Compositions and methods for improving library enrichment

Abstract

To provide compositions and methods for improving library enrichment.SOLUTION: This disclosure describes a hybridization buffer including a crowding agent, a method that includes using the hybridization buffer, and a kit including the hybridization buffer. This disclosure also describes blockers for use in hybrid capture methods, methods of using those blockers, and a kit including those blockers. Additionally, this disclosure describes a method of hybrid capture that does not include amplifying the library members using PCR prior to sequencing the library members.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 764,753, filed August 15, 2018, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0002] Summary of the Invention Despite the rapid advances in throughput of next-generation sequencing (NGS), traditional library preparation and enrichment methods can be time-consuming and limit throughput. The present disclosure describes compositions containing blockers and / or hybridization buffers, methods for using these compositions, and methods for improving the efficiency of nucleic acid selection before sequencing.

[0003] In one embodiment, the present disclosure describes a hybridization buffer comprising a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

[0004] In another aspect, the disclosure describes a method comprising using a hybridization buffer. The disclosure also describes a kit comprising the hybridization buffer.

[0005] In a further aspect, the present disclosure describes a blocker comprising an oligonucleotide, wherein the blocker can bind to an adaptor. The adaptor comprises a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI). The region of the blocker that can bind to the index region and / or UMI of the adaptor comprises at least three thymine or universal bases and at least one non-universal base.

[0006] In yet another aspect, the present disclosure describes a blocker comprising two unconnected oligonucleotides. The blocker can be bound to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI). The unconnected oligonucleotides comprise bases that do not correspond to the index region and / or UMI of the adaptor.

[0007] The present disclosure also describes methods that include using the blockers described herein and kits that include the blockers described herein.

[0008] In a further embodiment, the present disclosure describes a method comprising contacting a library with a blocker in the presence of a hybridization buffer; and contacting the library with a probe, wherein the probe hybridizes to a region of interest within the library members. The method does not include amplifying the library members using PCR prior to sequencing the library members.

[0009] As used herein, the term "nucleic acid" is intended to be consistent with art usage and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to nucleic acids in a sequence-specific manner or can be used as templates for replicating specific nucleotide sequences. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures may have other backbone linkages, including any of a variety known in the art. Naturally occurring nucleic acids typically have deoxyribose sugars (e.g., those found in deoxyribonucleic acid (DNA)) or ribose sugars (e.g., those found in ribonucleic acid (RNA)). Nucleic acids can contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can contain natural or unnatural bases. In this regard, natural deoxyribonucleic acids can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine, and ribonucleic acids can have one or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. Useful unnatural bases that can be included in nucleic acids are known in the art. The term "target," when used in reference to a nucleic acid, is intended as a semantic identifier for that nucleic acid in the context of the methods or compositions set forth herein and does not necessarily limit the structure or function of that nucleic acid beyond what is otherwise expressly set forth.

[0010] As used herein, the term "T m " refers to the temperature at which half of the DNA strands of the sample are in a double helix state and half of the DNA strands of the sample are in a random coil state.

[0011] As used herein, the term "adapter" and its derivatives (e.g., universal adapter, non-target adapter, etc.) generally refer to any linear, single-stranded oligonucleotide that can be ligated to a nucleic acid molecule of the present disclosure. In some embodiments, the adapter is not substantially complementary to the 3' or 5' end of any target sequence present in a sample. In some embodiments, suitable adapter lengths are within the ranges of 10-100 nucleotides, 12-60 nucleotides, and 15-50 nucleotides in length. Typically, an adapter can contain any combination of nucleotides and / or nucleic acids. In some embodiments, an adapter can contain one or more cleavable groups at one or more positions. In other embodiments, an adapter can contain a sequence substantially identical to or substantially complementary to at least a portion of a primer, e.g., a universal primer. In some embodiments, an adapter can contain an index or tag to assist in downstream error correction, identification, or sequencing.

[0012] As used herein, the term "universal sequence" refers to a sequence region common to two or more nucleic acid molecules, such as adapter-target adapter molecules, where the molecules also have sequence regions that differ from each other. The presence of a universal sequence in different members of a molecular population can enable the capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to a portion of the universal sequence, such as the universal extension primer binding site. Non-limiting examples of universal extension primer binding sites include sequences identical to or complementary to P5 and P7 primers. Similarly, the presence of a universal sequence in different members of a molecular population can enable the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, such as the universal primer binding site. Thus, the universal capture nucleic acid or universal primer contains a sequence that can specifically hybridize to the universal sequence. Target nucleic acid molecules can be modified to attach universal adapters, such as those described herein, to one or both ends of various target sequences.

[0013] The terms "P5" and "P7" can be used to refer to amplification primers, such as universal primer extension primers. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complementary strands of P5 and P7, respectively. It is understood that any suitable amplification primer can be used in the methods presented herein, and the use of P5 and P7 is merely an exemplary embodiment. The use of amplification primers such as P5 and P7 on flow cells is known in the art, as exemplified by the disclosures of WO2007 / 010251, WO2006 / 064199, WO2005 / 065814, WO2015 / 106941, WO1998 / 044151 and WO2000 / 018957. For example, any suitable forward amplification primer can be used in the method presented herein for hybridizing to complementary sequences and amplifying sequences, whether immobilized or in solution.Similarly, any suitable reverse amplification primer can be used in the method presented herein for hybridizing to complementary sequences and amplifying sequences, whether immobilized or in solution.Those skilled in the art will understand how to design and use primer sequences suitable for capture and nucleic acid amplification as presented herein.

[0014] As used herein, "amplify," "amplifying," or "amplification reaction," and derivatives thereof, generally refer to any act or process in which at least a portion of a nucleic acid molecule is duplicated or copied onto at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally comprises a sequence substantially identical to or substantially complementary to at least a portion of a template nucleic acid molecule. The template nucleic acid molecule may be single-stranded or double-stranded, and the additional nucleic acid molecules may independently be single-stranded or double-stranded. Amplification optionally involves linear or exponential replication of the nucleic acid molecule. In some embodiments, such amplification may be performed using isothermal conditions; in other embodiments, such amplification may involve thermocycling. In some embodiments, amplification is multiplex amplification, which involves simultaneously amplifying multiple target sequences in a single amplification reaction. In some embodiments, "amplification" includes amplifying at least a portion of DNA-based and RNA-based nucleic acids, alone or in combination. The amplification reaction may involve any amplification process known to those of skill in the art. In some embodiments, the amplification reaction involves polymerase chain reaction (PCR).

[0015] As used herein, "amplification conditions" and derivatives thereof generally refer to conditions suitable for the amplification of one or more nucleic acid sequences. Such amplification can be linear or exponential. In some embodiments, amplification conditions can include isothermal conditions, or can include thermocycling conditions or a combination of isothermal and thermocycling conditions. In some embodiments, conditions suitable for the amplification of one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions refer to a reaction mixture sufficient to amplify a nucleic acid, such as one or more target sequences, or amplified target sequences ligated to one or more adapters, e.g., adapter-ligated amplified target sequences. Amplification conditions typically include a catalyst for amplification or nucleic acid synthesis, such as a polymerase; a primer having some degree of complementarity to the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs), that facilitate the extension of primers hybridized to the nucleic acid. Amplification conditions may require hybridization or annealing of primers to nucleic acids, extension of the primers, and a denaturation step to separate the extended primers from the nucleic acid sequence being amplified. Typically, amplification conditions may include thermocycling, but this is not required; in some embodiments, amplification conditions include multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, amplification conditions include Mg 2+ or Mn 2+ and various regulators of ionic strength may also be included.

[0016] As used herein, the term "polymerase chain reaction" ("PCR") refers to a method such as that described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which describes a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying a polynucleotide of interest involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycles in the presence of a DNA polymerase. The two primers are complementary to each strand of the double-stranded polynucleotide of interest. The mixture is first denatured at high temperature, and then the primers are annealed to complementary sequences within the polynucleotide of the target molecule. After annealing, the primers are extended by a polymerase to form a new pair of complementary strands. Repeating the steps of denaturation, primer annealing, and polymerase extension multiple times (referred to as thermocycling) results in a highly concentrated amplified segment of the desired polynucleotide of interest. The length of the amplified segment (amplicon) of the desired polynucleotide of interest is determined by the relative positions of the primers with respect to each other, and therefore is a controllable parameter. Because this process is repeated, the method is referred to as a "polymerase chain reaction" (hereinafter "PCR"). The desired amplified segment of the polynucleotide of interest becomes the predominant nucleic acid sequence (in terms of concentration) in the mixture and is therefore said to be "PCR amplified." In a variation of the method discussed above, target nucleic acid molecules can be PCR amplified using multiple different primer pairs, in some cases one or more primer pairs, per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction.

[0017] As defined herein, "multiplex amplification" refers to the simultaneous amplification of two or more target sequences in a sample. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified in a single reaction vessel. The "plexity" or "plex" of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during a single multiplex amplification. Amplified target sequences can also be detected by several different methods (e.g., densitometry after gel electrophoresis, quantification by bioanalyzer or quantitative PCR, hybridization with labeled probes; incorporation of biotinylated primers followed by detection of avidin-enzyme conjugates; incorporation of 32P-labeled deoxynucleotide triphosphates into amplified target sequences).

[0018] As used herein, the term "primer" and its derivatives generally refer to any polynucleotide that can hybridize to a target sequence of interest. Typically, a primer serves as a substrate onto which nucleotides can be polymerized by a polymerase; however, in some embodiments, a primer can become incorporated into a synthesized nucleic acid strand and provide a site onto which another primer can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid molecule. A primer can contain any combination of nucleotides or their analogs. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to polymeric forms of nucleotides of any length and can include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. These terms should be interpreted as including DNA or RNA analogs made of nucleotide analogs as equivalents and applicable to single-stranded (e.g., sense or antisense) polynucleotides and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, which is a complementary or copy DNA produced from an RNA template, for example, by the action of reverse transcriptase. The term refers only to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA").

[0019] As used herein, the terms "ligate," "ligation," and their derivatives generally refer to a process for covalently joining two or more molecules, e.g., a process for covalently joining two or more nucleic acid molecules to one another. In some embodiments, ligation involves joining nicks between adjacent nucleotides of nucleic acids. In some embodiments, ligation involves forming a covalent bond between the end of a first nucleic acid molecule and the end of a second nucleic acid molecule. In some embodiments, ligation may involve forming a covalent bond between the 5' phosphate group of one nucleic acid and the 3' hydroxyl group of a second nucleic acid to form a ligated nucleic acid molecule. In some embodiments, a target sequence may be ligated to an adapter to generate an adapter-target sequence. Those skilled in the art will recognize that a ligation reaction may not join all molecules present in the reaction.

[0020] As used herein, "ligase" and its derivatives generally refer to any agent capable of catalyzing the ligation of two substrate molecules. In some embodiments, ligases include enzymes capable of catalyzing the joining of nicks between adjacent nucleotides of nucleic acids. In some embodiments, ligases include enzymes capable of catalyzing the formation of a covalent bond between the 5' phosphate of one nucleic acid molecule and the 3' hydroxyl of another nucleic acid molecule, thereby forming a ligated nucleic acid molecule. Suitable ligases may include, but are not limited to, T4 DNA ligase, T4 RNA ligase, thermostable T4 DNA ligase, and E. coli DNA ligase.

[0021] As used herein, term " flow cell " refers to the chamber that has a solid surface through which one or more fluid reagents can flow.The example of flow cell and related fluid system and detection platform that can be easily used in the method of the present disclosure are described in, for example, Bentley et al., Nature 456:53-59 (2008); WO04 / 018497; WO91 / 06678; WO07 / 123744; United States Patent No. 7,057,026; United States Patent No. 7,211,414; United States Patent No. 7,315,019; United States Patent No. 7,329,492; United States Patent No. 7,405,281; and United States Patent Application Publication No. 2008 / 0108082.

[0022] As used herein, the term "library" refers to a collection of members. In one embodiment, a library comprises a collection of nucleic acid members, such as a collection of whole genomic fragments, subgenomic fragments, cDNA, cDNA fragments, RNA, RNA fragments, or combinations thereof. In some embodiments, some or all of the library members comprise non-target adapter sequences. The adapter sequences can be located at one or both ends. The adapter sequences can be used, for example, in sequencing methods (e.g., NGS methods), for amplification, reverse transcription, or cloning into vectors.

[0023] As used herein, the term "member" or "library member" or other similar terms refers to a nucleic acid molecule, e.g., DNA, RNA, or a combination thereof, that is a member of a library. Typically, a member is a DNA molecule, e.g., genomic DNA or cDNA. A member may be fragmented, e.g., sheared or enzymatically prepared, genomic DNA. A member may include sequences derived from a subject, and may also include sequences not derived from the subject, e.g., non-target sequences, e.g., adapter sequences, primer sequences, and / or other sequences that enable identification, e.g., indexes.

[0024] As used herein, an "index" (also referred to as an "index region" or "index adapter") refers to a nucleic acid tag that can be used to identify a sample or source of nucleic acid material. When nucleic acid samples are derived from multiple sources, the nucleic acids in each nucleic acid sample can be tagged with different nucleic acid tags so that the source of the sample can be identified. Any suitable index or set of indexes can be used, as known in the art and as exemplified by the disclosures of U.S. Pat. No. 8,053,192, PCT Publication No. WO 05 / 068656, and U.S. Patent Application Publication No. 2013 / 0274117. In some embodiments, the index can include the 6-base Index1 (i7) sequence, the 8-base Index1 (i7) sequence, the 8-base Index2 (i5) sequence, the 10-base Index1 (i7) sequence, or the 10-base Index2 (i5) sequence of Illumina, Inc. (San Diego, CA).

[0025] As used herein, the term "unique molecular identifier" or "UMI" or "barcode" refers to a molecular tag that can be attached to a nucleic acid molecule. When incorporated into a nucleic acid molecule, the UMI can be used to correct for subsequent amplification bias by directly counting the unique molecular identifier (UMI) that is sequenced after amplification.

[0026] As used herein, the term "tagmentation" refers to the modification of DNA by a transposome complex containing a transposase enzyme complexed with an adapter containing a double-stranded recognition sequence (a hybridized transposon end sequence).Tagmentation simultaneously fragments DNA and ligates adapters to the 5'-ends of both strands of the double-stranded fragment.After purification to remove the transposase enzyme, gap filling and ligation, a complete double-stranded product is generated.Additional sequences can be added to the ends of the adapted fragments, for example, by PCR, ligation, or any other suitable method known to those skilled in the art.See, for example, US Patent Application Publication No. 2017 / 0044525.

[0027] The term "and / or" means one or more listed elements or a combination of any two or more of the listed elements.

[0028] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain advantages, in certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0029] The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0030] Whenever an embodiment is described herein using the language "include," "includes," or "including," etc., it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" is limited to what precedes the phrase "consisting of." That is, "consisting of" indicates that the listed elements are essential or required, and that no other elements may be present. The term "consisting essentially of" indicates that any elements listed before the phrase are included, and that other elements other than those listed may be included, provided that they do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements.

[0031] Unless otherwise identified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0032] As used herein, the term "each," when used in reference to a set of items, is intended to identify each individual term in the set, but does not necessarily refer to every term in the set, unless the context clearly dictates otherwise.

[0033] The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0034] For any method disclosed herein that includes separate steps, those steps can be performed in any practicable order, and, where appropriate, any combination of two or more steps can be performed simultaneously.

[0035] The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. In the following description, illustrative embodiments are illustrated in more detail. In several places throughout the application, guidance is provided by lists of examples, which examples can be used in various combinations. In each instance, the recited list serves merely as a representative group and should not be interpreted as an exclusive list.

[0036] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," or "some embodiments" or the like mean that the particular feature, arrangement, composition, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment of the present disclosure. Furthermore, particular features, arrangements, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and so forth used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0038] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless expressly stated to do so.

[0039] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviations found in their respective testing measurements. [Brief explanation of the drawings]

[0040] [Figure 1-1] 1A-1B are schematic diagrams illustrating an exemplary embodiment of hybrid capture and compared off-target capture during enrichment. Figure 1A. The probe contains a region designed to hybridize to a region of interest within the target genome and a ligand (e.g., a biotin group) that enables subsequent capture of the probe. Once hybridization is complete and a DNA template-probe hybrid is formed, a capture means (i.e., a component with affinity for the probe, such as streptavidin-coated magnetic beads) is used to bind to the probe hybridized to the DNA target and remove the target from the pool of oligonucleotides. Figure 1B. Due to interactions between terminal adapter sequences in the target sequence and terminal adapter sequences in the off-target library preparation products, unwanted or off-target oligonucleotides can be recovered during hybrid capture. [Figure 1-2] Same as above.

[0041] [Figure 2] Figure 2 shows an exemplary mechanism by which a blocker prevents hybridization between adapter sequences and prevents off-target capture.

