Methods for sequencing the natural strand of enriched genomic regions

Abstract

Methods for sequencing polynucleotides are provided herein in particular. In addition, methods for detecting base modifications in polynucleotides by sequencing only the native strand, for example, for methylation determination, are also provided herein. A key innovation in this method is the creation of a second strand after hybridization, configured such that adapter ligation is possible at only one end. This limits sequencing to the native strand, which is the only strand with potential modifications.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 450,472, filed Mar. 7, 2023, and U.S. Provisional Application No. 63 / 556,299, filed Feb. 21, 2024, which are hereby incorporated by reference in their entireties for all purposes.

Background Art

[0002] Quantifying base modifications in genomes, particularly the positions and frequencies of methylated cytosines, is an essential means for studying gene regulation, epigenetic inheritance, and other biological processes, as well as for diagnosing diseases clinically. Current array - based methods for identifying methylated cytosines are expensive, have limited accuracy and dynamic range, and cannot identify other base modifications or determine the types of methylation present. In response, researchers in laboratories and clinical settings are increasingly turning to reduced representation bisulfite sequencing (RRBS) or reduced representation methylation sequencing (RRMS). However, RRBS is also technically limited because enrichment of the target regions using bisulfite - converted DNA is difficult and inconsistent. In addition, RRMS often is not a practical alternative because it requires a large amount of sequencing because the entire genome has to be sequenced. Therefore, there is a need for improved methods that can accurately and cost - effectively enable the evaluation of modified bases in specific genomic regions. Ideally, this technique would avoid two major factors contributing to assay variability: amplification and bisulfite (or enzymatic) conversion of unmethylated cytosines. This disclosure addresses these and other concerns in the art.

Summary of the Invention

[0003] In one embodiment, a method for detecting base modifications in a target polynucleotide is provided, comprising: (a) preparing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) preparing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) preparing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting base modifications in a target polynucleotide, comprising: (d) preparing a pull-down complex; (e) optionally eluting an adapter-target polynucleotide from the pull-down complex; (f) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the preparation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) preparing a sequencing complex by contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide; and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence.

[0004] In another embodiment, a method for sequencing a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; (c) producing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support; and (d) A method is provided for isolating a pull-down complex, (e) optionally eluting an adapter-target polynucleotide from the pull-down complex, (f) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group, (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex, and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence, thereby sequencing the target polynucleotide, optionally comprising one or more base modifications.

[0005] In another embodiment, a method for quantifying base modifications in a target polynucleotide, comprising: (a) preparing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) preparing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) preparing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for quantifying base modifications in a target polynucleotide, comprising: (d) constructing a body; (e) isolating a pull-down complex; (f) optionally eluting an adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the construction of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide, thereby constructing a sequencing complex; and (h) sequencing the sequencing complex, which does not involve sequencing the complementary adapter-target polynucleotide but does involve sequencing the target polynucleotide sequence.

[0006] In another embodiment, a method for detecting multiple base modifications in multiple target polynucleotides, comprising: (a) producing multiple adapter-target polynucleotides by contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, wherein one or more adapters include a primer binding site; (b) producing multiple tagged adapter-target polynucleotides by contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; and (c) producing multiple pull-down complexes by contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting multiple base modifications in multiple target polynucleotides, comprising: (d) isolating multiple pull-down complexes; (e) optionally eluting multiple adapter-target polynucleotides from the multiple pull-down complexes; (f) contacting the multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to the multiple adapter-target polynucleotides to create multiple sequencing complexes; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing the multiple complementary adapter-target polynucleotides but does involve sequencing the multiple target polynucleotide sequences.

