further embodiments within the scope of the present disclosure.

[0154] Although the present disclosure has been described with reference to certain illustrative embodiments, those of ordinary skill in the art will recognize that changes can be made in form and detail without departing from the spirit and scope of the disclosure. More specifically, it will be apparent to one of skill in the art that the subject combinations, arrangements of components and / or arrangements can be resorted to in other ways, within the scope and spirit of the disclosure, without departing from the subject matter. Alternative uses will also become apparent to those of ordinary skill in the art, in light of the teachings herein. SEQUENCE LISTING <110> F. Hoffmann-La Roche Ltd. F. Hoffmann-La Roche Ltd. F. Hoffmann-La Roche Ltd. F. Hoffmann-La Roche Ltd. <120> Targeted depletion of non-target library molecules using toxic primers in the target capture process of next generation sequencing libraries <130> P35925-WO <140> <141> <150> US 63 / 049,255 <151> 2020-07-08 <160> 98 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence ​<220> <221> Source <223> / note=“synthetic toxic primer” <400> 1 gatccaccgc taagagtc 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 2 cattaattct cgcagctagc 20 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 3 tgtctgagcg tcgctt 16 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 4 gtggtatggc cgtagac 17 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 5 gggtgctgta ggcttt 16 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 6 ggcaggatca accaggta 18 <210> 7 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 7 cactgtaccg gccgtg 16 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 8 ataactgtgg taattctaga gcta 24 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 9 aaatgcacgc atcccc 16 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 10 taacctcggg ccgatc 16 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 11 gcgactacca tcgaaagt 18 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 12 ggggaggtag tgacgaa 17 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 13 caattacagg gcctcgaa 18 <210> 14 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 14 gatcttggga gcgggc 16 <210> 15 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 15 aagagcatcg aggggg 16 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 16 ctattttgtt ggttttcgga ac 22 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 17 cgccggtcca agaattt 17 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 18 ataaacgatg ccgaccg 17 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 19 ccggaaccca aagacttt 18 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 20 ggacacggac aggattg 17 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 21 aaagagctat caatctgtca atc 23 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 22 tgctaactag ttacgcgac 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 23 catcacagac ctgttattgc 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 24 ttgcaattat tccccatgaa c 21 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 25 gcacttactg ggaattcct 19 <210> 26 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 26 gagcgctgag aagacg 16 <210> 27 <211> 25 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 27 cttttacttc ctctagatag tcaag 25 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 28 ctgatctgag gtcgcg 16 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 29 gggaatcctg gttagtttct t 21 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 30 gcccaagtcc ttctgatc 18 <210> 31 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 31 ccaagcaacc cgactc 16 <210> 32 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 32 taaacgggtg gggtcc 16 <210> 33 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / NOTE= "synthetic toxic primer" <400> 33 gccgggttga atcctc 16 <210> 34 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 34 cctcctcctc ctcccc 16 <210> 35 <211> 16 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 35 ggggggctgt aacact 16 <210> 36 <211> 16 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 36 gtgggtagcc gacgtc 16 <210> 37 <211> 16 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 37 tctccagtcc gccgag 16 <210> 38 <211> 16 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 38 ctcgtgctcc acctcc 16 <210> 39 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 39 cgtccgacct gggtata 17 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 40 cagctatcct gagggaaac 19 <210> 41 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 41 ggaatgcgag tgcctag 17 <210> 42 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 42 ccttaacccg gcgttc 16 <210> 43 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 43 cacctgccga atcaact 17 <210> 44 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 44 gctcccgtcc actctc 16 <210> 45 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 45 ctcctactcg tcgcgg 16 <210> 46 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 46 gagaagggtt ccatgtgaa 19 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 47 ccgactgacc catgttc 17 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 