[0042] [Figure 3-1]Figures 3A-3I show diagrams of exemplary blockers. Figure 3A. Diagram of a NEXTERA blocker containing bases complementary to the 8- or 10-base index region of the adapter. Figure 3B. Diagram of a NEXTERA blocker modified with 8 or 10 deoxyinosine bases in the region of the blocker corresponding to the index region of the adapter. Figure 3C. Diagram of a TRUSEQ blocker modified with 6, 8, or 10 deoxyinosine bases in the region of the blocker corresponding to the index region of the adapter. Figure 3D. Diagram of NEXTERA and TRUSEQ blockers containing 6 or 8 thymines in the region of the blocker corresponding to the index region of the adapter. Figure 3E. Diagram of a split NEXTERA blocker containing a component corresponding to the region 5' to the 8- or 10-base index region and a component corresponding to the region 3' to that index region. These constructs do not contain any bases corresponding to the index region of the adapter (indicated by the 8- or 10-base gap corresponding to the index region). Figure 3F. Diagram of a split TRUSEQ blocker containing a component corresponding to a region 5' to the 8- or 10-base index region and a component corresponding to a region 3' to the index region. These constructs do not contain any bases corresponding to the adapter index region (indicated by the 8- or 10-base gap corresponding to the index region). Figure 3G. Diagram of a NEXTERA blocker corresponding only to a portion of the adapter region 3' to the index region. Figure 3H. Diagram of a TRUSEQ blocker that pairs with only a portion of the adapter region 3' to the index region. Figure 3I. Diagram of a NEXTERA or TRUSEQ blocker corresponding only to a portion of the adapter region 5' to the index region (e.g., p5 or p7 sequence). [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0043] [Figure 4]FIG. 4 is a graph of Padded Read Enrichment (a measure of the amount of on-target DNA captured) results obtained using blockers as described in Example 2.

[0044] [Figure 5] FIG. 5 shows a diagram of blockers containing a modified G (+G) or random (A, T, G, or C) (N) nucleotide at the fifth position of the portion of the blocker sequence that corresponds to the index sequence, as described in Example 3.

[0045] [Figure 6] Figures 6A-6B show blocker constructs and results as described in Example 4. Figure 6A shows diagrams of various blocker constructs, including (1) a split blocker pair containing a first sequence complementary to a 25-30 mer region 5' of the index region and a second sequence complementary to a 34-35 mer region 3' of the index region, (2) a blocker that pairs only with the 3' portion of the adapter ("Inner" blocker), (3) a blocker that pairs only with the 5' portion of the adapter ("Outer" blocker), and (4) a short (8-nucleotide) blocker ("Short8nt") that corresponds to the index region. Figure 6B shows padded read enrichment results achieved using XGEN Blocking Oligos (Integrated DNA Technologies, Coralville, IA), split blockers, or blockers that bind only to a portion of the adapter.

[0046] [Figure 7-1]Figures 7A-7F show results using an enhanced hybridization buffer containing dextran sulfate, as described in Example 5. Figure 7A shows padded read enrichment results achieved using various concentrations of dextran sulfate in the hybridization buffer. Figure 7B shows padded read enrichment results achieved using a 100 μL hybridization volume, four different probe panels, and three different buffer conditions (IDT hybridization buffer, Illumina non-enhanced hybridization buffer, and Illumina enhanced hybridization buffer). Figure 7C shows padded read enrichment and coverage uniformity results for all four probe panels at various hybridization incubation times, with and without an additional temperature gradient step. The black dotted line in the left panel indicates hybridization results in a buffer without dextran sulfate. Shown are the sequencing results (percent identified reads (PF) for NOVASEQ and NEXTSEQ, and percent indexed (%) for ISEQ) for three sets of runs, including NOVASEQ (Figure 7D), NEXTSEQ (Figure 7E), and ISEQ (Figure 7F), as well as the associated coefficient of variation (CV) for runs using dextran sulfate hybridization buffer and a library volume of 30 μL. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above.

[0047] [Figure 8-1]Figures 8A-8C show the results of the hybridization assay described in Example 6. Figure 8A shows padded read enrichment results when using modified blockers, increased washing temperatures, and / or a crowding agent (dextran sulfate) in the hybridization buffer. Figure 8B shows padded read enrichment results achieved using the method described in Example 6 on a 9-probe panel using an enhanced hybridization buffer containing xGEN blockers, compared to the results of an IDT exome panel when using the IDT XGEN LOCKDOWN kit protocol. Figure 8C shows somatic variant detection by a single hybridization enrichment protocol using a 12-gene 535-probe panel to enrich a library generated from the Horizon Discover HD701 quantitative multiplex (QM) DNA standard. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0048] [Figure 9-1]9A-9D show schematic diagrams of exemplary workflows. eBBN indicates bead-based tagmentation using bead-bound transposome complexes; H indicates hybridization; C indicates capture; W indicates wash; E indicates elution; PCR indicates PCR amplification; S indicates sequencing; Q indicates quantification; and SpVac indicates Speed ​​Vacuum enrichment. FIG. 9A shows a schematic diagram of an exemplary workflow for hybrid capture illustrating library preparation using a bead-based tagmentation protocol. Any suitable library preparation method may be used prior to enrichment. Notably, only one hybridization and capture is involved. FIG. 9B shows a schematic diagram of an exemplary workflow for hybrid capture. FIG. 9C shows a schematic diagram of an exemplary workflow for PCR-free hybrid capture. FIG. 9D shows a schematic diagram of an exemplary workflow for PCR-free hybrid capture with reduced hybridization time. [Figure 9-2] Same as above.

[0049] [Figure 10] Figure 10 shows the results of Padded Read Enrichment achieved with BN001 and BN002 (unmodified blockers); BX003 and BX007 (G-Clamp modified blockers); BN007 and BN008 (BNA and deoxyinosine modified blockers); BN005 and BN006 (BNA and sequence-specific modified blockers); or BN023, BN024, BN025, and BN026 (LNA modified split blockers), as described in Example 8.

[0050] [Figure 11]Figure 11 shows the results of padded read enrichment achieved with BN001 and BN002 (unmodified blockers) or BN023, BN024, BN025 and BN026 (LNA-modified split blockers) when the corresponding adapters contain 8-nucleotide or 10-nucleotide indices as described in Example 9. DETAILED DESCRIPTION OF THE INVENTION

[0051] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present disclosure describes compositions comprising blockers and / or hybridization buffers, as well as methods for improving the efficiency of nucleic acid selection prior to sequencing, including methods using those compositions and methods involving hybrid capture. Enrichment by hybrid capture

[0052] Various methods can be used to enrich desired sequences from a complex pool of nucleic acids. These methods include polymerase chain reaction (PCR), molecular inversion probes (MIPs) or sequence capture by hybridization ("hybrid capture"). For example, see Mamanova et al., Nat. Methods 7:111-118 (2010); U.S. Patent Application Publication No. 2014 / 0031240; and U.S. Patent Application Publication No. 2017 / 0114404.

[0053] Next-generation sequencing (NGS) applications typically use enrichment via hybrid capture. Prepared NGS template pools, or libraries, are heat-denatured and mixed with a pool of capture probe oligonucleotides ("probes"). These probes are designed to hybridize to regions of interest within the target genome, typically 60-200 bases in length, and are further modified to contain ligands that allow subsequent capture of the bound probes. A common capture method incorporates biotin groups onto the probes. As shown in one embodiment in Figure 1A, after hybridization to form DNA template-probe hybrids is complete, capture is performed using a component with affinity exclusively for the probes. For example, streptavidin-coated magnetic beads can be used to bind the biotin moiety of biotinylated probes hybridized to the desired DNA targets from the library. Washing removes unbound nucleic acids, reducing the complexity of the retained material. The retained material is then eluted from the magnetic beads and submitted to an automated sequencing process.

[0054] Although DNA hybridization with probes can be highly specific, after the completion of hybrid capture method, unwanted sequences remain in the enriched pool.The majority of these unwanted sequences are due to unwanted hybridization events between library members that do not have complementarity to the probe and library members that have complementarity to the probe (i.e., on-target library members).Two types of sequences cause unwanted hybridization during hybrid capture method: (1) highly repetitive DNA elements found in endogenous genomic DNA; and (2) the terminal adapter sequences engineered into each library member.

[0055] A repetitive endogenous DNA element (e.g., an Alu sequence or a long interspersed repeat (LINE)) present in one DNA fragment in a complex pool may hybridize to a similar element present in another, unrelated DNA fragment. These fragments may originally originate from completely different locations in the genome and become linked during the hybridization process of the hybrid capture method. If one of these DNA fragments is the desired fragment containing the binding site for the probe, the unwanted fragment will also be captured along with the desired fragment. This class of off-target library members can be reduced by adding excess repetitive elements to the hybridization buffer of the hybridization reaction. Most commonly, human Cot-1 DNA (which binds to Alu, LINE, and other repetitive sites in the target and thereby blocks the ability of NGS templates to interact with each other) is added to the hybridization buffer.

[0056] Due to interactions between the terminal adapter sequences in individual library members, off-target (also referred to as non-target) library members can also be captured. Typically, library members contain a sequence segment of a gene of interest, such as a segment for sequencing. When a member is on-target, the sequence of the gene of interest forms a duplex with the capture probe. On-target sequences can include, for example, exons or introns (or fragments thereof), coding or non-coding regions, enhancers, untranslated regions, specific SNPs, etc. Typically, library members also contain one or more non-target sequences. These non-target sequences usually do not contain the target sequence of interest, but do contain, for example, adapters. Because a pool of library members usually contains at least some identical terminal adapter sequences, these adapter sequences are present at a very high effective concentration in the hybridization solution. As a result, as shown in one embodiment in Figure 1B, library members containing off-target sequences can anneal to the captured target sequence via a portion of the added adapter sequence, thereby capturing the off-target sequence along with the on-target library member (e.g., by concatenation or "daisy chain" of sequences linked together). Because the annealed target sequence contains a binding site for the probe, the entire daisy chain can be captured. Thus, capturing a single desired fragment can bring with it numerous undesired fragments, reducing the overall efficiency of enrichment for the desired segment.

[0057] In some embodiments, the present disclosure describes methods and compositions for minimizing the selection of off-target nucleic acids by hybrid capture. Blocker Oligonucleotides

[0058] In one aspect, the present disclosure describes "blocker oligonucleotides" (also referred to herein as "blockers") and methods for preventing the selection of off-target nucleic acids during hybrid capture by using blockers to bind to at least a portion of an adapter sequence (e.g., form a duplex with at least a portion of the adapter sequence). When used, as shown in one embodiment in Figure 2, binding of the blocker to the adapter sequence prevents interactions between adapter sequences in the off-target library preparation (e.g., formation of concatenated strands), which could result in unwanted library members being recovered during hybrid capture.

[0059] In some embodiments, at least two blockers may be used, with the first blocker binding to a first adapter sequence (e.g., forming a duplex with the first adapter sequence) and the second blocker binding to a second adapter sequence (e.g., forming a duplex with the second adapter sequence). In some embodiments, the first adapter sequence may be at the 5' end of the library member and the second adapter sequence may be at the 3' end of the library member. In some embodiments, multiple different blockers may be used.

[0060] In some embodiments, the blocking oligonucleotide forms a duplex with the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members, and the resulting duplex is a T of the duplex formed by the background nucleic acid (e.g., the complement of the adapter sequence) and the adapter sequence. m Higher T m It has.

[0061] In some embodiments, the blocker-adaptor complex has a higher T than the adaptor-adaptor complex. m Such a high T mmeans that the blocker-adapter complex is likely to form before the adaptor-adaptor complex, which may prevent off-target capture (e.g., by preventing daisy chain formation). In some embodiments, the T of the blocker-adapter oligonucleotide duplex m T of the adapter duplex with its exact complementary strand m In some embodiments, the T of the blocker-adapter oligonucleotide duplex is at least 1.5° C., at least 2° C., at least 3° C., at least 4° C., at least 5° C., at least 10° C., at least 15° C., at least 20° C., or at least 25° C. higher than the T of the blocker-adapter oligonucleotide duplex. m T of the adapter duplex with its exact complementary strand m 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C higher than

[0062] In some embodiments, the association rate of a blocking oligonucleotide to the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members is higher than the association rate of background nucleic acid (e.g., the complement of the adapter sequence) to the adapter sequence. In some embodiments, the association rate is at least 2-fold higher, at least 4-fold higher, at least 6-fold higher, at least 8-fold higher, or at least 10-fold higher. In some embodiments, the association rate is up to 4-fold higher, up to 6-fold higher, up to 8-fold higher, up to 10-fold higher, or up to 12-fold higher.

[0063] In some embodiments, the dissociation rate between the blocking oligonucleotide and the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members is lower than the dissociation rate between background nucleic acid (e.g., the complementary strand of the adapter sequence) and the adapter sequence. In some embodiments, the dissociation rate is at least 2-fold lower, at least 4-fold lower, at least 6-fold lower, at least 8-fold lower, or at least 10-fold lower. In some embodiments, the association rate is at most 4-fold lower, at most 6-fold lower, at most 8-fold lower, at most 10-fold lower, or at most 12-fold lower.

[0064] In some embodiments, the duplex formed between the blocking oligonucleotide and the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members is longer than the duplex formed between the adapter sequence and its complementary strand, e.g., the duplex formed between the Watson-Crick strand of a double-stranded adapter. In some embodiments, the duplex between the blocking oligonucleotide and the adapter sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 nucleotides longer than the duplex formed between the adapter sequence and its complementary strand.

[0065] In some embodiments, a blocker preferably has a higher Tg of that blocker T than an oligonucleotide that does not contain the modification and has the same sequence as the blocker. m For example, in some embodiments, the blocker includes a modification that increases "T mThe oligonucleotide may be a "high-temperature oligonucleotide" which contains at least one modified group that increases the thermal melting temperature value of a duplex nucleic acid containing the oligonucleotide as a hybridization partner compared to a duplex nucleic acid containing an oligonucleotide having the same nucleobase composition and unmodified groups as a hybridization partner. m High temperature oligonucleotides are further described in U.S. Patent Application Publication No. 2014 / 0031240.

[0066] In some embodiments, the blocker comprises one or more non-naturally occurring nucleotides. In some embodiments, the duplexes formed between the blocking oligonucleotide having the non-naturally occurring nucleotide and the adapter sequence of at least 1, at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 200 library members exhibit a value of a parameter (e.g., affinity, association rate, inverse of dissociation rate, or T) related to the binding interaction with background nucleic acids (e.g., other complementary non-target nucleic acid sequences) and non-target nucleic acid sequences. m ) has a higher value of that parameter.

[0067] In some embodiments, the blocker comprises a modified base. Any suitable modified base may be included in the blocker. In some embodiments, the modification preferably includes a T m a modification that increases the T of a blocker-adapter complex that has the same sequence but does not contain the modified base. m Compared with the T of the blocker-adapter complex containing the modified base, m These include modifications that increase T mModifications that enhance nucleotide sequence may include, for example, DNA or RNA oligonucleotides modified to capture low GC regions; bridged oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids (LNA); bridged nucleic acids (also called bicyclic nucleic acids or BNAs); tricyclic nucleic acids; peptide nucleic acids (PNAs); C5-modified pyrimidine bases; propynyl pyrimidines; morpholinos; phosphoramidites; 5'-pyrene caps; and the like. In some embodiments, LNAs comprise a ribose moiety of a nucleotide modified with an additional bridge connecting the 2' oxygen and the 4' carbon. Exemplary BNAs are disclosed, for example, in U.S. Patent Nos. 7,399,845, 7,427,672, 7,547,684, 7,696,345, 7,741,457, 8,022,193, 8,268,980, 8,278,425, 8,278,426, 8,846,637, 8,846,639, and 9,546,368. In some embodiments, BNAs can include constrained ethyl nucleic acids from Ionis Pharmaceuticals (Carlsbad, CA) or 2'-O,4'-aminoethylene-bridged nucleic acids from Biosynthesis, Inc. (Lewisville, TX). In some embodiments, PNAs include N-(2-aminoethyl)-glycine repeat units linked by peptide bonds. In some embodiments, tricyclic nucleic acids include, for example, tricyclic nucleic acids disclosed in US Pat. No. 9,221,864.Exemplary phosphoramidites include 1-[5'-O-(4,4'-dimethoxytrityl)-β-D-2'-deoxyribofuranosyl]-9-(2-trifluoroacetamidoethoxy)-1,3-diaza-2-oxophenoxazine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (AP-dC-CE phosphoramidite), 5'-dimethoxytrityl-uridine,2'-O-methyl,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (2' -OMe-U-CE phosphoramidite), 5'-dimethoxytrityl-N-acetyl-5-methyl-cytidine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2'-OMe-5-Me-C-CE phosphoramidite), 5'-dimethoxytrityl-N-acetyl-cytidine, 2'-O-methyl, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2'-OMe-Ac-C-CE phosphoramidite).

[0068] In some embodiments, the T of the blocker is increased relative to an oligonucleotide having the same sequence as the blocker without the modification. m Modifications that increase ATP can include non-basic modifications, such as minor groove binders (MGBs), spermine, G-clamps, or Uaq anthraquinone caps.