[0007] In another embodiment, a method for sequencing multiple target polynucleotides, comprising: (a) contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, thereby producing multiple adapter-target polynucleotides, wherein one or more adapters include a primer binding site; (b) contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, thereby producing multiple tagged adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; (c) contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support, thereby producing multiple pull-down complexes; and (d) isolating the multiple pull-down complexes. A method is provided for sequencing multiple target polynucleotides, comprising: (e) optionally eluting multiple adapter-target polynucleotides from multiple pull-down complexes; (f) contacting multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to multiple adapter-target polynucleotides to create multiple sequencing complexes; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing multiple complementary adapter-target polynucleotides but does involve sequencing multiple target polynucleotide sequences.

[0008] In another embodiment, a method for detecting multiple base modifications in a target polynucleotide, comprising: (a) preparing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) preparing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) preparing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting multiple base modifications in a target polynucleotide, comprising: (d) constructing a body; (e) isolating a pull-down complex; (f) optionally eluting an adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the construction of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide, thereby constructing a sequencing complex; and (h) sequencing the sequencing complex, which does not involve sequencing the complementary adapter-target polynucleotide but does involve sequencing the target polynucleotide sequence.

[0009] In another embodiment, a method for detecting base modifications in multiple target polynucleotides, comprising: (a) contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, thereby producing multiple adapter-target polynucleotides, wherein one or more adapters include a primer binding site; (b) contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, thereby producing multiple tagged adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; and (c) contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support, thereby producing multiple pull-down complexes. A method is provided for detecting base modifications in the multiple target polynucleotides, comprising: (d) isolating multiple pull-down complexes; (e) optionally eluting multiple adapter-target polynucleotides from the multiple pull-down complexes; (f) contacting the multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to the multiple adapter-target polynucleotides; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing the multiple complementary adapter-target polynucleotides but does involve sequencing the multiple target polynucleotide sequences. [Brief explanation of the drawing]

[0010] [Figure 1] An exemplary schematic diagram of a concentration method for determining natural methylation is shown. Step 1: Ligate the input DNA with an adapter containing a primer binding site (orange) and a barcode (purple). Step 2: Hybridize the DNA input with a biotinylated DNA polynucleotide complementary to the region(s) of interest. Step 3: Use streptavidin beads to pull down and isolate the desired region. Step 4: Elute the DNA from the beads. Step 5: Synthesize the complementary strand using a primer and polymerase at the 3' end of the DNA. Polymerase activity also leaves A at the other end. Step 6: Attach a sequencing adapter to the end of the DNA opposite to the primer site. As a result, only the natural strand is sequenced. [Figure 2A] The experimental results using innate methylation analysis after enrichment are shown. A: Histogram of methylation levels in the enriched region, showing that methylation was identified across the entire range. B: Correlation between microarray methylation frequency and Nanohyb enriched region based on nanopore sequencing. The correlation was comparable to the level observed between whole-genome methylation sequencing and the array. [Figure 2B] Same as above. [Figure 3A] The results of the analysis of methylation patterns in sperm are shown. [Figure 3B] Same as above. [Figure 4] The copy number variations (CNVs) detected using the methods described herein are shown. [Modes for carrying out the invention]

[0011] After reading this description, methods for implementing the disclosure in various alternative embodiments and applications will become apparent to those skilled in the art. However, not all of the various embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented only as examples and not as limitations. Accordingly, the detailed description of these various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0012] Before disclosing and describing this technology, it should be understood that the embodiments described below are not limited to any particular composition, any method of preparing such composition, or any use thereof, and are therefore naturally subject to change. It should also be understood that the terms used herein are intended solely to describe and not to limit any particular embodiment.

[0013] Detailed explanations are divided into various sections for the convenience of the reader, and disclosures found in any section may be combined with those in other sections. Headings or subheadings may be used herein for the convenience of the reader and are not intended to affect the scope of this disclosure.

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. In this specification and the subsequent claims, certain terms are used with the following meanings:

[0015] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Where used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless otherwise indicated by the context.

[0016] "Optional" or "optional" means that the event or matter described thereafter may or may not occur, and that the description includes both the cases in which the event or matter occurs and the cases in which it does not occur.