48 ggattcgggg atctgaac 18 <210> 49 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 49 gttcgccccg agagag 16 <210> 50 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 50 gacgctttcc aaggca 16 <210> 51 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 51 ccaaggtgaa cagcctc 17 <210> 52 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 52 ggcttgccga cttccc 16 <210> 53 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / note=“synthetic toxic primer” <400> 53 ctcgcgtcca gagtcg 16 <210> 54 <211> 17 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 54 taagtcggct gctaggc 17 <210> 55 <211> 19 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 55 gcgggagtaa ctatgactc 19 <210> 56 <211> 19 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 56 catgcgcgtc actaattag 19 <210> 57 <211> 17 <212> DNA <213> artificial sequence <220> <221> source <223> / note=“synthetic toxic primer” <400> 57 gaataagtgg gaggccc 17 <210> 58 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 58 accgggtcag tgaaaaaa 18 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 59 tacacctgtc aaacggtaac 20 <210> 60 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 60 ttctgtcctc cctgagc 17 <210> 61 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 61 ccaagcgttc atagcga 17 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 62 tcacaatgat aggaagagcc 20 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 63 gttttaccct actgatgatg tg 22 <210> 64 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 64 ctcagccaag cacataca 18 <210> 65 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 65 gttggcctcg gatagc 16 <210> 66 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 66 gttccccacg aacgtg 16 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> SOURCE <223> / NOTE= "synthetic toxic primer" <400> 67 tcgacacaag ggtttgtc 18 <210> 68 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE= "Synthetic toxic primer" <400> 68 aggctaggac caaacctat 19 <210> 69 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE= "Synthetic toxic primer" <400> 69 cagctcaaaa cgcttagc 18 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE= "Synthetic toxic primer" <400> 70 aaggttaatc actgctgttt c 21 <210> 71 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> NOTE= "Synthetic toxic primer" <400> 71 atagaagccg gcgtaaag 18 <210> 72 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 72 ttattgggga gggggtg 17 <210> 73 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 73 gcttagccct aaacctcaa 19 <210> 74 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 74 ggctcgtagt gttctgg 17 <210> 75 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 75 atataccgcc atcttcagc 19 <210> 76 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 76 tgtagccttc atcagggt 18 <210> 77 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 77 gaaaactacg atagccctta tg 22 <210> 78 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 78 gtgtgtacgc gcttca 16 <210> 79 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 79 agtgcacttg gacgaac 17 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 80 ggtttggggc taggtttag 19 <210> 81 <211> 22 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 81 gcaatagata tagtaccgca ag 22 <210> 82 <211> 27 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 82 cttgctatat tatgcttggt tataatt 27 <210> 83 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 83 ctaaaagagc acacccgt 18 <210> 84 <211> 19 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 84 cttggacaac cagctatca 19 <210> 85 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 85 ctaggaaaaa accttgtaga gag 23 <210> 86 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note= "synthetic toxic primer" <400> 86 tgagcttgaa cgctttct 18 <210> 87 <211> 28 <212> DNA <213> artificial sequence <220> <221> source <223> / note= "synthetic toxic primer" <400> 87 aatgttagta taagtaacat gaaaacat 28 <210> 88 <211> 20 <212> DNA <213> artificial sequence <220> <221> source <223> / note= "synthetic toxic primer" <400> 88 ttaatctgac gcaggcttat 20 <210> 89 <211> 26 <212> DNA <213> artificial sequence <220> <221> source <223> / note= "synthetic toxic primer" <400> 89 gaaaggttaa aaaaagtaaa aggaac 26 <210> 90 <211> 17 <212> DNA <213> artificial sequence <220> <221> source <223> / note= "synthetic toxic primer" <400> 90 aacaggcggg gtaagat 17 <210> 91 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 91 gttcagctgt ctcttacttt taa 23 <210> 92 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 92 tgttatgccc gcctct 16 <210> 93 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 93 gagcagaacc caacctc 17 <210> 94 <211> 17 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 94 tgtactgctc ggaggtt 17 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“synthetic toxic primer” <400> 95 acaatagggt ttacgacctc 20 <210> 96 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 96 ctgcaccatc gggatg 16 <210> 97 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 97 gtacgaaagg acaagagaaa taa 23 <210> 98 <211> 25 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note=“Synthetic toxic primer” <400> 98 gtgggtataa tactaagttg agatg 25