[0069] In some embodiments, the blocking oligonucleotide is at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length, hi some embodiments, the blocking oligonucleotide is up to 45, up to 50, up to 55, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150, or up to 200 nucleotides in length.

[0070] In some embodiments, the blocking oligonucleotide has a Tm In some embodiments, the number of modifications is preferably such that the optimal T under stringent conditions (0.1×SSC) of at least about 1.4° C. is achieved for a duplex DNA containing the modifications as one complementary strand. m Increase in value (optimal high temperature T m In some embodiments, T m The preferred number of modifications in the high-temperature blocking oligonucleotide is determined by the T m T of the adapter duplex with its exact complementary strand m In some embodiments, T is at least 1.5°C, at least 2°C, at least 3°C, at least 4°C, at least 5°C, at least 10°C, at least 15°C, at least 20°C, or at least 25°C higher than T m The preferred number of modifications in the high-temperature blocking oligonucleotide is determined by the T m T of the adapter duplex with its exact complementary strand m The number is such that the temperature is up to 2°C, up to 3°C, up to 4°C, up to 5°C, up to 10°C, up to 15°C, up to 20°C, up to 25°C or up to 30°C higher than the reference temperature.

[0071] In some embodiments, the blocking oligonucleotide comprises at least 2 percent (%), at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the bases in the blocking oligonucleotide, hi some embodiments, the blocking oligonucleotide comprises up to 5%, up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 100% of the bases in the blocking oligonucleotide.

[0072] In some embodiments, modifications to blockers can be included in a specific pattern. For example, modified bases can be included every second base (see, e.g., Table 2A, BN021 and BN022), every third base (see, e.g., Table 2A, BN035 and BN036), every fourth base, or every fifth base, or some combination thereof (see, e.g., Table 2A, BN036). In some embodiments, each base in a blocker can be modified (see, e.g., Table 2A, BN027 and BN028). In some embodiments, guanine, which has a higher affinity than other nucleotides, can be preferentially modified when modified. For example, in some embodiments, LNA or BNA type guanine can be included in a blocker.

[0073] In some embodiments, a blocker may include a terminal modification. For example, a blocker may include a 3'-terminal group that prevents the blocker from serving as a primer for DNA synthesis. Such 3'-terminal groups may include, for example, 3'-dC, 2',3'-ddC (also referred to herein as 3ddc), inverted dT, 3'-spacer C3 (also referred to herein as 3SpC3), and the like.

[0074] A blocker may bind to (e.g., form a duplex with) any suitable adapter sequence (including any portion thereof). As used herein, a portion of a blocker sequence that binds to (e.g., forms a duplex with) a portion of an adapter sequence is referred to as a portion of the blocker sequence that "corresponds to" that portion of the adapter sequence. In some embodiments, the corresponding sequence may be the exact complement of the adapter sequence. However, in some embodiments, at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 bases in the corresponding sequence may not be complementary.

[0075] In some exemplary embodiments, the blocker may bind to an adapter such as those shown in Table 1. (The adapter sequences are shown in Table 1A, and further information regarding the sequence components is shown in Table 1B.) In some exemplary embodiments, the blocker may bind to an adapter comprising a sequence disclosed in Illumina Adapter Sequences (available on the World Wide Web at support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / experiment-design / illumina-adapter-sequences-1000000002694-07.pdf).

[0076] In some embodiments, the blocker binds to an adapter, and the adapter comprises a universal sequence, such as, for example, a universal adapter and / or a universal primer sequence. In some embodiments, the universal primer sequence can comprise P5, P7, P5', and / or P7'. In some embodiments, the universal primer sequence can comprise the V2.A14.METS sequence, the V2.B15.METS sequence, the complement of the V2.A14.METS sequence, and / or the complement of the V2.B15.METS sequence. For example, BN001 as shown in Table 2A comprises P5 and the V2.A14.METS sequence; BN002 as shown in Table 2A comprises P7 and the V2.B15.METS sequence; BN003 as shown in Table 2A comprises P5' and the reverse complement of the V2.A14.METS sequence; and BN004 as shown in Table 2A comprises P7' and the reverse complement of the V2.B15.METS sequence.

[0077] In some sequencing applications, it is desirable for library members to contain at least one index and / or UMI sequence. Thus, in some embodiments, the blocker binds to an adapter, and the adapter contains at least one of an index and a UMI. U.S. Patent Application Publication No. 2014 / 0031240 explains that for adapters containing an index (referred to in U.S. Patent Application Publication No. 2014 / 0031240 as a "barcode domain"), "blocking oligonucleotides can be generated using three approaches: 1) synthesizing a series of blockers that perfectly match each adapter; 2) synthesizing a single blocker with an N-mer domain that pairs with the adapter's barcode domain; or 3) synthesizing a single blocker with a universal base domain that pairs with the adapter's barcode domain." Because each different index and / or UMI has a unique sequence, a perfectly complementary blocker, as in Approach 1, can be a sequence that contains a perfect match complementarity to each index and / or UMI sequence present (see FIG. 3A). However, if many different indexes and / or UMIs are used, including blockers with perfect match complementarity may require tens or hundreds of unique blockers for multiplex amplification, making it a less cost-effective approach.

[0078] In some embodiments, the disclosure describes blockers for adapters that include an index and / or a UMI and methods of using such blockers, e.g., blockers that include a thymine in the region of the blocker that corresponds to the index and / or UMI ( FIG. 3D and Example 2 ); blockers that include a universal base and at least one non-universal base ( FIG. 5 and Example 3 ); or blockers that do not include an index region (e.g., including “split blockers” or blockers that pair with only a portion of the adapter) ( FIGS. 3E-3I , FIG. 6A , and Examples 4, 8, and 9 ).

[0079] In some embodiments, the blocker comprises at least one of the sequences in Table 2A, Table 2B, Table 2C, or Table 3. In some embodiments, the sequences in Table 2A can be used according to tagmentation (e.g., NEXTERA) library preparation (Illumina, Inc., San Diego, CA). In some embodiments, the sequences in Table 2B can be used according to ligation-based library preparation and / or TRUSEQ preparation (Illumina, Inc., San Diego, CA). In some embodiments, the sequences in Table 2C can be used according to tagmentation (e.g., NEXTERA) library preparation (Illumina, Inc., San Diego, CA). In some embodiments, when a sequence in Table 2 comprises a BNA-modified nucleic acid, the BNA-modified nucleic acid can comprise a 2'-O,4'-aminoethylene-bridged nucleic acid from Biosynthesis, Inc. (Lewisville, TX). In some embodiments, when a sequence in Table 2 comprises an LNA-modified nucleic acid, the LNA-modified nucleic acid may comprise an LNA-containing oligonucleotide from Qiagen (Hilden, Germany). Further information regarding the components of the sequences in Tables 2A, 2B, and 2C can be found in Tables 1B and 2D.

[0080] In another aspect, the present disclosure describes a kit including a blocking oligonucleotide. The blocking oligonucleotide may include a blocker as described herein. In some embodiments, the blocking oligonucleotide is a plurality of blocking oligonucleotides. The kit may further include one or more of a hybridization buffer, a probe, a panel of probes, and a flow cell. The hybridization buffer may include a hybridization buffer as described elsewhere in this disclosure. In some embodiments, the probe is as described elsewhere in this disclosure. In some embodiments, the components may be provided in separate containers. For example, the blocking oligonucleotide may be provided in a first container, and another component, such as a hybridization buffer, or a probe or panel of probes, may be provided in a different container. Alternatively, the blocking oligonucleotide and hybridization buffer may be provided in a first container, and the probe or panel of probes may be provided in a different container. Table 1A - Exemplary adapter sequences [Table 1A] [Table 1B] Table 2A - Exemplary Blocker Sequences [Table 2A-1] [Table 2A-2] [Table 2A-3] Table 2B - Exemplary Blocker Sequences [Table 2B-1] [Table 2B-2] Table 2C - Exemplary Blocker Sequences [Table 2C] [Table 2D] Thymine-containing blockers

[0081] In some embodiments, the blocker may include at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases in the index region of the adapter and / or the region of the blocker that corresponds to the UMI of the adapter (e.g., the region of the blocker that binds to the index region of the adapter and / or the UMI).

[0082] In some embodiments, the region of the blocker that corresponds to (e.g., binds to) the index region and / or UMI of the adapter contains the same number of thymine bases as the number of bases in the index region and / or UMI of the adapter.

[0083] In some embodiments, the blocker comprises at least one sequence of Table 2A, Table 2B, Table 2C, or Table 3. Some exemplary embodiments of such blockers are described in Example 2. Blockers containing a universal base and at least one non-universal base

[0084] In some embodiments, a blocker may contain a universal base and at least one non-universal base in the region of the blocker corresponding to an index region. As used herein, a "universal base" is a modified nucleobase that hybridizes to at least two of the common bases (e.g., deoxyadenosine (A), deoxythymidine (T), deoxycytidine (C), deoxyguanosine (G), and uridine (U)). In some embodiments, a universal base hybridizes to any of the common bases. As used herein, a "non-universal base" is a nucleobase (including, in some embodiments, a modified nucleobase) that hybridizes only through conventional Watson-Crick base pairing interactions (e.g., deoxyadenosine (A)-deoxythymidine (T) or deoxyguanosine (G)-deoxycytidine (C)).

[0085] In some embodiments, the at least one non-universal base may be included in every third base of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base may be located at the third and / or fifth position (from the 5' end) of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base may be located at the second and / or fourth position of the blocker sequence corresponding to the index region. In some embodiments, the at least one non-universal base comprises a modified guanine. In some embodiments, the modified guanine may be modified with an LNA or a BNA. In some embodiments, the at least one non-universal base comprises a random (A, T, G, or C) nucleotide.

[0086] Without wishing to be bound by theory, it is believed that including modified guanines or random nucleotides in addition to universal bases in the regions of the blocker corresponding to the index regions (as shown in one embodiment in Figure 5) improves the affinity of the blocker for library members. Some exemplary embodiments of such blockers are described in Example 3.

[0087] The universal base may include any known universal base. In some embodiments, the universal base used in the index region may include, for example, 2'-deoxyinosine, 2'-deoxynebularine, 3-nitropyrrole 2'-deoxynucleoside, or 5-nitroindole 2'-deoxynucleoside. In some embodiments, a combination of universal bases may be used in the index region.

[0088] The modified guanine can be a guanine modified in any suitable manner (including, for example, an LNA-modified guanine, a BNA-modified guanine, etc.) In some embodiments, the modified guanine is preferably an LNA-modified guanine.

[0089] In some embodiments, the blocker comprises at least one sequence of Table 2A, Table 2B, Table 2C, or Table 4. Split Blocker

[0090] In some embodiments, a blocker can include at least two unconnected oligonucleotides, which include bases that do not correspond to the index region and / or UMI of the adapter (e.g., include bases that do not bind to the index region and / or UMI of the adapter) or include bases that only partially correspond to the index region and / or UMI of the adapter (e.g., include bases that bind to only a portion of the index region and / or UMI of the adapter). In some embodiments, a blocker includes four unconnected oligonucleotides.

[0091] In some embodiments, at least two unconnected oligonucleotides hybridize to the same strand of a target sequence. In some embodiments, at least a portion of a blocker corresponds to the 5' end of an adapter, and at least a portion of a blocker corresponds to the 3' end of the same adapter. In some embodiments, such as when a blocker includes four unconnected oligonucleotides, two unconnected oligonucleotides can hybridize to the same strand of a first target sequence, and two unconnected oligonucleotides can hybridize to the same strand of a second target sequence. Exemplary embodiments of such blockers are shown in Figures 3E-3I and 6A and described in Examples 4, 8, and 9.

[0092] In some embodiments, at least a portion of the blockers may correspond to a universal extension primer. In some embodiments, at least a portion of the blockers may correspond to P5, P7, P5', and / or P7'. In some embodiments, at least a portion of the blockers may correspond to V2.A14.METS, V2.B15.METS, the complement of V2.A14.METS, and / or the complement of V2.B15.METS.

[0093] In some embodiments, the blocker can include the same number of bases as the adapter, excluding the portion of the adapter that includes bases in the index region and / or UMI of the adapter. In some embodiments, the blocker can include 1, 2, 3, 4, or 5 fewer bases than the number of bases that make up the portion of the adapter, excluding bases in the index region and / or UMI of the adapter.

[0094] In some embodiments, blockers containing more bases may be preferred (e.g., when high enrichment performance is desired), and in some embodiments, blockers containing fewer bases may be preferred (e.g., when blocker cost, synthetic purity, and / or yield are factors).

[0095] In some embodiments, up to 5 bases of a blocker, up to 4 bases of a blocker, up to 3 bases of a blocker, up to 2 bases of a blocker, or only 1 base of a blocker may pair with the index region of an adapter and / or the UMI of an adapter, or no bases of a blocker may pair with the index region of an adapter and / or the UMI of an adapter.

[0096] In some embodiments, the blocker comprises at least one sequence (or portion of a sequence) of Table 2A, Table 2B, Table 2C, or Table 5. In some embodiments, the blocker comprises at least one of BN023, BN024, BN025, and BN026 of Table 2A. In some embodiments, the blocker comprises BN023 and BN025 of Table 2A. In some embodiments, the blocker comprises BN024 and BN026 of Table 2A. Hybridization buffer

[0097] In another aspect, the present disclosure describes a hybridization buffer containing a crowding agent and a method for using the hybridization buffer. As used herein, "crowding agent" includes a compound that enables, enhances, or promotes molecular crowding. In some embodiments, the crowding agent includes an inert macromolecule used at a concentration that alters DNA-protein interactions. A "hybridization buffer" is defined as any buffer in which a nucleotide-probe hybrid can form as part of a hybrid capture assay.

[0098] In some embodiments, the crowding agent may include at least one of dextran, dextran sulfate, polyethylene glycol (PEG), ficoll, glycerol, betaine, etc. Dextran and dextran sulfate may include high molecular weight dextran (e.g., dextran having a molecular weight of at least 500,000 Da) and / or low molecular weight dextran (e.g., dextran having a molecular weight of up to 10,000 Da, up to 50,000 Da, or up to 100,000 Da). In some embodiments, the crowding agent preferably includes dextran sulfate.

[0099] In some embodiments, the crowding agent may be included in the hybridization buffer in an amount of at least 0.5% weight / volume (w / v), at least 1% (w / v), at least 1.5% (w / v), at least 2% (w / v), at least 3% (w / v), or at least 4% (w / v). In some embodiments, the crowding agent may be included in the hybridization buffer in an amount of up to 1% (w / v), up to 1.5% (w / v), up to 2% (w / v), up to 3% (w / v), up to 4% (w / v), up to 5% (w / v), up to 6% (w / v), up to 8% (w / v), or up to 10% (w / v).

[0100] As described in some embodiments in Example 5 (see, e.g., Figures 7D-7F), including a crowding agent (e.g., dextran sulfate) in the hybridization buffer can, in some embodiments, shorten the hybridization time and / or allow hybridization to occur in a larger volume of hybridization buffer (e.g., greater dilution of the target oligonucleotide) than without the crowding agent. For example, including dextran sulfate in the hybridization buffer can shorten the hybridization time from overnight at a single temperature to approximately 80 minutes, or to 60 minutes with an additional temperature gradient step.

[0101] In some embodiments, the inclusion of a crowding agent can improve enrichment specificity. For example, one embodiment is shown in Example 5 (see FIG. 8A), where enrichment specificity increased from 45% to 80-95%. In some embodiments, the improvement in enrichment specificity resulting from the inclusion of a crowding agent can be more pronounced with shorter incubation times.

[0102] In some embodiments, including a crowding agent in the hybridization buffer allows enrichment reactions to occur more quickly at lower DNA concentrations (e.g., reactions in a larger relative volume of hybridization buffer). Such an increase can facilitate "pooling-by-volume" and / or elimination of sample concentration steps. Achieving a rapid hybridization reaction generally requires the use of high concentrations of DNA and probes from library preparations, which in some embodiments requires a correspondingly small volume of hybridization buffer (e.g., less than 20 μL). Achieving this small volume requires a sample concentration step after library preparation (resulting in longer overall assay times and higher costs). Including a crowding agent in the hybridization buffer allows enrichment reactions to be achieved more quickly at lower DNA and / or probe concentrations (and in some embodiments, in larger volumes). In some embodiments, including a crowding agent in the hybridization buffer allows multiple library preparation samples to be combined without a concentration step prior to hybridization. In some embodiments, samples from different library preparations can be pooled based on volume, allowing for multiplex enrichment, as further described in this disclosure. The "plexi" or "plex" of a given multiplex enrichment refers to the number of different library preparations combined prior to enrichment (e.g., hybridization and capture). In some embodiments, the plexi of a multiplex enrichment can be up to 2-plex, up to 4-plex, up to 6-plex, up to 12-plex, up to 24-plex, up to 48-plex, up to 96-plex, or more.