[0017] The term "approximately," when used before a numerical expression that includes a range, such as temperature, time, quantity, or concentration, indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value between them. Preferably, when the term "approximately" is used in reference to a quantity, it means that the quantity may vary by + / - 10%.

[0018] As used herein, the term “each” is intended to identify individual articles in a set of articles, but not necessarily all articles in the set. Exceptions may exist where express disclosure or context clearly indicates otherwise.

[0019] "Comprising" or "comprises" is intended to mean that a composition or method includes the elements described, but does not exclude other elements. "Essentially consisting of" means, when used to define a composition or method, to exclude other elements that are even slightly essential to the combination relating to the described purpose. Thus, a composition essentially consisting of the elements defined herein does not exclude other materials or steps that do not substantially affect the basic and novel features of the claimed invention. "Consists of" means to exclude elements and substantial method steps that exceed trace amounts of other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0020] Where used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment,” and “polynucleotide” are intended to be interchangeable and include, but are not limited to, polymeric forms of covalently linked nucleotides, which may have varying lengths, that are either deoxyribonucleotides or ribonucleotides, or analogues, derivatives, or modifications thereof. Different polynucleotides may have different three-dimensional structures and may exhibit a variety of known or unknown functions. Non-exclusive examples of polynucleotides include genes, gene fragments, exons, introns, intergenetic DNA (including, but not limited to, heterochromatin DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA sequences, isolated RNA sequences, nucleic acid probes, and primers. Polynucleotides useful in the methods of this disclosure may include natural nucleic acid sequences and their variants, artificial nucleic acid sequences, or combinations of such sequences.

[0021] Polynucleotides typically consist of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (or uracil (U) instead of thymine (T) if the polynucleotide is RNA). Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule, or the term may apply to the polynucleotide molecule itself. This alphabetical representation can be entered into a database on a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searches. Polynucleotides may optionally include one or more non-standard nucleotides, one or more nucleotide analogs, and / or more modified nucleotides.

[0022] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides), in any of single-stranded, double-stranded, or multi-stranded forms. In a plurality of embodiments, "nucleic acid" does not include nucleosides. Terms such as "polynucleotide", "oligonucleotide", "oligo" refer, in their ordinary and customary meanings, to linear sequences of nucleotides. The term "nucleoside" refers, in its ordinary and customary meaning, to a glycosylamine containing a nucleobase and a pentose (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" refers, in its ordinary and customary meaning, to a single unit, i.e., a monomer, of a polynucleotide. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides, contemplated herein include any kind of RNA, e.g., mRNA, siRNA, miRNA, and guide RNA, and any kind of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "double-stranded" in the context of a polynucleotide refers, in its ordinary and customary meaning, to being double-stranded. A nucleic acid can be linear or branched. For example, a nucleic acid can be a linear sequence of nucleotides, or a nucleic acid can be branched, e.g., by including one or more arms or branches of nucleotides. Optionally, a branched nucleic acid can branch repeatedly to form higher-order structures, such as dendrimers, etc.

[0023] The term "nucleotide" according to the present disclosure particularly relates to ribonucleotides, 2'-deoxyribonucleotides, or 2',3'-dideoxyribonucleotides.

[0024] The term "nucleobase" refers to either a natural or unnatural purine or pyrimidine base. Nucleobases include adenine, cytosine, guanine, thymine, uracil, hypoxanthine, xanthine, 7-deaza-adenine and 7-deazaguanine, inosine.

[0025] As used herein, the phrase "dNTP" means a 2'-deoxynucleotide triphosphate, where the nucleotide contains a natural or unnatural nucleobase.

[0026] As used herein, the term "double-stranded", when used in connection with a polynucleotide, means that some or all of the nucleotides between the complementary strands of the polynucleotide are hydrogen-bonded together to form a partial or complete double helix. A partially double-stranded polynucleotide may have at least 10%, 25%, 50%, 60%, 70%, 80%, 90% or 95% of its nucleotides hydrogen-bonded to complementary nucleotides.