Claims

1. A method for enriching at least one target nucleic acid molecule in a nucleic acid molecular library, the method comprising: (a) Hybridize a first target capture primer with a first target nucleic acid molecule in the nucleic acid molecule library, wherein the first target capture primer contains a capture portion; (b) Hybridize the first toxic primer with a first non-target nucleic acid molecule in the nucleic acid molecular library, wherein the first toxic primer does not contain any capture portion; (c) Extending the target capture primer and the toxic primer of the first hybridization, wherein the extension of the target capture primer of the first hybridization provides a first target capture primer extension complex comprising the first target nucleic acid molecule and the extended first target capture primer; and (d) Enriching the first target nucleic acid molecule in the nucleic acid molecular library relative to the nucleic acid molecular library.

2. The method of claim 1, wherein the first target-capturing primer and the first toxic primer are added to the library as a primer pool.

3. The method according to claim 1 or 2, wherein the extension of the toxic primer of the first hybridization prevents the first target capture primer from hybridizing with the first non-target nucleic acid molecule.

4. The method according to claim 1 or 2, wherein the extension of the toxic primer of the first hybridization replaces the first target primer that hybridizes with the first non-target nucleic acid molecule.

5. The method according to claim 1 or 2, wherein each of the nucleic acid molecules in the nucleic acid molecular library comprises a first end including a first adaptor and a second end including a second adaptor.

6. The method of claim 5, further comprising amplifying the first target nucleic acid molecule with a first amplification primer and a second amplification primer, wherein the first amplification primer comprises a 3' end complementary to the first adaptor, and wherein the second amplification primer comprises a 3' end complementary to the second adaptor.

7. The method of claim 1, wherein enriching the first target nucleic acid molecule relative to the nucleic acid molecule library comprises (i) capturing the first target capture primer extension complex; (ii) removing uncaptured non-target nucleic acid molecules; and (iii) releasing the first target nucleic acid molecule from the captured first target capture primer extension complex.

8. The method of claim 7, wherein capturing the first target capture primer extension complex comprises contacting the first target capture primer extension complex with a functionalized substrate.

9. The method of claim 7, wherein capturing the first target-capturing primer extension complex comprises: (i) Hybridizing a universal capture oligonucleotide with the capture portion of the first target capture primer extension complex to form a universal capture oligonucleotide complex, wherein the universal capture oligonucleotide comprises (a) a first member of a specific binding entity pair and (b) a nucleotide sequence complementary to at least a portion of the capture sequence of the capture portion; (ii) contacting the universal capture complex with a functionalized substrate, wherein the functionalized substrate comprises a second member of the specific binding entity pair.

10. The method according to any one of claims 7 to 9, wherein removing uncaptured nucleic acid molecules comprises washing away said uncaptured non-target nucleic acid molecules.

11. The method according to any one of claims 7 to 9, wherein releasing the captured first target-capturing primer extension complex comprises: (i) Hybridize the release primer with the first target nucleic acid molecule; And (b) the release primer for extended hybridization.

12. The method according to any one of claims 1, 2, and 6 to 9, wherein the melting temperature of the first target-capturing primer is greater than the melting temperature of the first toxic primer.

13. The method according to any one of claims 1, 2, and 6 to 9, wherein the nucleic acid molecular library comprises a plurality of target nucleic acid molecules and a plurality of non-target nucleic acid molecules, wherein the plurality of target nucleic acid molecules are present in low abundance compared with the plurality of non-target nucleic acid molecules.

14. A reagent kit comprising: (a) A first target capture primer, the first target capture primer being complementary to a target nucleic acid molecule in a nucleic acid molecule library, and wherein the first target capture primer comprises a capture portion; as well as (b) A first toxic primer, which is complementary to a non-target nucleic acid molecule in the nucleic acid molecular library, and wherein the first toxic primer does not contain a capture portion.

15. A composition comprising (a) A nucleic acid library, wherein the nucleic acid library contains a variety of target nucleic acid molecules and a variety of non-target nucleic acid molecules, wherein the target nucleic acid molecules are present in low abundance compared with the variety of non-target nucleic acid molecules; (b) A first target capture primer, the first target capture primer being complementary to a first target nucleic acid molecule in a nucleic acid molecule library, and wherein the first target capture primer contains a capture portion; (c) A first toxic primer, which is complementary to a first non-target nucleic acid molecule in the nucleic acid molecule library, and wherein the first toxic primer does not contain a capture portion; and (d) First polymerase.

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