[0103] In some embodiments, the hybridization buffer containing the crowding agent further comprises at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker. In some embodiments, the components of the hybridization buffer, such as the Cot-1 DNA and the blocker, are kept separate from the components of the hybridization buffer containing the destabilizing agent and the crowding agent until the hybridization reaction is performed.

[0104] In some embodiments, the hybridization buffer may contain human Cot-1 DNA in an amount of at least 0.05 mg / mL, at least 0.1 mg / mL, at least 0.2 mg / mL, or at least 0.3 mg / mL. In some embodiments, the hybridization buffer may contain human Cot-1 DNA in an amount of up to 0.1 mg / mL, up to 0.2 mg / mL, up to 0.3 mg / mL, or up to 0.5 mg / mL. In some embodiments, the hybridization buffer may preferably contain human Cot-1 DNA in an amount of 0.2 mg / mL.

[0105] In some embodiments, the hybridization buffer may contain a destabilizing agent. In some embodiments, the destabilizing agent may include formamide and / or urea. In some embodiments, the hybridization buffer may contain at least 1% (v:v), at least 5% (v:v), or at least 10% (v:v) formamide. In some embodiments, the hybridization buffer may contain up to 5% (v:v), up to 10% (v:v), or up to 15% (v:v) formamide. In some embodiments, the hybridization buffer may contain at least 1 M urea, at least 2 M urea, or at least 4 M urea. In some embodiments, the hybridization buffer may contain up to 2 M urea, up to 4 M urea, or up to 6 M urea. In some embodiments, the hybridization buffer may preferably contain 10% formamide.

[0106] In some embodiments, the hybridization buffer may include a buffer. Any suitable buffer may be used. In some embodiments, the buffer may include phosphate (e.g., including potassium phosphate or sodium phosphate); Tris-HCl; piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); piperazine-N,N'-bis(3-propanesulfonic acid) (PIPPS); 2-(N-morpholino)ethanesulfonic acid (MES); (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), etc. In some embodiments, the hybridization buffer may include at least 40 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, or at least 70 mM phosphate. In some embodiments, the hybridization buffer may contain up to 50 mM, up to 55 mM, up to 60 mM, up to 65 mM, up to 70 mM, up to 75 mM, or up to 80 mM phosphate. In some embodiments, the phosphate-containing buffer may include dihydrogen phosphate and / or monohydrogen phosphate. In some embodiments, the phosphate-containing buffer may include KH2PO4-K2HPO4. In some embodiments, the hybridization buffer may preferably contain 66.6 mM KH2PO4-K2HPO4.

[0107] In some embodiments, the hybridization buffer may contain a salt. Any suitable salt may be included. In some embodiments, the salt may be, for example, NaCl or sodium citrate (e.g., trisodium citrate). In some embodiments, the hybridization buffer may contain at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, or at least 1 M salt. In some embodiments, the hybridization buffer may contain up to 0.2 M, up to 0.3 M, up to 0.4 M, up to 0.5 M, up to 0.6 M, up to 0.7 M, up to 0.8 M, up to 0.9 M, up to 1 M, up to 2 M, up to 3 M, or up to 4 M salt. In some embodiments, the hybridization buffer may preferably contain 0.8 M NaCl.

[0108] In some embodiments, the hybridization buffer may contain a detergent (e.g., an anionic detergent (e.g., sodium dodecyl sulfate, sulfonates, alcohol sulfates, alkylbenzene sulfonates, phosphate esters, or carboxylates), a nonionic detergent (e.g., a polyoxyethylene or glycoside detergent, e.g., Tween®, Triton, or Brij detergent), a cationic detergent (e.g., a quaternary ammonium cationic surfactant), or a zwitterionic detergent (e.g., CHAPS), etc.). In some embodiments, the detergent is an anionic detergent. In some embodiments, the detergent is a nonionic detergent. In some embodiments, the detergent is TWEEN® 20, TWEEN® 80, sodium dodecyl sulfate (SDS), etc. In some embodiments, the detergent may comprise at least 0.001% (v / v), at least 0.01% (v / v), at least 0.05% (v / v), at least 0.1% (v / v), at least 1% (v / v), or at least 5% (v / v) of the hybridization buffer. In some embodiments, the detergent may comprise up to 0.01% (v / v), up to 0.05% (v / v), up to 0.1% (v / v), up to 1% (v / v), up to 5% (v / v), or up to 10% (v / v) of the hybridization buffer. In some embodiments, the hybridization buffer may preferably contain 0.04% (v / v) TWEEN® 20.

[0109] For example, in some embodiments, the hybridization buffer may include 0.5% to 10% dextran sulfate, 0.05 mg / mL to 0.5 mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40 mM to 80 mM KH2PO4-K2HPO4, 0.1 M to 4 M NaCl, and 0.001% to 10% (v / v) Tween® 20. In some embodiments, the hybridization buffer may include (e.g., in the final reaction) 1.5% dextran sulfate, 0.2 mg / mL Cot-1, 10% (v / v) formamide, 66.6 mM KH2PO4-K2HPO4, 0.8 M NaCl, and 0.04% (v / v) TWEEN® 20.

[0110] In some embodiments, the hybridization buffer may include a blocker. In some embodiments, the blocker is as described elsewhere in this disclosure. In some embodiments, the blocker is present at a concentration of at least 0.001 mM, at least 0.005 mM, at least 0.01 mM, at least 0.05 mM, or at least 0.1 mM. In some embodiments, the blocker is present at a concentration of up to 0.01 mM, up to 0.05 mM, up to 0.1 mM, up to 0.2 mM, or up to 0.5 mM.

[0111] In another aspect, the present disclosure describes a kit comprising a hybridization buffer, e.g., a hybridization buffer as described herein. In certain embodiments, the kit further comprises at least one of a blocker and a probe. In embodiments, the components are provided in separate containers. For example, the hybridization buffer may be provided in a first container, and another component, e.g., a blocker or probe, may be provided in a different container. In some embodiments, the blocker and / or probe are as described elsewhere in this disclosure. Hybrid Acquisition

[0112] In a further aspect, the present disclosure describes a method of hybrid capture. In some embodiments, the method comprises a step comprising hybridizing a probe to a library member and capturing the probe. In some embodiments, the method further comprises pooling the library before hybridizing the probe. In some embodiments, the method further comprises amplifying the captured sequence after capture. In some embodiments, the method can be used in combination with a blocker oligonucleotide as described in the present disclosure. In some embodiments, the method can include using a hybridization buffer containing a crowding agent as described in the present disclosure. Library Pool

[0113] In some embodiments, samples from different library preparations are combined prior to hybridization, and each combined library preferably contains sequences that contain index regions that are distinct from the index regions of sequences in any other combined library.

[0114] In some embodiments, samples from different library preparations may be pooled based on volume, allowing for multiplex enrichment. The "plexi" or "plex" of a given multiplex enrichment refers to the number of different library preparations combined prior to enrichment (e.g., hybridization and capture). In some embodiments, the plexi of a multiplex enrichment may be up to 2-plex, up to 4-plex, up to 6-plex, up to 12-plex, up to 24-plex, up to 48-plex, up to 96-plex, or more.

[0115] In some embodiments, a sample from a library preparation is derived from a tagmentation reaction. See, e.g., U.S. Patent No. 9,574,226. In some embodiments, a sample from a library preparation is derived from a NEXTERA library preparation (Illumina, Inc., San Diego, CA). In some embodiments, a library preparation can be added directly to a hybridization reaction.

[0116] In some embodiments, the DNA concentration of each sample from the library preparations to be combined can be at least 0.1 ng / μL, at least 0.3 ng / μL, at least 0.4 ng / μL, at least 0.5 ng / μL, at least 1 ng / μL, at least 2 ng / μL, at least 4 ng / μL, at least 6 ng / μL, at least 8 ng / μL, at least 10 ng / μL, at least 12 ng / μL, at least 20 ng / μL, at least 50 ng / μL, at least 60 ng / μL, at least 100 ng / μL, or at least 200 ng / μL. In some embodiments, the DNA concentration of each sample derived from the combined library preparations may be at most 0.4 ng / μL, at most 0.5 ng / μL, at most 1 ng / μL, at most 2 ng / μL, at most 4 ng / μL, at most 6 ng / μL, at most 8 ng / μL, at most 10 ng / μL, at most 12 ng / μL, at most 15 ng / μL, at most 20 ng / μL, at most 50 ng / μL, at most 100 ng / μL, at most 120 ng / μL, at most 200 ng / μL, at most 300 ng / μL, or at most 400 ng / μL. For example, in some embodiments, the DNA concentration of each sample derived from the library preparations may be in the range of 0.3 ng / μL to 400 ng / μL. For example, in some embodiments, the DNA concentration of each sample derived from the library preparations may be in the range of 0.1 ng / μL to 120 ng / μL. For example, in some embodiments, the DNA concentration of the combined samples derived from the library preparation may be in the range of 0.1 ng / μL to 120 ng / μL (e.g., in 100 μL). For example, in some embodiments, the DNA concentration of each sample derived from the library preparation may be in the range of 0.5 ng / μL to 12 ng / μL. In some embodiments, the DNA concentration of the combined samples derived from the library preparation may be in the range of 0.5 ng / μL to 12 ng / μL (e.g., in 100 μL). For example, in some embodiments, the DNA concentration of each sample derived from the library preparation may be in the range of 0.5 ng / μL to 120 ng / μL.In some embodiments, the DNA concentration of the combined samples from the library preparation can be in the range of 0.5 ng / μL to 120 ng / μL (e.g., in 100 μL).

[0117] In some embodiments, at least 1 μL, at least 2 μL, at least 3 μL, at least 5 μL, at least 10 μL, at least 15 μL, at least 20 μL, or at least 25 μL of each sample from a library preparation may be combined, hi some embodiments, up to 2 μL, up to 3 μL, up to 5 μL, up to 10 μL, up to 15 μL, up to 20 μL, up to 25 μL, up to 30 μL, up to 40 μL, or up to 50 μL of each sample from a library preparation may be combined.

[0118] In some embodiments, at least 10 ng, at least 15 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 500 ng, at least 1,000 ng, or at least 5,000 ng of DNA from each library preparation may be combined. In some embodiments, up to 15 ng, up to 25 ng, up to 50 ng, up to 100 ng, up to 200 ng, up to 500 ng, up to 1,000 ng, up to 5,000 ng, up to 10,000 ng, or up to 12,000 ng of DNA from each library preparation may be combined. For example, in some embodiments, 10 ng to 12,000 ng of DNA from each library preparation may be combined. Probe hybridization

[0119] In some embodiments, hybrid capture methods involve contacting a library with a probe, where the probe hybridizes to a region of interest within the library members, the region of interest being distant from the adapter region and containing the genomic material of interest. The probe contains a ligand that allows for subsequent capture of the probe. In some embodiments, the ligand preferably contains a biotin group.

[0120] The library is contacted with the probe in the presence of a hybridization buffer. In some embodiments, the hybridization buffer may include a crowding agent, as described herein. In some embodiments, the hybridization buffer may include a blocker, as described herein.

[0121] In some embodiments, the hybrid capture method includes a heat denaturation step. For example, a hybridization mixture containing library members, blockers, and probes in a hybridization buffer can be heated to at least 90°C, at least 92°C, or at least 95°C. In some embodiments, the hybridization mixture can be heated to up to 92°C, up to 95°C, up to 97°C, or up to 100°C. In some embodiments, the hybridization mixture preferably further includes Cot-1 DNA. In some embodiments, the hybridization buffer can include a blocker before being added to the hybridization mixture. In some embodiments, the blocker can be added to the hybridization mixture independently of the hybridization buffer.

[0122] In some embodiments, the DNA concentration in the hybridization mixture (e.g., the final hybridization reaction) can be at least 0.1 ng / μL, at least 0.3 ng / μL, at least 0.4 ng / μL, at least 0.5 ng / μL, at least 1 ng / μL, at least 2 ng / μL, at least 4 ng / μL, at least 6 ng / μL, at least 8 ng / μL, at least 10 ng / μL, at least 12 ng / μL, at least 15 ng / μL, at least 20 ng / μL, at least 50 ng / μL, at least 75 ng / μL, at least 100 ng / μL, at least 120 ng / μL, at least 150 ng / μL, or at least 200 ng / μL. In some embodiments, the DNA concentration in the hybridization mixture (e.g., the final hybridization reaction) can be up to 0.3 ng / μL, up to 0.4 ng / μL, up to 0.5 ng / μL, up to 1 ng / μL, up to 2 ng / μL, up to 4 ng / μL, up to 6 ng / μL, up to 8 ng / μL, up to 10 ng / μL, up to 12 ng / μL, up to 15 ng / μL, up to 20 ng / μL, up to 50 ng / μL, up to 75 ng / μL, up to 100 ng / μL, up to 120 ng / μL, up to 150 ng / μL, up to 200 ng / μL, or up to 500 ng / μL. For example, in some embodiments, the DNA concentration in the hybridization mixture (e.g., the final hybridization reaction) can be in the range of 0.1 ng / μL to 120 ng / μL.

[0123] In some embodiments, the methods may involve the use of a hybridization mixture volume of up to 150 μL, up to 140 μL, up to 130 μL, up to 120 μL, up to 110 μL, up to 100 μL, up to 90 μL, up to 80 μL, up to 70 μL, up to 60 μL, or up to 50 μL, hi some embodiments, the methods may involve the use of a hybridization mixture volume of at least 10 μL, at least 20 μL, at least 30 μL, at least 40 μL, at least 50 μL, at least 60 μL, or at least 70 μL.

[0124] In some embodiments, the hybridization mixture may be heated at a rate of at least 1°C / min, at least 1.5°C / min, at least 2°C / min, at least 2.5°C / min, at least 3°C / min, or at least 5°C / min. In some embodiments, the hybridization mixture may be heated at a rate of up to 1.5°C / min, up to 2°C / min, up to 2.5°C / min, up to 3°C / min, up to 5°C / min, or up to 10°C / min. In some embodiments, the hybridization mixture may be maintained at the temperature for thermal denaturation for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 20 minutes. In some embodiments, the hybridization mixture may be maintained at the temperature for thermal denaturation for up to 2 minutes, up to 5 minutes, up to 10 minutes, up to 20 minutes, or longer.

[0125] After the heat denaturation step, the hybrid capture method includes cooling the hybridization mixture. When the hybridization mixture is cooled from the heat denaturation step temperature to the hybridization temperature, probe:target hybrids (i.e., probe:library member hybrids) are formed.

[0126] In some embodiments, the hybridization temperature can be at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, or at least 70°C. In some embodiments, the hybridization temperature can be up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C, or up to 70°C. In some embodiments, hybridization can involve a decrease in temperature from the denaturation temperature to the hybridization temperature. In some embodiments, the temperature gradient can include a rate of at least -5°C / min, at least -2°C / min, at least -1°C / min, at least -0.5°C / min, or at least -0.2°C / min.

[0127] In some embodiments, the method may include maintaining the library in contact with the probes and / or maintaining the hybridization mixture at the hybridization temperature for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 90 minutes, up to 1 hour, or up to 30 minutes. In some embodiments, the method may include maintaining the library in contact with the probes and / or maintaining the hybridization mixture at the hybridization temperature for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours, or at least 24 hours. m is the adapter T m In some embodiments, including higher cases, the blocker:adapter hybrid forms before the adaptor:adapter hybrid forms, thereby preventing the formation of "daisy chains" or concatemers of adaptor:adapter hybrids. capture

[0128] In some embodiments, the hybrid capture method includes capturing probes hybridized to library members using a capture means. The probes can be captured by any suitable means. For example, when the probes contain biotin groups, the probes can be captured using streptavidin beads, such as streptavidin-coated magnetic beads. In some embodiments, the probes can contain FITC and the capture means can contain anti-FITC. In some embodiments, the probes can contain a hapten and the capture means can contain a molecule (e.g., an antibody) that binds to the hapten. Other capture means, such as isotachophoresis, size exclusion chromatography, liquid chromatography, magnetic means, etc., can also be used.

[0129] In some embodiments, such as when the capture means comprises streptavidin-coated beads, the method may include forming a capture mixture comprising the probe (e.g., probe:target hybrid, etc.) and the capture means.

[0130] In some embodiments, the method can include maintaining the capture mixture at a capture temperature. In some embodiments, the capture temperature can be at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, or at least 70°C. In some embodiments, the capture temperature can be up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C, or up to 70°C. In some embodiments, the capture mixture can be maintained at the capture temperature for at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes. In some embodiments, the capture mixture can be maintained at the capture temperature for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 30 minutes, up to 45 minutes, or longer. In some embodiments, additional shaking and / or mixing can be performed every 5 or 10 minutes.

[0131] In some embodiments, the method may further comprise washing the captured probe (the captured probe may comprise, for example, a probe:target hybrid and / or a captured library member). In some embodiments, the captured probe may be washed at least once, at least twice, at least three times, or more. In some embodiments, the captured probe may be washed at most once, at most twice, at most three times, or at most five times.