[0027] A single-stranded polynucleotide refers to a polynucleotide that has little or no hydrogen bonding to another polynucleotide and thus does not form or is unstable in forming a double helix under a given set of hybridization conditions.

[0028] A "polymerase" is generally an enzyme that catalyzes the reaction between 3'-OH and 5'-triphosphate in nucleotides, oligomers, and their analogues, thereby forming nucleic acid polymers. Polymerases are not limited to these, but include DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, template-independent DNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, DNA polymerase 1, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, Phusion DNA polymerase, SuperFi DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Bst DNA polymerase large fragment, Stoeffel fragment, 9°N DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, Phi29 polymerase, Tli DNA polymerase, eukaryotic DNA polymerase beta, telomerase, KOD HiFi, KOD1 DNA polymerase, Q-beta replicase, terminal transferase (TdT), AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, Thermo Sequenase (Thermo Fisher Scientific) is one example. These polymerases include wild-type, mutant isoforms, chimeric forms, and genetically modified variants, such as exopolymerases and other mutants, such as those that tolerate modified nucleotides and incorporate them into nucleic acid chains.

[0029] As used herein, the term “base modification” refers to a nucleotide that is modified by the addition of a chemical group or substituted with a non-natural nucleotide or non-natural nucleoside. In several embodiments, base modification includes methylation. In several embodiments, base modification includes 5-methylcytosine (5mC), N6-methyladenine (6mA), 7-methylguanine (m7G), or 2'-O-methylation (2'-O-methyl). In several embodiments, base modification includes oxidation of a methylated nucleotide. In several embodiments, base modification includes 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), 5-hydroxymethyluracil (5hmU), 5-formyluracil (5hmU), or β-D-glucosyl-hydroxymethyluracil (base J). In several embodiments, base modification includes bromodeoxyuridine (BrdU).

[0030] As used herein, the term "methylation" refers to the process by which a methyl group is added to a nucleotide. In some embodiments, methylation includes the addition of a methyl group to adenine, cytosine, or guanine. In some embodiments, methylation includes 2'-O-methylation (2'-O-methyl).

[0031] As used herein, the term “unnatural nucleotide” refers to nucleotide analogs, synthetic nucleotides, and nucleotide mimes not found in nature. In several embodiments, unnatural nucleotides can be substituted for natural nucleotides found in polynucleotides.

[0032] As used herein, the term “unnatural nucleoside” refers to nucleoside analogs, synthetic nucleosides, and nucleoside mimetic compounds not found in nature. In several embodiments, the unnatural nucleoside can be substituted for a natural nucleoside found in a polynucleotide. In several embodiments, the unnatural nucleoside is bromodeoxyuridine (BrdU).

[0033] As used herein, the term “target polynucleotide” refers to a polynucleotide selected for sequencing. For example, a target polynucleotide may be a polynucleotide that has or is expected to have base modifications. In some embodiments, a target polynucleotide may be a polynucleotide that has or is expected to have base modifications in a given cell type. In some embodiments, a target polynucleotide may be a genomic polynucleotide. In some embodiments, a target polynucleotide may be genomic DNA. In some embodiments, a target polynucleotide may be a part of a cell’s genome. In some embodiments, a target polynucleotide may be a fragment of a cell’s genome. In some embodiments, a target polynucleotide may be DNA derived from a particular cell type. In some embodiments, a target polynucleotide may be used to identify a cell type. In some embodiments, a target polynucleotide may originate from any intracellular compartment. In some embodiments, a target polynucleotide may be a nuclear polynucleotide. In some embodiments, a target polynucleotide may be nuclear DNA. In some embodiments, a target polynucleotide may be a mitochondrial polynucleotide. In some embodiments, a target polynucleotide may be mitochondrial DNA (mDNA). In several embodiments, the target polynucleotide may be a part of cellular mitochondrial DNA. In several embodiments, the target polynucleotide may be a mitochondrial DNA fragment. In several embodiments, the target polynucleotide may be a DNA fragment. In several embodiments, the target polynucleotide may be chromosomal DNA or a fragment thereof. In several embodiments, the target polynucleotide may be extracellular. In several embodiments, the target polynucleotide may be cell-free DNA (cfDNA). In several embodiments, the target polynucleotide may be contained in a biological sample. In several embodiments, the target polynucleotide may be isolated from a biological sample. In several embodiments, the target polynucleotide may be animal-derived DNA. In several embodiments, the target polynucleotide may be vertebrate-derived DNA.In several embodiments, the target polynucleotide may be mammalian DNA. In several embodiments, the target polynucleotide may be human DNA. In several embodiments, the target polynucleotide may be rodent DNA. In several embodiments, the target polynucleotide may be mouse DNA. In several embodiments, the target polynucleotide may be rat DNA. In several embodiments, the target polynucleotide may be invertebrate DNA. In several embodiments, the target polynucleotide may be plant DNA. In several embodiments, the target polynucleotide may be pathogen DNA. In several embodiments, the target polynucleotide may be viral DNA. In several embodiments, the target polynucleotide may be bacterial DNA. In several embodiments, the target polynucleotide may be parasitic DNA. In several embodiments, the target polynucleotide may be archaeal DNA.