[0132] In some embodiments, washing the captured library members may include heating the captured probes to a washing temperature in a wash buffer. In some embodiments, the wash buffer may contain a detergent. Any suitable buffer may be used, including, for example, Tris-HCl; piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); piperazine-N,N'-bis(3-propanesulfonic acid) (PIPPS); 2-(N-morpholino)ethanesulfonic acid (MES); (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and the like. Any suitable detergent may be used, including, for example, TWEEN® 20, TWEEN® 80, sodium dodecyl sulfate (SDS), and the like. In some embodiments, the wash buffer may contain a compound, such as betaine, that is intended to reduce the formation of secondary structure in GC-rich regions. In some embodiments, the wash buffer may contain an iron chelator, including, for example, deferoxamine mesylate, EGTA, EDTA, and the like.

[0133] The wash temperature can be at least 20° C., at least 23° C., at least 25° C., at least 30° C., at least 35° C., at least 40° C., at least 45° C., at least 50° C., at least 55° C., at least 56° C., at least 57° C., at least 58° C., at least 59° C., at least 60° C., at least 61° C., at least 62° C., at least 63° C., at least 64° C., at least 65° C., or at least 70° C. In some embodiments, the wash temperature can be up to 30° C., up to 35° C., up to 40° C., up to 45° C., up to 50° C., up to 55° C., up to 56° C., up to 60° C., up to 61° C., up to 62° C., up to 63° C., up to 64° C., up to 65° C., or up to 70° C.

[0134] In some embodiments, the method may further include eluting the captured library members from the capture means and / or probes. For example, the captured library members may be eluted from streptavidin beads and / or biotinylated probes. In embodiments in which the streptavidin beads comprise streptavidin magnetic beads, elution may include the use of a magnet. In some embodiments, elution may include the use of a strong base, including, for example, NaOH, KOH, Ca(OH)2, etc.

[0135] In some embodiments, a solid support may be used as a capture means, such as a solid support comprising streptavidin. In some embodiments, the capture means captures the putative T of the probe:target under conditions of successively increasing stringency. m Temperature lower than the value and / or T of the adapter m The washing temperature may be higher than the above value.

[0136] In some embodiments, captured library members can be eluted from the ligand (e.g., eluted from a biotinylated probe) or from the capture means (e.g., eluted from streptavidin beads) and loaded directly onto a flow cell.

[0137] In some embodiments, at least 60 femtomoles, at least 80 femtomoles, at least 90 femtomoles, at least 100 femtomoles, or at least 150 femtomoles of DNA can be eluted. In some embodiments, up to 150 femtomoles, up to 500 femtomoles, up to 1 picomole, up to 2 picomoles, or up to 3 picomoles of DNA can be eluted. For example, in some embodiments, 100 femtomoles to 2 picomoles of DNA can be eluted.

[0138] In some embodiments, eluted library members can be loaded onto a flow cell in a volume of less than 100 μL, less than 90 μL, less than 80 μL, less than 70 μL, or less than 60 μL, hi some embodiments, captured library members can be loaded onto a flow cell in a volume of approximately 55 μL.

[0139] In some embodiments, after elution, the eluate may have a DNA concentration of at least 1.1 picomolar (pM), at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM. In some embodiments, the eluate may have a DNA concentration of up to 100 pM, up to 200 pM, up to 250 pM, or up to 300 pM. For example, in some embodiments, the eluate may have a DNA concentration in the range of 1.3 pM to 250 pM. In some embodiments, the eluate may be loaded onto a flow cell without further dilution and / or alteration of the DNA concentration. amplification

[0140] In another embodiment, the present disclosure describes a method that includes amplifying the library members using PCR after hybrid capture but before sequencing the captured library members. Alternatively, the present disclosure describes a method that does not include subjecting the captured library members to amplification conditions after hybrid capture and before sequencing the library members (e.g., a method that does not include subjecting the captured library members to a PCR step to amplify the captured library members before sequencing the library members). Methods of sequencing captured library members typically include amplifying the library members (e.g., using a PCR step) before sequencing the library members.

[0141] The amount of DNA captured during hybridization and hybrid capture may be too small to be directly quantified by fluorometry or analytical methods (e.g., bioanalyzers). Library amplification allows for quantification and quality control of the process.

[0142] Furthermore, it is common to dilute the PCR-amplified sample after hybrid capture and before sequencing. For example, typical quantification of Illumina, Inc.'s NEXTERA Rapid Capture samples after PCR is performed in the mid-nanomolar range, while sequencing concentrations are in the low picomolar range. Therefore, amplifying the captured library members generates several orders of magnitude more molecules than are required for sequencing.

[0143] In some embodiments, the present disclosure describes methods for sequencing captured library members after hybrid capture. Such methods may include loading captured and / or eluted library members directly onto a flow cell (e.g., using a direct flow cell loading jig, such as described in U.S. Provisional Patent Application No. 62 / 564,466, filed September 28, 2017).

[0144] In some embodiments, the method may further comprise sequencing the captured library members after hybrid capture. Embodiment Exemplary Blocker Embodiments

[0145] 1. A blocker comprising an oligonucleotide, The blocker can bind to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); The region of the blocker that can bind to the index region and / or UMI of the adapter is at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or A blocker comprising a universal base and at least one non-universal base.

[0146] 2. The blocker of embodiment 1, wherein the region of the blocker that can bind to the index region or UMI of the adapter comprises the same number of thymine bases as the number of bases in the index region and / or UMI of the adapter.

[0147] 3. The blocker of embodiment 1, wherein the at least one non-universal base is located in a region of the blocker sequence corresponding to the index region and / or UMI, and the non-universal base is located at the third or fifth position, or both, of the blocker sequence relative to the 5' end of the index region.

[0148] 4. The blocker of blocker embodiment 1 or blocker embodiment 3, wherein the at least one non-universal base comprises a modified guanine.

[0149] 5. The blocker of any one of embodiments 1, 3, or 4, wherein the at least one non-universal base comprises a random nucleotide.

[0150] 6. The blocker of any one of embodiments 1, 3, 4, or 5, wherein the universal base comprises 2'-deoxyinosine, 2'-deoxynebularine, 3-nitropyrrole 2'-deoxynucleoside, or 5-nitroindole 2'-deoxynucleoside.

[0151] 7. The blocker of any one of the preceding blocker embodiments, wherein said universal primer sequence comprises at least one of P5, P7, P5', P7', V2.A14.METS, V2.B15.METS, the complement of V2.A14.METS, and the complement of V2.B15.METS.

[0152] 8. The blocker has a T of the blocker compared to the same blocker that does not contain the modification. m The blocker of any one of the preceding blocker embodiments, comprising a modification that increases

[0153] 9. The blocker of any one of the preceding blocker embodiments, wherein the blocker comprises at least one of: a DNA or RNA oligonucleotide modified to capture a low GC region; a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap.

[0154] 10. The blocker of any one of the preceding blocker embodiments, wherein the blocker comprises a modified base in every 1st base, at least every 2nd base, at least every 3rd base, at least every 4th base, or at least every 5th base.

[0155] 11. The blocker of any one of the preceding blocker embodiments, wherein the blocker comprises a 3' end group that prevents the blocker from being available to act as a primer for DNA synthesis.

[0156] 12. The blocker of embodiment 11, wherein the 3'-end group comprises 3'-dC, 2',3'-ddC, inverted dT, or a 3'-spacer C3.

[0157] 13. The blocker of any one of the preceding blocker embodiments, wherein said blocker comprises a sequence in Table 2A, Table 2B, Table 2C, or Table 3.

[0158] 14. A kit comprising a blocker according to any one of the preceding blocker embodiments. Exemplary Split Blocker Embodiments

[0159] 1. A blocker comprising two unconnected oligonucleotides, the blocker is capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); The unconnected oligonucleotide comprises a base that does not correspond to the index region and / or UMI of the adapter, a blocker.

[0160] 2. The split blocker of embodiment 1, wherein said unconnected oligonucleotide does not comprise a base corresponding to an index region and / or UMI of said adapter.

[0161] 3. The blocker of any one of the preceding split blocker embodiments, wherein the universal primer sequence comprises at least one of P5, P7, P5', P7', V2.A14.METS, V2.B15.METS, the complement of V2.A14.METS, and the complement of V2.B15.METS.

[0162] 4. The blocker has a T of the blocker compared to the same blocker that does not contain the modification. m The blocker of any one of the preceding split blocker embodiments, comprising a modification that increases

[0163] 5. The blocker of any one of the preceding split blocker embodiments, wherein the blocker comprises at least one of: a DNA or RNA oligonucleotide modified to capture a low GC region; a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap.

[0164] 6. The blocker of any one of the preceding split blocker embodiments, wherein said blocker comprises a modified base in every 1st base, at least every 2nd base, at least every 3rd base, at least every 4th base, or at least every 5th base.

[0165] 7. The blocker of any one of the preceding split blocker embodiments, wherein said blocker comprises a 3' end group that prevents the blocker from being available to act as a primer for DNA synthesis.

[0166] 8. The blocker of embodiment 11, which is a split blocker, wherein the 3'-end group comprises 3'-dC, 2',3'-ddC, inverted dT, or a 3'-spacer C3.

[0167] 9. The blocker of any one of the preceding split blocker embodiments, wherein said blocker comprises a sequence in Table 2A, Table 2B, Table 2C, or Table 3.

[0168] 10. The blocker of any of the preceding split blocker embodiments, wherein said blocker comprises at least one of BN023, BN024, BN027 and BN028 of Table 2A.

[0169] 11. A kit comprising a blocker according to any one of the preceding split blocker embodiments. Exemplary Hybridization Buffer Embodiments

[0170] 1. Hybridization buffer containing a crowding agent.

[0171] 2. The hybridization buffer of embodiment 1, wherein said crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), ficoll, glycerol, and betaine.

[0172] 3. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the crowding agent is included in the hybridization buffer in an amount of at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%, or at least 4%.

[0173] 4. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the crowding agent is included in the hybridization buffer in an amount of up to 1%, up to 1.5%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 8%, or up to 10%.

[0174] 5. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein said hybridization buffer comprises at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

[0175] 6. The hybridization buffer of embodiment 5, wherein said hybridization buffer comprises Cot-1 DNA in an amount of at least 0.05 mg / mL, at least 0.1 mg / mL, at least 0.2 mg / mL, or at least 0.3 mg / mL.

[0176] 7. The hybridization buffer of embodiment 5 or 6, wherein said hybridization buffer comprises Cot-1 DNA in an amount of up to 0.1 mg / mL, up to 0.2 mg / mL, up to 0.3 mg / mL or up to 0.5 mg / mL.

[0177] 8. The hybridization buffer according to any one of embodiments 5 to 7, wherein the destabilizing agent constitutes at least 1% (v / v), at least 5% (v / v), or at least 10% (v / v) of the hybridization buffer.

[0178] 9. The hybridization buffer according to any one of embodiments 5 to 8, wherein the destabilizing agent constitutes up to 5% (v / v), up to 10% (v / v), or up to 15% (v / v) of the hybridization buffer.

[0179] 10. The hybridization buffer of any one of embodiments 5 to 9, wherein the destabilizing agent comprises formamide.

[0180] 11. The hybridization buffer of any one of embodiments 5 to 10, wherein the hybridization buffer comprises at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, or at least 1 M salt.

[0181] 12. The hybridization buffer of any one of embodiments 5-11, wherein the hybridization buffer comprises at most 0.2M, at most 0.3M, at most 0.4M, at most 0.5M, at most 0.6M, at most 0.7M, at most 0.8M, at most 0.9M, at most 1M, at most 2M, at most 3M, or at most 4M salt.

[0182] 13. The hybridization buffer according to any one of embodiments 5 to 11, wherein the salt comprises NaCl.

[0183] 14. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises phosphate.

[0184] 15. The hybridization buffer of embodiment 14, wherein the hybridization buffer comprises at least 40 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, or at least 70 mM phosphate.

[0185] 16. The hybridization buffer of embodiment 14 or 15, wherein the hybridization buffer comprises up to 50 mM, up to 55 mM, up to 60 mM, up to 65 mM, up to 70 mM, up to 75 mM, or up to 80 mM phosphate.

[0186] 17. The hybridization buffer according to any one of embodiments 14 to 16, wherein the phosphate comprises KH2PO4—K2HPO4.

[0187] 18. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises a surfactant.

[0188] 19. The hybridization buffer of embodiment 18, wherein the hybridization buffer comprises at least 0.001% (v / v), at least 0.01% (v / v), at least 0.05% (v / v), at least 0.1% (v / v), at least 1% (v / v), or at least 5% (v / v) of a surfactant.

[0189] 20. The hybridization buffer according to embodiment 18 or 19, wherein the hybridization buffer comprises up to 0.01% (v / v), up to 0.05% (v / v), up to 0.1% (v / v), up to 1% (v / v), up to 5% (v / v), or up to 10% (v / v) of a surfactant.

[0190] 21. The hybridization buffer according to any one of embodiments 18 to 20, wherein the surfactant comprises TWEEN® 20.

[0191] 22. The hybridization buffer comprises: 0.5% to 10% dextran sulfate, 0.05mg / mL to 0.5mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40mM~80mM KH2PO4-K2HPO4, 0.1M to 4M NaCl, and 0.001% to 10% (v / v) Tween® 20 The hybridization buffer of any one of the preceding hybridization buffer embodiments, comprising:

[0192] 23. The hybridization buffer comprises: 1.5% dextran sulfate, 0.2mg / mL human Cot-1 DNA, 10% (v / v) formamide, 66.6mM KH2PO4-K2HPO4, 0.2mM Enhanced Adapter Blocker (0.1mM for each adapter) 0.8M NaCl, and The hybridization buffer of any one of the preceding hybridization buffer embodiments, comprising 0.04% (v / v) Tween® 20.

[0193] 24. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein the hybridization buffer comprises a blocker.

[0194] 25. The blocker is T m 25. The hybridization buffer according to embodiment 24, comprising a high temperature oligonucleotide.

[0195] 26. The blocker is T m 26. The hybridization buffer according to embodiment 24 or 25, comprising a plurality of modifications that enhance

[0196] 27. T of the blocker m 27. The hybridization buffer of embodiment 26, wherein the multiple modifications that increase I include at least one of: bridged oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acid (LNA); bridged nucleic acid (BNA); tricyclic nucleic acid; peptide nucleic acid (PNA); C5-modified pyrimidine base; propynyl pyrimidine; morpholino; phosphoramidite; and 5'-pyrene cap.

[0197] 28. The hybridization buffer comprises a blocker; the blocker comprises an oligonucleotide; The blocker can bind to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); The region of the blocker that can bind to the index region and / or UMI of the adapter is at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or a universal base and at least one non-universal base The hybridization buffer of any one of the preceding hybridization buffer embodiments, comprising:

[0198] 29. The hybridization buffer comprises a blocker; the blocker comprises two unconnected oligonucleotides; the blocker is capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); 10. The hybridization buffer of any one of the preceding hybridization buffer embodiments, wherein said unligated oligonucleotide comprises a base that does not correspond to an index region of an adapter and / or a UMI of an adapter.

[0199] 30. The blocker is T m 30. The hybridization buffer according to embodiment 28 or 29, comprising a plurality of modifications that enhance

[0200] 31. A kit comprising the hybridization buffer of any one of the preceding hybridization buffer embodiments. Exemplary Methods Using Hybridization Buffer (Buffer Method Embodiments)

[0201] 1. A method comprising using a hybridization buffer containing a crowding agent for hybrid capture.

[0202] 2. The method of buffer method embodiment 1, wherein said crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), ficoll, glycerol, and betaine.

[0203] 3. The method according to any one of the preceding buffer method embodiments, wherein the crowding agent is included in the hybridization buffer in an amount of at least 0.5% (w / v), at least 1% (w / v), at least 1.5% (w / v), at least 2% (w / v), at least 3% (w / v), or at least 4% (w / v).

[0204] 4. The method of any one of the preceding buffer method embodiments, wherein the crowding agent is included in the hybridization buffer in an amount of up to 1% (w / v), up to 1.5% (w / v), up to 2% (w / v), up to 3% (w / v), up to 4% (w / v), up to 5% (w / v), up to 6% (w / v), up to 8% (w / v), or up to 10% (w / v).

[0205] 5. The method of any one of the preceding buffer method embodiments, wherein said hybridization buffer comprises at least one of human Cot-1 DNA, a destabilizing agent, and a salt.

[0206] 6. The method of buffer method embodiment 5, wherein said hybridization buffer comprises human Cot-1 DNA in an amount of at least 0.05 mg / mL, at least 0.1 mg / mL, at least 0.2 mg / mL, or at least 0.3 mg / mL.

[0207] 7. The method of buffer method embodiment 5 or 6, wherein said hybridization buffer comprises human Cot-1 DNA in an amount of up to 0.1 mg / mL, up to 0.2 mg / mL, up to 0.3 mg / mL or up to 0.5 mg / mL.

[0208] 8. The method according to any one of embodiments 5 to 7, wherein the destabilizing agent constitutes at least 1% (v / v), at least 5% (v / v), or at least 10% (v / v) of the hybridization buffer.

[0209] 9. The method according to any one of embodiments 5 to 8, wherein the destabilizing agent constitutes up to 5% (v / v), up to 10% (v / v), or up to 15% (v / v) of the hybridization buffer.

[0210] 10. The method of any one of embodiments 5 to 9, wherein the destabilizing agent comprises formamide.

[0211] 11. The method of any one of methods 5 to 10, wherein the hybridization buffer comprises at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, or at least 1 M salt.

[0212] 12. The method of any one of methods 5-11, wherein the hybridization buffer comprises at most 0.2M, at most 0.3M, at most 0.4M, at most 0.5M, at most 0.6M, at most 0.7M, at most 0.8M, at most 0.9M, at most 1M, at most 2M, at most 3M, or at most 4M salt.

[0213] 13. The method of any one of embodiments 5 to 12, wherein the salt comprises NaCl.

[0214] 14. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises phosphate.

[0215] 15. The method according to buffer method embodiment 14, wherein the hybridization buffer comprises at least 40 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, or at least 70 mM phosphate.

[0216] 16. The method of buffer method embodiment 14 or 15, wherein the hybridization buffer comprises up to 50 mM, up to 55 mM, up to 60 mM, up to 65 mM, up to 70 mM, up to 75 mM, or up to 80 mM phosphate.

[0217] 17. The method according to any one of embodiments 14 to 16, wherein the phosphate comprises KH2PO4—K2HPO4.

[0218] 18. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises a surfactant.

[0219] 19. The method according to buffer method embodiment 17, wherein the hybridization buffer comprises at least 0.001% (v / v), at least 0.01% (v / v), at least 0.05% (v / v), at least 0.1% (v / v), at least 1% (v / v), or at least 5% (v / v) of a surfactant.

[0220] 20. The method according to buffer method embodiment 18 or 19, wherein the hybridization buffer comprises up to 0.01% (v / v), up to 0.05% (v / v), up to 0.1% (v / v), up to 1% (v / v), up to 5% (v / v), or up to 10% (v / v) of a surfactant.

[0221] 21. The method according to any one of embodiments 18 to 20, wherein the hybridization buffer comprises TWEEN® 20.

[0222] 22. The hybridization buffer comprises: 0.5% to 10% dextran sulfate, 0.05mg / mL~0.5mg / mL Cot-1, 1% to 15% (v / v) formamide, 40mM~80mM KH2PO4-K2HPO4, 0.1M to 4M NaCl, and 0.001% to 10% (v / v) TWEEN® 20 The method according to any one of the preceding buffer method embodiments, comprising:

[0223] 23. The hybridization buffer comprises: 1.5% dextran sulfate, 0.2 mg / mL Cot-1, 10% (v / v) formamide, 66.6mM KH2PO4-K2HPO4, 0.8 M NaCl, and 0.04% (v / v) TWEEN® 20 The method according to any one of the preceding buffer method embodiments, comprising:

[0224] 24. The method of any one of the preceding buffer method embodiments, wherein the hybridization buffer comprises a blocker.

[0225] 25. The method according to buffer method embodiment 24, wherein the blocker is present at a concentration of at least 0.001 mM, at least 0.005 mM, at least 0.01 mM, at least 0.05 mM, or at least 0.1 mM.

[0226] 26. The method according to buffer method embodiment 24 or 25, wherein the blocker is present at a concentration of at most 0.01 mM, at most 0.05 mM, at most 0.1 mM, at most 0.2 mM or at most 0.5 mM.

[0227] 27. The method of any one of the preceding buffer method embodiments, wherein said method comprises forming said hybridization buffer from a first composition comprising human Cot-1 DNA and a blocker, and a second composition comprising formamide and dextran sulfate.

[0228] 28. The method of any one of the preceding buffer method embodiments, wherein the method comprises contacting a library with the hybridization buffer, and the hybridization buffer comprises a blocker.

[0229] 29. The method of embodiment 28, wherein the method comprises combining samples from at least two library preparations prior to contacting the library with the blocker in the presence of the hybridization buffer.

[0230] 30. The method of buffer method embodiment 29, wherein at least 1 μL, at least 2 μL, at least 3 μL, at least 5 μL, at least 10 μL, at least 15 μL, or at least 20 μL of sample from each library preparation is combined.

[0231] 31. The method of method embodiment 29 or 30, wherein up to 2 μL, up to 3 μL, up to 5 μL, up to 10 μL, up to 15 μL, up to 20 μL, up to 25 μL, up to 30 μL, up to 40 μL, or up to 50 μL of buffered sample from each library preparation is combined.

[0232] 32. The method of any one of methods 29-31, wherein at least 10 ng, at least 15 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 500 ng, at least 1,000 ng, or at least 5,000 ng of DNA from each library preparation is combined.

[0233] 33. The method of any one of methods 29-32, wherein up to 15 ng, up to 25 ng, up to 50 ng, up to 100 ng, up to 200 ng, up to 500 ng, up to 1,000 ng, up to 5,000 ng, up to 10,000 ng, or up to 12,000 ng of DNA from each library preparation is combined.

[0234] 34. The method of any one of buffer methods embodiments 29-33, wherein the DNA concentration of each sample from the library preparations to be combined is at least 0.1 ng / μL, at least 0.3 ng / μL, at least 0.4 ng / μL, at least 0.5 ng / μL, at least 1 ng / μL, at least 2 ng / μL, at least 4 ng / μL, at least 6 ng / μL, at least 8 ng / μL, at least 10 ng / μL, at least 12 ng / μL, at least 20 ng / μL, at least 50 ng / μL, at least 60 ng / μL, at least 100 ng / μL, or at least 200 ng / μL.

[0235] 35. The method of any one of methods 29-34, wherein the DNA concentration of each sample from the combined library preparations is at most 0.4 ng / μL, at most 0.5 ng / μL, at most 1 ng / μL, at most 2 ng / μL, at most 4 ng / μL, at most 6 ng / μL, at most 8 ng / μL, at most 10 ng / μL, at most 12 ng / μL, at most 15 ng / μL, at most 20 ng / μL, at most 50 ng / μL, at most 100 ng / μL, at most 120 ng / μL, at most 200 ng / μL, at most 300 ng / μL, or at most 400 ng / μL.

[0236] 36. The method according to any one of embodiments 29 to 35, wherein the DNA concentration after combining the library preparations is in the range of 0.3 ng / μL to 400 ng / μL.

[0237] 37. The method of any one of embodiments 29 to 36, wherein the method further comprises contacting the library members with a probe, the probe hybridizing to a region of interest within the library members, and the probe comprising a ligand.

[0238] 38. The method according to embodiment 37, wherein the ligand comprises a biotin group.

[0239] 39. The method of any one of embodiments 29 to 38, wherein the method comprises forming a hybridization mixture comprising library members, the hybridization buffer (wherein the hybridization buffer comprises a blocker), and a probe.

[0240] 40. The method of embodiment 39, wherein the hybridization mixture further comprises human Cot-1 DNA.

[0241] 42. The method of buffer method embodiment 39 or 40, wherein the method comprises using a volume of hybridization mixture of at most 150 μL, at most 140 μL, at most 130 μL, at most 120 μL, at most 110 μL, at most 100 μL, at most 90 μL, at most 80 μL, at most 70 μL, at most 60 μL, or at most 50 μL.

[0242] 42. The method of any one of embodiments 39-41, wherein the method comprises using a volume of hybridization mixture of at least 10 μL, at least 20 μL, at least 30 μL, at least 40 μL, at least 50 μL, at least 60 μL, or at least 70 μL.

[0243] 43. The method of any one of methods 39-42, wherein the DNA concentration in the hybridization mixture is at least at least 0.1 ng / μL, at least 0.3 ng / μL, at least 0.4 ng / μL, at least 0.5 ng / μL, at least 1 ng / μL, at least 2 ng / μL, at least 4 ng / μL, at least 6 ng / μL, at least 8 ng / μL, at least 10 ng / μL, at least 12 ng / μL, at least 15 ng / μL, at least 20 ng / μL, at least 50 ng / μL, at least 75 ng / μL, at least 100 ng / μL, at least 120 ng / μL, at least 150 ng / μL, at least 200 ng / μL.

[0244] 44. The method of any one of methods 39-43, wherein the DNA concentration in the hybridization mixture is at most 0.3 ng / μL, at most 0.4 ng / μL, at most 0.5 ng / μL, at most 1 ng / μL, at most 2 ng / μL, at most 4 ng / μL, at most 6 ng / μL, at most 8 ng / μL, at most 10 ng / μL, at most 12 ng / μL, at most 15 ng / μL, at most 20 ng / μL, at most 50 ng / μL, at most 75 ng / μL, at most 100 ng / μL, at most 120 ng / μL, at most 150 ng / μL, at most 200 ng / μL, or at most 500 ng / μL.

[0245] 45. The method of any one of embodiments 37-44, wherein the library is contacted with the probes for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 90 minutes, up to 1 hour, or up to 30 minutes.

[0246] 46. ​​The method of any one of embodiments 37-45, wherein the library is contacted with the probes for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours, or at least 24 hours.

[0247] 47. The method of any one of buffer methods 39-46, wherein the method comprises maintaining the hybridization mixture at a hybridization temperature, wherein the hybridization temperature is at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, or at least 70°C.

[0248] 48. The method of any one of embodiments 39-47, wherein the method comprises maintaining the hybridization mixture at a hybridization temperature, wherein the hybridization temperature is at most 56°C, at most 58°C, at most 60°C, at most 61°C, at most 62°C, at most 63°C, at most 64°C, at most 65°C, or at most 70°C.

[0249] 49. The method of any one of embodiments 39-48, wherein the method comprises maintaining the hybridization mixture at the hybridization temperature for up to 3 days, up to 2 days, up to 24 hours, up to 20 hours, up to 16 hours, up to 12 hours, up to 6 hours, up to 3 hours, up to 2 hours, up to 90 minutes, up to 1 hour, or up to 30 minutes.

[0250] 50. The method of any one of embodiments 39-49, wherein the method comprises maintaining the hybridization mixture at the hybridization temperature for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, at least 2 hours, at least 3 hours, at least 6 hours, at least 12 hours, at least 16 hours, at least 20 hours, or at least 24 hours.

[0251] 51. The method according to any one of embodiments 37 to 50, wherein the method further comprises capturing the probe after hybridization using a capture means.

[0252] 52. The method of embodiment 51, wherein capturing the probe comprises using streptavidin beads.

[0253] 53. The method according to any one of embodiments 37 to 52, wherein the method comprises forming a capture mixture comprising the probe and a capture means.

[0254] 54. The method of buffer method embodiment 53, wherein the method comprises maintaining the capture mixture at a capture temperature of at least 50°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, or at least 70°C.

[0255] 55. The method of buffer method embodiment 53 or 54, wherein the method comprises maintaining the capture mixture at a capture temperature of up to 56°C, up to 58°C, up to 60°C, up to 61°C, up to 62°C, up to 63°C, up to 64°C, up to 65°C, or up to 70°C.

[0256] 56. The method according to any one of embodiments 53-55, wherein the method comprises maintaining the capture mixture at the capture temperature for at least 2 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes.

[0257] 57. The method of any one of buffer methods 53-56, wherein the method comprises maintaining the capture mixture at the capture temperature for up to 5 minutes, up to 10 minutes, up to 15 minutes, up to 20 minutes, up to 30 minutes, or up to 45 minutes.

[0258] 58. The method according to any one of embodiments 53 to 57, wherein the method comprises washing the captured probe.

[0259] 59. The method of any one of embodiments 53 to 58, wherein the method further comprises eluting the captured library members from the capture means into an eluent.

[0260] 60. The method of any one of embodiments 53-59, wherein the method further comprises eluting the captured library members from the probes into an eluent.

[0261] 61. The method according to buffer method embodiment 60, wherein at least 60 femtomoles, at least 80 femtomoles, at least 90 femtomoles, at least 100 femtomoles, or at least 150 femtomoles of DNA are eluted.

[0262] 62. The method according to buffer method embodiment 60 or 61, wherein up to 150 femtomoles, up to 500 femtomoles, up to 1 picomole, up to 2 picomoles, or up to 3 picomoles of DNA are eluted.

[0263] 63. The method according to any one of buffer methods 60-62, wherein the DNA concentration of the eluent is at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM.

[0264] 64. The method according to any one of buffer methods 60 to 63, wherein the DNA concentration of the eluent is at most 100 pM, at most 200 pM, at most 250 pM or at most 300 pM.

[0265] 65. The method according to any one of buffer methods 60 to 64, wherein the DNA concentration of the eluent is in the range of 1.3 pM to 250 pM.

[0266] 66. The method according to any one of embodiments 37 to 65, wherein the method further comprises sequencing the library members.

[0267] 67. The method according to embodiment 66, wherein the method comprises amplifying the library members before sequencing the library members.

[0268] 68. The method according to buffer method embodiment 66, wherein the method does not include amplifying the library members before sequencing the library members.

[0269] 69. The blocker is T m 69. The method according to any one of embodiments 24 to 68, wherein the buffer comprises a high temperature oligonucleotide.

[0270] 70. The blocker is T m 70. The method of any one of embodiments 24 to 69, wherein the buffer comprises multiple modifications that enhance

[0271] 71. T of the blocker m 71. The method of buffer method embodiment 70, wherein the plurality of modifications that increase β-aminopurine include at least one of: bridged oligonucleotides; modified 5-methyldeoxycytidine (5-methyl-dc); 2,6-diaminopurine; locked nucleic acids (LNA); bridged nucleic acids (BNA); tricyclic nucleic acids; peptide nucleic acids (PNA); C5-modified pyrimidine bases; propynyl pyrimidines; morpholinos; phosphoramidites; and 5'-pyrene caps.

[0272] 72. The blocker comprises an oligonucleotide; the blocker can bind to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); The region of the blocker that can bind to the index region and / or UMI of the adapter is at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or a universal base and at least one non-universal base 72. The method according to any one of embodiments 24 to 71, comprising:

[0273] 73. The blocker comprises two unconnected oligonucleotides; the blocker is capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); 73. The method according to any one of embodiments 24 to 72, wherein the unligated oligonucleotide comprises a base that does not correspond to an index region of the adapter and / or a UMI of the adapter. Exemplary Methods for PCR-Free Hybrid Capture (Capture Method Embodiments)

[0274] 1. contacting the library with a blocker in the presence of a hybridization buffer; and contacting the library with a probe. wherein the probe hybridizes to a region of interest within a library member; The method does not include amplifying the library members using PCR prior to sequencing the library members.

[0275] 2. The method of capture method embodiment 1, wherein the probe further comprises a ligand, and the library members are eluted from the ligand prior to sequencing the library members.

[0276] 3. The method of capture method embodiment 2, wherein the ligand comprises biotin.

[0277] 4. The method of any of capture methods embodiments 2 or 3, wherein said method comprises loading said library members onto a flow cell after elution from said ligand.

[0278] 5. The method according to any one of embodiments 1 to 4, wherein the method further comprises capturing the probe after hybridization using a capture means.

[0279] 6. The method of embodiment 5, wherein the capture means comprises streptavidin.

[0280] 7. The method of any of capture methods 5 or 6, wherein said method comprises loading said library members onto a flow cell after elution from said capture means.

[0281] 8. The method of capture method embodiment 4 or 7, wherein the method comprises loading the library members onto a flow cell in a volume of less than 100 μL, less than 90 μL, less than 80 μL, less than 70 μL, or less than 60 μL.

[0282] 9. The method of capture method embodiment 4 or 7, wherein said method comprises loading said library members onto a flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM.

[0283] 10. The method of capture method embodiment 4, 7 or 9, wherein said method comprises loading said library members onto a flow cell at a concentration of at most 100 pM, at most 200 pM, at most 250 pM or at most 300 pM.

[0284] 11. The method of any one of capture methods 4 or 7-10, wherein the method comprises loading the library members onto a flow cell using a direct flow cell loading jig.

[0285] 12. The method of any one of the preceding capture method embodiments, wherein the hybridization buffer comprises a crowding agent.

[0286] 13. The hybridization buffer comprises: 0.5% to 10% dextran sulfate, 0.05mg / mL to 0.5mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40mM~80mM KH2PO4-K2HPO4, 0.1M to 4M NaCl, and 0.001% to 10% (v / v) Tween® 20 13. The method of claim 12, comprising:

[0287] 14. The blocker is T m The method according to any one of the preceding capture method embodiments, comprising a high temperature oligonucleotide.

[0288] 15. The blocker is T m The method of any one of the preceding capture method embodiments, comprising a plurality of modifications that enhance

[0289] 16. T of the blocker m The method of capture method embodiment 15, wherein the plurality of modifications that increase β-methylcytidine include at least one of: a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap.

[0290] 17. The blocker comprises an oligonucleotide; the blocker can bind to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); a region of the blocker that can bind to the index region and / or UMI of the adapter, at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or a universal base and at least one non-universal base The method of any one of the preceding capture method embodiments, comprising:

[0291] 18. The blocker comprises two unconnected oligonucleotides; the blocker is capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); 10. The method of any one of the preceding capture method embodiments, wherein the unligated oligonucleotide comprises a base that does not correspond to an index region of the adapter and / or a UMI of the adapter.