[0034] As used herein, the term “adapter” generally refers to any oligonucleotide that can be added to, for example, ligated to, a nucleic acid molecule, thereby producing a nucleic acid product that can be sequenced on a sequencing platform. In some embodiments, the adapter comprises two complementary oligonucleotides that form a double-stranded structure. In several embodiments, the adapter is an oligonucleotide that can be ligated to the ends of a target polynucleotide.

[0035] As used herein, the term "ligation" refers to the process of joining two biomolecules (e.g., oligonucleotides, polynucleotides) by forming a chemical bond between their ends. In some embodiments, the chemical bond is a non-covalent chemical bond. In some embodiments, ligation is catalyzed by a ligase.

[0036] As used herein, the term "ligase" refers to an enzyme that catalyzes the ligation of two biomolecules (e.g., oligonucleotides, polynucleotides) by forming a new chemical bond.

[0037] As used herein, the term “adapter-target polynucleotide” refers to a target polynucleotide to which one or more adapters are ligated at one or more ends (or both ends) of the target polynucleotide.

[0038] As used herein, the term “primer binding site” refers to a nucleotide sequence of a polynucleotide to which a primer binds and which functions as the starting site for replication of the polynucleotide.

[0039] As used herein, the term “complementary adapter-target polynucleotide” refers to a polynucleotide that is complementary to and hybridizes with an adapter-target polynucleotide. In some embodiments, the complementary adapter-target polynucleotide includes a 3' protecting group. In some embodiments, the complementary adapter-target polynucleotide includes a 5' protecting group. In some embodiments, the complementary adapter-target polynucleotide is complementary to the adapter-target polynucleotide in whole or substantially in whole. In some embodiments, the complementary adapter-target polynucleotide is complementary to a portion of the adapter-target polynucleotide.

[0040] As used herein, the term “3' protecting group” refers to a terminal portion of a polynucleotide that prevents sequencing of the polynucleotide. In several embodiments, the 3' protecting group prevents sequencing of the complementary adapter-target polynucleotide. In several embodiments, the 3' protecting group prevents ligation of the sequencing adapter to the complementary adapter-target polynucleotide. In several embodiments, the 3' protecting group comprises a nucleotide overhang or a 3' phosphate group. In several embodiments, the 3' protecting group comprises a nucleotide overhang. In several embodiments, the 3' protecting group comprises a 3' phosphate group.

[0041] As used herein, the term “5' protecting group” refers to the 5' end portion of a polynucleotide that prevents sequencing of the polynucleotide. In several embodiments, the 5' protecting group prevents sequencing of the complementary adapter-target polynucleotide. In several embodiments, the 5' protecting group prevents ligation of the sequencing adapter to the complementary adapter-target polynucleotide. In several embodiments, the 5' protecting group comprises an aldehyde, an amine, or a thiol. In several embodiments, the 5' protecting group comprises an aldehyde. In several embodiments, the 5' protecting group comprises an amine. In several embodiments, the 5' protecting group comprises a thiol. In several embodiments, the 5' protecting group comprises a 5' aldehyde. In several embodiments, the 5' protecting group comprises a 5' amine. In several embodiments, the 5' protecting group comprises a 5' thiol.