[0292] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention set forth herein. Embodiment

[0293] The following numbered items provide embodiments as described herein, but the embodiments described herein are not limiting.

[0294] Item 1. A hybridization buffer comprising a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker.

[0295] Item 2. The hybridization buffer of item 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), ficoll, glycerol, and betaine.

[0296] Item 3. The hybridization buffer according to any one of the preceding items, wherein the hybridization buffer comprises 0.5% to 10% dextran sulfate, 0.05 mg / mL to 0.5 mg / mL Cot-1, 1% to 15% (v / v) formamide, 40 mM to 80 mM KH2PO4-K2HPO4, 0.1 M to 4 M NaCl, and 0.001% to 10% (v / v) TWEEN® 20.

[0297] Item 4. The blocker is T m The hybridization buffer of any one of the preceding items, comprising multiple modifications that enhance

[0298] Item 5. A method comprising using the hybridization buffer according to any one of the preceding items.

[0299] Item 6. The method of Item 5, wherein the method comprises forming a hybridization mixture containing library members, the hybridization buffer, human Cot-1 DNA, a blocker, and a probe.

[0300] Item 7. The method of Item 6, wherein the hybridization mixture comprises samples from at least two library preparations, combining at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, or at least 500 ng of DNA from each library preparation.

[0301] Item 8. The method according to Item 6 or Item 7, wherein the DNA concentration of the hybridization mixture is within the range of 0.1 ng / μL to 120 ng / μL.

[0302] Item 9. The method according to any one of Items 6 to 8, wherein the method comprises the steps of: contacting the library members with the probes, wherein the probes hybridize to target regions within the library members and contain a ligand; capturing the hybridized probes using a capture means; and eluting the captured library members from the capture means into an eluent, wherein the DNA concentration of the eluent is within the range of 1.3 pM to 250 pM.

[0303] Item 10. The method according to any one of Items 6 to 9, wherein the method further comprises a step of sequencing the library members, and the method does not comprise a step of amplifying the library members prior to sequencing the library members.

[0304] Item 11. The method of item 10, wherein the method comprises loading the library members onto a flow cell using a direct flow cell loading jig.

[0305] Item 12. A blocker comprising an oligonucleotide, wherein the blocker is capable of binding to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), wherein the region of the blocker that can bind to the index region and / or UMI of the adapter comprises at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or a universal base and at least one non-universal base.

[0306] Item 13. A blocker comprising two unconnected oligonucleotides, wherein the blocker is capable of binding to at least a portion of an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), and the unconnected oligonucleotides comprise bases that do not correspond to the index region and / or UMI of the adapter.

[0307] Item 14. The blocker of items 12 or 13, wherein the universal primer sequence comprises at least one of P5, P7, P5', P7', V2.A14.METS, V2.B15.METS, the complement of V2.A14.METS, and the complement of V2.B15.METS.

[0308] Item 15. The blocker has a T of the blocker compared to the same blocker not containing the modification. m 15. The blocker according to any one of items 12 to 14, comprising a modification that increases

[0309] Item 16. The blocker of any one of items 12 to 15, wherein the blocker comprises at least one of: a DNA or RNA oligonucleotide modified to capture a low GC region; a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap.

[0310] Item 17. A method comprising the steps of contacting a library with a blocker in the presence of a hybridization buffer; and contacting the library with a probe, wherein the probe hybridizes to a region of interest within the library members, the method does not include amplifying the library members using PCR prior to sequencing the library members.

[0311] Item 18. The method of item 17, wherein the method comprises loading the library members onto a flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM and at most 100 pM, at most 200 pM, at most 250 pM, or at most 300 pM.

[0312] Item 19. The blocker is T m 19. The method of claim 17 or 18, comprising multiple modifications that enhance

[0313] Item 20. The method according to any one of Items 17 to 19, wherein the hybridization buffer contains a crowding agent.

[0314] Item 21. The method according to any one of Items 17 to 20, wherein the hybridization buffer comprises 0.5% to 10% dextran sulfate, 0.05 mg / mL to 0.5 mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40 mM to 80 mM KH2PO4-K2HPO4, 0.1 M to 4 M NaCl, and 0.001% to 10% (v / v) Tween® 20.

[0315] Item 22. The method according to any one of Items 17 to 21, wherein the method comprises loading the library members onto a flow cell using a direct flow cell loading jig.

[0316] Item 23. A kit comprising the hybridization buffer according to any one of Items 1 to 4.

[0317] Item 24. A kit comprising the blocker according to any one of Items 12 to 16. [Example]

[0318] Example 1 - Hybridization and Capture Hybridization The non-strengthened hybridization buffer contains the following components / final concentrations in 100 μL of hybridization reaction: human Cot-1 0.2 mg / ml, formamide 10% (v / v), KH2PO4-K2HPO4 66.6 mM, NaCl 0.8 M, TWEEN® 20 0.04% (v / v).

[0319] The enhanced hybridization buffer contains the following components / final concentrations in 100 μL of hybridization reaction: dextran sulfate 1.5% (w / v), human Cot-1 0.2 mg / ml, formamide 10% (v / v), KH2PO4-K2HPO4 66.6 mM, NaCl 0.8 M, TWEEN® 20 0.04% (v / v).

[0320] In each case, the 100 μL final reaction also contains probes (at least 500 probes and up to 500,000 probes) at a concentration of 125 pM per probe (total probe concentration of at least 30 nM), and up to 30 μL (50 ng to 6 μg) of DNA sample. Blockers, if included, are present at 0.2 mM.

[0321] Hybridization reactions are performed with precise temperature control in a thermocycler or HYBEX system. After denaturation at 95°C for 5 minutes, the hybridization reactions are incubated at 58°C or 62°C for 90 minutes (total hybridization time approximately 100 minutes) or up to 24 hours. Depending on the probe design, a ramp of 2°C per minute starting from 95°C to 58°C or 62°C (an additional 20 minutes) can be used, followed by incubation at 58°C or 62°C (for the experiments described herein, samples are incubated at 62°C). capture

[0322] For each 100 μL hybridization reaction, 250 μL of streptavidin beads SMB (Streptavidin Magnetic Beads, Illumina, Inc., San Diego, CA) are used to capture the target DNA for 15 minutes at the hybridization incubation temperature (e.g., 62°C if the hybridization reaction is incubated at 62°C for 90 minutes).The target DNA is then washed four times in total with wash buffer EEW (Enhanced Enrichment Wash buffer, Illumina, Inc., San Diego, CA (Tris-HCl, deferoxamine mesylate (DFO), betaine, and TWEEN® 20)) at the same temperature (e.g., 62°C) for 5 minutes. The targeted DNA was eluted with fresh denaturing solution (0.1% (v / v) TWEEN® 20 (EE1) and 2N NaOH (HP3)), neutralized with ET2 (Elution Target Buffer 2, Illumina, Inc., San Diego, CA (Tris base, Tris acetate)), and further amplified using a primer cocktail (PPC; sequencing adapter primer set containing 5 μM P5 and P7 primers, Illumina, Inc.) and Enhanced PCR Mix (EPM) (Illumina, Inc., San Diego, CA), followed by cleanup with 0.9× Solid Phase Reversible Immobilization (SPRI) beads. Example 2 - Thymine-containing blockers

[0323] This example describes blockers that contain a thymine in the region of the blocker that corresponds to the index and / or UMI (see Figure 3D).

[0324] Hybridization and capture are performed as described in Example 1 using the blockers in Table 3, with enhanced hybridization buffer and 0.2 mM blocker. The resulting captured DNA is sequenced and Padded Read Enrichment is calculated using the Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, CA).

[0325] Blockers containing a stretch of Ts at the index sequence position require at least 8 or 10 modified nucleotides in the region of the blocker that does not correspond to the index to increase capture of on-target DNA (as measured by Padded Read Enrichment).

[0326] Oligos containing sequences complementary to the index region required fewer modified nucleotides (8 and 10 nucleotides were tested) and showed higher Padded Read Enrichment. The results are shown in Figure 4. When the blockers contained the same number of modifications (8 or 10 nucleotides), the blocker containing sequences complementary to the index region (last bar) showed higher Padded Read Enrichment than the blocker containing a stretch of Ts in the index region (labeled "10 modifications and (T)8" in Figure 4). [Table 3] [Table 4-1] [Table 4-2] [Table 5] Example 3 - Blockers containing universal and modified bases

[0327] Hybridization and capture are performed as described in Example 1 using 0.2 mM of the blockers in Table 4 in enhanced hybridization buffer. As shown in Table 4, the regions of the blockers corresponding to the index regions contain a universal base (deoxyinosine) and an LNA-modified guanine or random nucleotides.

[0328] Without wishing to be bound by theory, it is believed that the inclusion of modified Gs or random nucleotides at every third base in a blocker sequence corresponding to an index region (e.g., at the third and / or fifth position (from the 5' end) of a blocker sequence corresponding to an index region, as shown in one embodiment in Figure 5, or at the second and / or fourth position of a blocker sequence corresponding to an index region) improves the affinity of the blocker for library members. Example 4 - Split Blocker

[0329] In this example, we demonstrate that split LNA blockers work well when used with libraries containing an 8-nucleotide index.

[0330] Hybridization and capture were performed as described in Example 1 using the blockers in Table 5 and enhanced hybridization buffer. As shown in Table 5, blockers TiLNAS1 (BN025), TiLNAS2 (BN026), P5LNA (BN023), and P7LNA (BN024) do not contain regions corresponding to the index region. P5LNA (BN023) and P7LNA (BN024) each contain eight LNA-modified bases. TiLNAS1 (BN025) and TiLNAS2 (BN026) each contain ten LNA-modified bases. TiLNA17 (BN027) and TiLNA18 (BN028) contain a universal base (deoxyinosine) in the region of the blocker corresponding to the index region, but the regions of the blocker corresponding to other regions of the adapter are truncated.

[0331] The resulting captured DNA was sequenced and padded read enrichment was calculated using the Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, CA). Diagrams of the blockers are shown in Figure 6A and Figures 3E-3I, and the results are shown in Figure 6B. Sample "Split 4pc" includes TiLNAS1 (BN025), TiLNAS2 (BN026), P5LNA (BN023) and P7LNA (BN024) in Table 5; sample "Inner" includes TiLNAS1 (BN025) and TiLNAS2 (BN026) in Table 5; sample "Outer" includes P5LNA (BN023) and P7LNA (BN024) in Table 5; sample Short 8nt includes TiLNA17 (BN027) and TiLNA18 (BN028) in Table 5. Two two-piece blockers ("split blockers"), including BN023, BN024, BN025, and BN026, performed similarly to XGEN Blocking Oligos (Integrated DNA Technologies, Coralville, IA) as measured by Padded Read Enrichment, and experiments using blockers that blocked only part of the adapter did not perform as well. Example 5 - Hybridization Buffer

[0332] This example shows a comparison of on-target capture using non-enhanced and enhanced hybridization buffers.

[0333] Hybridization and capture are performed as described in Example 1 using XGEN Blocking Oligos (Integrated DNA Technologies, Coralville, IA) and enhanced hybridization buffer with the dextran sulfate concentrations shown in Figure 7A. The resulting captured DNA is sequenced, and Padded Read Enrichment is calculated using the Enrichment v3.0 BASESPACE App (Illumina, Inc., San Diego, CA). The results are shown in Figure 7A. The inclusion of dextran sulfate allows for rapid hybridization (60-90 min) in a high volume (100 μL) of hybridization buffer.

[0334] Enrichment using a 100 μL hybridization buffer volume is evaluated using four different probe panels: Coding Exome Oligos (CEX) (Illumina, Inc., San Diego, CA), IDT Exome Research Panel (IDT Exome) (Integrated DNA Technologies, Coralville, IA), TRUSIGHT Cancer Panel (Cancer) (Illumina, Inc., San Diego, CA), and TRUSIGHT One Sequencing Panel (TSO) (Illumina, Inc., San Diego, CA). Hybridization is tested in three hybridization buffers: IDT buffer from the XGEN LOCKDOWN kit (Integrated DNA Technologies, Coralville, IA), and hybridization buffer with and without dextran sulfate as described in Example 1. The results are shown in Figure 7B. The enhanced hybridization buffer produced similar or higher on-target results compared to the IDT buffer for all four probe panels (ranging from 4,000 to 450,000 probes).

[0335] Two key enrichment metrics (Padded Read Enrichment and Coverage Uniformity) for four panels (CEX, IDT Exome, Cancer, and TSO) are obtained using Illumina Enhanced Hybridization Buffer containing IDT XGEN Blocker at various hybridization incubation times, with and without an additional temperature gradient step. The results are shown in Figure 7C. The black dotted line in the left panel indicates the hybridization results in buffer without dextran sulfate.

[0336] Three runs of 12-plex library input were pooled to a total volume of 30 μL and enriched using Illumina Enhanced Hybridization Buffer containing IDT XGEN blocker. Samples were run on various Illumina, Inc. sequencing instruments (NOVASEQ, NEXTSEQ, and ISEQ). No sample concentration step was required. The results are shown in Figures 7D-7F. The results demonstrate that using Enhanced Hybridization Buffer containing dextran sulfate, up to 30 μL of sample can be accommodated in a single hybridization reaction (e.g., a single 15 μL sample or 12-plex runs at 2.5 μL each). Using Enhanced Hybridization Buffer, up to 12 samples can be enriched in a single reaction without the need for a speed vacuum, centrifugation columns, or SPRI beads to further reduce the pool volume. Example 6 - Improved Adapter + Enhanced Hybridization Buffer

[0337] The enrichment performance of the IDT Exome Panel (Cat# 1056114, Integrated DNA Technologies, Coralville, IA) was evaluated using the following combinations: (1) IDT buffer (17 μL) from the XGEN LOCKDOWN kit (Integrated DNA Technologies, Coralville, IA); (2) non-enhanced adapter blockers (NEXTERA Blocker1 i5 (BN001) and NEXTERA Blocker2 in Table 2) in a small (11 μL) reaction volume; (1) a non-enhanced hybridization buffer containing i7 (BN002) (TiE0); (2) a non-enhanced hybridization buffer containing enhanced adapter blockers (XGEN blockers) in a small (11 μL) reaction volume (TiE3); (3) a non-enhanced hybridization buffer containing enhanced adapter blockers (XGEN blockers) in a large (100 μL) reaction volume (TiE4); and (4) an enhanced hybridization buffer containing enhanced adapter blockers (XGEN blockers) in a large (100 μL) reaction volume (TiE9). The results are shown in Figure 8A and demonstrate that the use of modified blockers, increased wash temperatures, and / or crowding agents (dextran sulfate) in the hybridization buffer improves enrichment performance.

[0338] Enrichment performance (measured using Padded Read Enrichment) is evaluated in nine probe panels using an IDT enrichment assay (Catalog No. 1080584, Integrated DNA Technologies, Coralville, IA) and in an Illumina enrichment assay using enhanced hybridization buffer. High Padded Read Enrichment was measured in 100 μL reaction volumes containing various probe panels (500 to 450,000 probes). Enrichment is achieved using probe panels including: (1) XGEN Exome Research Panel (IDT Exome) (Catalog No. 1056114, Integrated DNA Technologies, Coralville, IA); (2) Twist Human Core Exome (Twist Bioscience, Inc., Catalog No. 100254); (3) Coding Exome Oligos (CEX) (Catalog No. 15034575, Illumina, Inc., San Diego, CA); (4) TruSight One (Illumina Inc., San Diego, CA, Catalog No. FC-141-1007; Probe Catalog No. 15046658); (5) TruSight One Expanded (Illumina, Inc., San Diego, CA, Catalog No. FC-141-2007; Probe Catalog No. 20015656); and (6) TruSight (6) TruSight Cancer (Illumina, Inc., San Diego, CA, Catalog No. FC-121-0202; Probe Catalog No. 15035642); (7) TruSight Cardio (Illumina, Inc., San Diego, CA, Catalog No. FC-141-1010; Probe Catalog No. 15069654); (8) TruSight RNA Fusion Panel (Illumina Inc., San Diego, CA, Catalog No. 20000906); (9) Oncology DNA Probes Master Pool (OPD1) (Illumina, Inc., Catalog No. OP-101-1004; Probe Catalog No. 20001565). The results are shown in Figure 8B. The white bars (column 1) represent Nextera Rapid The gray bars show the performance of the IDT exome panel in the IDT lockdown enrichment workflow using Capture library preparation (Illumina, Inc., San Diego, CA). The gray bars show the performance of the IDT exome panel in the IDT lockdown enrichment workflow using Illumina Enrichment with enhanced hybridization buffer and IDT xGEN adapter blockers. The figures show the performance of the panels when used with the Illumina Enrichment Assay. The black horizontal lines indicate the comparative results obtained with the corresponding panels using the Twist Enrichment Assay (Twist Bioscience, Inc.) (column 3), Nextera Rapid Capture library preparation (columns 4-8), or the TruSight Tumor 170 workflow with the OPD1 panel (column 10). The performance of each panel in the Illumina Enrichment Assay with enhanced hybridization buffer is evaluated in at least three independent experiments; error bars indicate the standard deviation.

[0339] Somatic variants were detected by a single hybridization enrichment protocol using enhanced hybridization buffer and XGEN blocker on a panel of 12 genes and 535 probes to enrich libraries generated from the Horizon Discover HD701 quantitative multiplex (QM) DNA standard. The data shown in Figure 8C plots the expected variant frequency (x-axis) against the called variant frequency (y-axis). Each panel in Figure 8C represents various concentrations of high-affinity adapter blocker (IDT XGEN adapter blocker, 0.1x (0.02 mM) and 0.5x (0.1 mM)) and a negative control (no high-affinity adapter blocker). Each point represents a somatic variant call colored by read depth. The dashed red line represents the predicted value of a perfect correlation between known and called variant frequencies. Blue and gray shading represent the best-fit line and 95% confidence interval, respectively, for the called variant frequency compared to the known variant frequency. Expected variant frequency (x-axis) is calculated using IDT XGEN adapter blocker. Correlates with known (called) variant frequencies (y-axis) for XGEN adapter blockers (0.1x (0.02mM), 0.5x (0.1mM)), but not for negative control (no high-affinity adapter blocker). Example 7A - PCR-free hybrid capture using TRUSEQ adapter-ligated libraries

[0340] This example demonstrates hybrid capture without PCR.

[0341] Hybridization is performed using XGEN LOCKDOWN reagent (Integrated DNA Technologies, Coralville, IA) according to the package instructions with the following modifications: Targeted DNA is generated using the TRUSEQ adapter ligation approach and captured using 100 μL of DYNABEADS M-270 Streptavidin (Integrated DNA Technologies, Coralville, IA). The hybridization is incubated for only 30 minutes instead of the recommended 4 hours. The following protocol is then followed: 1. Incubate the beads with 10 μL of 0.1 N NaOH to denature the captured library from the probes and release them into solution. 2. Remove the captured library solution from the beads (using a magnet) and neutralize with 10 μL of 200 mM Tris.HCl pH 7.0. 3. (Optional) Add denatured PhiX Control v3 (Illumina, Inc., San Diego, CA) to the sample and up to 7.5 μL of resuspension buffer (RSB) (Illumina, Inc., San Diego, CA). 4. Add 27.5 uL of 2x HT1 (hybridization buffer, Illumina, Inc., San Diego, CA).

[0342] The resulting sample (55 μL in 1× HT1 buffer) was loaded directly onto a flow cell using a pipette or a flow cell loading jig, as described, for example, in U.S. Provisional Patent Application No. 62 / 564,466, filed September 28, 2017. The results are shown in Table 6A. The control (column 1) was performed using the same conditions as the PCR-free method, except that PCR was performed after capture. The first attempt at PCR-free enrichment is shown in column 2, and shows that there were too few reads to achieve an acceptable level of coverage for the exome. While the goal was greater than approximately 50×, only an average target coverage of approximately 18× was achieved. The second attempt (column 3) shows that using a direct flow cell loading jig and increasing the DNA input to the hybridization improved the sequencing rate of the PCR-free enrichment method to approximately 140×, far exceeding the target of approximately 40×. Downsampling the no-PCR data to 70 million reads (column 4) yields metrics similar to (or better than) the control. Example 7B - PCR-free hybrid capture using bead-based NEXTERA libraries improves exome quality with short hybridization times

[0343] A control experiment was performed using conditions similar to those in Example 7A, but with libraries generated using a bead-based tagmentation library preparation method and corresponding blocking oligonucleotides. In this experiment, both hybridization duration and PCR amplification were variables. The recommended 4-hour hybridization reaction protocol (7.5 hours total, see IDT XGEN protocol) without direct flow cell loading, sequencing using the NEXTSEQ instrument, and post-capture PCR was used as a control (a schematic of this workflow is shown in Figure 9B) and compared with two conditions using only 30 minutes of hybridization (with and without post-capture PCR) and direct flow cell loading (Figure 9C). By shortening the hybridization duration and eliminating PCR, the enrichment protocol can be performed in only approximately 2 hours. A schematic of this workflow with shortened hybridization time and no PCR is shown in Figure 9D.

[0344] The results are shown in Table 6B. All data are downsampled to approximately 23 million clusters. In the first column (control), the data show good quality exomes, but this is using a long workflow of approximately 7.5 hours. Reducing hybridization to 30 minutes shortens the protocol duration to a total of approximately 4 hours (column 2). However, the number of PCR replicates increases compared to the control. Increasing PCR replicates also reduces library diversity and the corresponding reduction in coverage at approximately 20×. Eliminating PCR from the 30-minute workflow (column 3) significantly reduces PCR replicates, increases diversity to levels above the control, and significantly shortens the workflow (2 hours versus 7.5 hours). This example highlights that eliminating PCR can improve the quality of hybridization capture reactions by minimizing template duplication during PCR. [Table 6A] [Table 6B] Example 8 - Blocker Comparison

[0345] Hybridization and capture are performed as described in Example 1 using 0.2 mM blockers BN001 and BN002; BX003 and BX007; BN007 and BN008; BN005 and BN006; or BN023, BN024, BN025, and BN026 (as listed in Table 2A) in enhanced hybridization buffer. "Unmodified" blockers BN001 and BN002 contain thymine in the region of the blocker corresponding to the index region of the adapter and a base complementary to the adapter in the region of the blocker corresponding to the non-index region of the adapter. Blockers BN001 and BN002 do not contain modified bases and contain 3'-ddC end groups. Blockers BX003 and BX007 are modified versions of BN001 and BN002, each with two AP-dC-CE phosphoramidite (G-Clamp) substitutions in the region of the blocker corresponding to the non-index region of the adapter. Blockers BN007 and BN008 are modified versions of BN001 and BN002 with a universal base (deoxyinosine) in the region of the blocker corresponding to the index region of the adapter and a BNA-modified base in the region of the blocker corresponding to the non-index region of the adapter. Blockers BN023 and BN025 are modified versions of BN001 in the region of the blocker corresponding to the non-index region of the adapter, including an LNA-modified base; these blockers also contain a 3'-spacer C3 instead of 3'-ddC. Blockers BN024 and BN026 are modified versions of the region of BN002 that corresponds to the non-index region of the adapter and contain LNA modified bases; these blockers also contain a 3'-spacer C3 instead of 3'-ddC.

[0346] The resulting captured DNA is sequenced and the Padded Read Enrichment, a measure of the amount of captured on-target DNA, is calculated. The results are shown in Figure 10 and demonstrate that various modifications of the "unmodified" blocker improve Padded Read Enrichment in hybridization assays. Example 9 - Split Blocker Comparison

[0347] Hybridization and capture were performed as described in Example 1 using 0.2 mM BN001 and BN002; or BN023, BN024, BN025, and BN026 (as listed in Table 2A) in enhanced hybridization buffer. The corresponding adapters contained 8-nucleotide or 10-nucleotide indices. The resulting captured DNA was sequenced, and padded read enrichment was calculated. The results are shown in Figure 11 and demonstrate that the use of "split blockers" (i.e., blockers that do not contain bases corresponding to the index region of the adapter) allows these blockers to be used with adapters with various index sequences and lengths. These results demonstrate that split blockers can be used in situations where accommodating changes in index design may be disadvantageous.

[0348] The entire disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (e.g., nucleotide sequence entries in GenBank and RefSeq, and amino acid sequence entries in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference. In the event of a discrepancy between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples have been provided merely for clarity of understanding. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to those skilled in the art which are encompassed within the scope of the invention as defined by the claims. In certain embodiments, for example, the following items are provided: (Item 1) A hybridization buffer comprising a crowding agent and at least one of human Cot-1 DNA, a destabilizing agent, a salt, and a blocker. (Item 2) 2. The hybridization buffer of claim 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), Ficoll, glycerol, and betaine. (Item 3) 3. The hybridization buffer according to item 1 or 2, comprising a crowding agent and a blocker. (Item 4) 3. The hybridization buffer according to item 1 or 2, comprising a crowding agent, human Cot-1 DNA, a blocker, a destabilizing agent, a buffer, a salt, and a surfactant. (Item 5) 5. The hybridization buffer according to any one of items 1 to 4, wherein the hybridization buffer comprises a buffer, and the buffer is a phosphate buffer. (Item 6) 6. The hybridization buffer according to any one of items 1 to 5, wherein the hybridization buffer contains a salt, and the salt is NaCl or sodium citrate. (Item 7) 7. The hybridization buffer according to any one of items 1 to 6, wherein the hybridization buffer comprises a destabilizing agent, and the destabilizing agent is formamide or urea or a mixture thereof. (Item 8) 8. The hybridization buffer according to any one of items 1 to 7, wherein the hybridization buffer comprises a surfactant, and the surfactant is TWEEN® 20, TWEEN® 80, or sodium dodecyl sulfate (SDS). (Item 9) The hybridization buffer is 0.5% to 10% dextran sulfate, 0.05mg / mL to 0.5mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40mM~80mM KH2PO4-K2HPO4, 0.1M to 4M NaCl, and 0.001% to 10% (v / v) TWEEN® 20 2. The hybridization buffer according to item 1, comprising: (Item 10) The hybridization buffer comprises a blocker, the blocker comprising: (a)T m Multiple modifications to enhance; (b) T of said blockers comprising at least one of: a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap. m Multiple modifications that increase; (c) an oligonucleotide, wherein the blocker is capable of binding to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); the region of the blocker capable of binding to the index region and / or UMI of the adapter comprises at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or an oligonucleotide comprising a universal base and at least one non-universal base; or (d) two unconnected oligonucleotides, wherein the blocker is capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI); and the unconnected oligonucleotides comprising bases that do not correspond to the index region and / or the UMI of the adaptor. 10. The hybridization buffer according to any one of items 1 to 9, comprising at least one of: (Item 11) 11. A method for preparing an enriched library, the method comprising forming a hybridization mixture comprising library members, the hybridization buffer of any one of items 1 to 10, and a probe under conditions sufficient for the probe to hybridize to a region of interest within the library members, wherein the probe comprises a ligand, to form a hybridization mixture. (Item 12) capturing the hybridized probes using a capture means; and eluting the captured library members from the capture means into an eluate containing enriched library members, wherein the DNA concentration of the eluate is in the range of 1.3 pM to 250 pM. Item 12. The method according to item 11, comprising: (Item 13) 13. The method of claim 11 or 12, wherein the hybridization mixture comprises samples from at least two library preparations, wherein at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, or at least 500 ng of DNA from each library preparation is combined. (Item 14) 14. The method according to any one of items 11 to 13, wherein the DNA concentration of the hybridization mixture is in the range of 0.1 ng / μL to 120 ng / μL. (Item 15) 15. The method of any one of items 11 to 14, comprising sequencing the enriched library members, wherein the method does not comprise amplifying the library members prior to capturing the library members. (Item 16) 16. The method of claim 15, wherein the sequencing step is performed on a flow cell, and the method comprises loading the enriched library members onto the flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM and at most 100 pM, at most 200 pM, at most 250 pM, or at most 300 pM. (Item 17) 17. The method of claim 16, comprising loading the enriched library members onto a flow cell using a direct flow cell loading jig. (Item 18) A kit comprising the hybridization buffer according to any one of items 1 to 10 and instructions for use. (Item 19) A blocker comprising an oligonucleotide, the blocker capable of binding to an adapter, the adapter comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), wherein the region of the blocker capable of binding to the index region and / or UMI of the adapter comprises at least three thymine bases, at least four thymine bases, at least five thymine bases, at least six thymine bases, at least seven thymine bases, at least eight thymine bases, at least nine thymine bases, or at least ten thymine bases; or a blocker comprising a universal base and at least one non-universal base. (Item 20) A blocker comprising two unconnected oligonucleotides, the blocker capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), the unconnected oligonucleotides comprising bases that do not correspond to the index region and / or the UMI of the adaptor. (Item 21) 21. The blocker of claim 19 or claim 20, wherein the universal primer sequence comprises at least one of P5, P7, P5', P7', V2.A14.METS, V2.B15.METS, the complement of V2.A14.METS, and the complement of V2.B15.METS. (Item 22) The blocker has a T of the blocker compared to the same blocker without the modification. m 22. The blocker according to any one of items 19 to 21, comprising a modification that increases (Item 23) 23. The blocker of any one of items 19 to 22, wherein the blocker comprises at least one of: a DNA or RNA oligonucleotide modified to capture a low GC region; a bridged oligonucleotide; a modified 5-methyldeoxycytidine (5-methyl-dc); a 2,6-diaminopurine; a locked nucleic acid (LNA); a bridged nucleic acid (BNA); a tricyclic nucleic acid; a peptide nucleic acid (PNA); a C5-modified pyrimidine base; a propynyl pyrimidine; a morpholino; a phosphoramidite; and a 5'-pyrene cap.

Claims

1. A hybridization buffer comprising a crowding agent, human Cot-1 DNA, a destabilizing agent, a salt, and a blocker, wherein the blocker comprises two distinct oligonucleotides, the blocker being capable of binding to at least a portion of an adaptor, the adaptor comprising a universal primer sequence and at least one of an index region or a unique molecular identifier (UMI), wherein the two distinct oligonucleotides each bind to a respective portion of the adaptor such that the two distinct oligonucleotides are separated by a gap when bound to the adaptor, and wherein the gap corresponds to the index region and / or the UMI of the adaptor; A hybridization buffer wherein the blocker comprises a plurality of modifications that increase the T m of the blocker, the plurality of modifications comprising at least one of a bridged oligonucleotide, a modified 5-methyldeoxycytidine (5-methyl-dc), a 2,6-diaminopurine, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a tricyclic nucleic acid, a peptide nucleic acid (PNA), a C5-modified pyrimidine base, a propynyl pyrimidine, a morpholino, a phosphoramidite, and a 5'-pyrene cap.

2. 2. The hybridization buffer of claim 1, wherein the crowding agent comprises at least one of dextran, dextran sulfate, polyethylene glycol (PEG), ficoll, glycerol, and betaine.

3. The hybridization buffer according to claim 1 or 2, comprising a crowding agent, human Cot-1 DNA, a blocker, a destabilizing agent, a buffer, a salt, and a surfactant.

4. The hybridization buffer according to any one of claims 1 to 3, wherein the hybridization buffer comprises a buffer, and the buffer is a phosphate buffer.

5. The hybridization buffer according to any one of claims 1 to 4, wherein the salt is NaCl or sodium citrate.

6. The hybridization buffer according to any one of claims 1 to 5, wherein the destabilizing agent is formamide or urea or a mixture thereof.

7. 7. The hybridization buffer according to claim 1, wherein the hybridization buffer contains a surfactant, and the surfactant is TWEEN® 20, TWEEN® 80, or sodium dodecyl sulfate (SDS).

8. The hybridization buffer is 0.5% to 10% dextran sulfate, 0.05 mg / mL to 0.5 mg / mL human Cot-1 DNA, 1% to 15% (v / v) formamide, 40mM~80mM KH ２ PO ４ -K ２ HPO ４ 、 0.1 M to 4 M NaCl, and 0.001% to 10% (v / v) TWEEN® 20 The hybridization buffer of claim 1 , comprising:

9. 10. A method for preparing an enriched library, the method comprising forming a hybridization mixture comprising library members, a hybridization buffer according to any one of claims 1 to 8, and a probe under conditions sufficient for the probe to hybridize to a region of interest within the library members, wherein the probe comprises a ligand, to form a hybridization mixture.

10. capturing the hybridized probes using a capture means; and eluting the captured library members from the capture means into an eluate containing enriched library members, wherein the DNA concentration of the eluate is in the range of 1.3 pM to 250 pM.

10. The method of claim 9, comprising:

11. 11. The method of claim 9 or claim 10, wherein the hybridization mixture comprises samples from at least two library preparations, wherein at least 10 ng, at least 25 ng, at least 50 ng, at least 100 ng, at least 200 ng, or at least 500 ng of DNA from each library preparation is combined.

12. The method of any one of claims 9 to 11, wherein the DNA concentration of the hybridization mixture is in the range of 0.1 ng / μL to 120 ng / μL.

13. 11. The method of claim 10, further comprising sequencing the enriched library members, wherein the method does not include amplifying the library members prior to capturing the library members.

14. 14. The method of claim 13, wherein the sequencing step is performed on a flow cell, and the method comprises loading the enriched library members onto the flow cell at a concentration of at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 10 pM, or at least 100 pM and at a concentration of up to 100 pM, up to 200 pM, up to 250 pM, or up to 300 pM.

15. 15. The method of claim 14, comprising loading the enriched library members onto a flow cell using a direct flow cell loading jig.

16. A kit comprising the hybridization buffer of any one of claims 1 to 8 and instructions for use.

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