[0042] As used herein, the term nucleotide overhang refers to a sequence of unpaired nucleotides at the end of a polynucleotide.

[0043] As used herein, the terms “complementary” or “substantially complementary” refer to hybridization, base pairing, or double-strand formation between nucleotides or nucleic acids. For example, complementarity exists between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer-binding site of a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides can base-pair with a nucleotide of the same family or a sequence of nucleotides of the same family. As described herein and as is commonly known in the art, the complementary (compatible) nucleotide of adenosine (A) is thymidine (T), and the complementary (compatible) nucleotide of guanosine (G) is cytosine (C). Thus, a complement may include a sequence of nucleotides that base-pair with the corresponding complementary nucleotide of a second nucleic acid sequence. The nucleotides of the complement may be partially or completely compatible with the nucleotides of the second nucleic acid sequence. If the nucleotides of the complement are perfectly matched to each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. If the nucleotides of the complement are partially matched to the nucleotides of the second nucleic acid sequence, only some of the nucleotides of the complement form base pairs with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence contains nucleotides complementary to the coding sequence and thus forms a complement to the coding sequence. Further examples of complementary sequences are sense sequences and antisense sequences, where the sense sequence contains nucleotides complementary to the antisense sequence and thus forms a complement to the antisense sequence. "Double-stranded" means that at least two fully or partially complementary oligonucleotides and / or polynucleotides undergo Watson-Crick base pairing between all or most of their nucleotides, resulting in the formation of a stable complex. In several embodiments, the first template polynucleotide and the second template polynucleotide of the overlapping cluster are substantially not complementary (e.g., at least 50%, 75%, 90%, or more discomplementary to each other).

[0044] As described herein, sequence complementarity may be partial (only a portion of the nucleic acid fits according to base pairing) or complete (all nucleic acids fit according to base pairing). Thus, two complementary sequences may have a specified percentage of complementary nucleotides (e.g., about 60% over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, two sequences are complementary if they are fully complementary and have 100% complementarity. In some embodiments, the sequences in a pair of complementary sequences may form a single polynucleotide portion having non-base-pairing nucleotides (e.g., in a hairpin or loop structure with or without an overhang) or separate polynucleotide portions. In some embodiments, one or both sequences in a pair of complementary sequences form a longer polynucleotide portion that may or may not include additional complementary regions.

[0045] As used herein, the term “contact” is used in its plain and ordinary sense and refers to a process that allows at least two separate species (e.g., chemical compounds or cells containing biomolecules) to be in close proximity to react, interact, or physically come into contact. However, the resulting reaction product may be produced from the reaction between the added reagents or directly from intermediates derived from one or more of the added reagents that may be produced in the reaction mixture. The term “contact” may also include allowing two species, which may be compounds, nucleic acids, proteins, or enzymes (e.g., DNA polymerase), to react, interact, or physically come into contact.

[0046] As used herein, the term “barcode” refers to a known nucleic acid sequence that enables the identification of a certain characteristic of the nucleic acid to which the barcode is associated. In some embodiments, the characteristic of the nucleic acid to be identified is the sample or source from which the nucleic acid is obtained. Simply as an example, some embodiments described herein describe the attachment of multiple barcodes (e.g., two, three, four, five, six, or more) to a nucleic acid of interest in a single cell present in a cell population. The unique combination of barcodes attached to the nucleic acid of each individual cell can advantageously enable the identification of the cell from which the tagged nucleic acid of interest was obtained. In some embodiments, the barcodes are at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides long or more. In some embodiments, the barcodes are less than 10, 9, 8, 7, 6, 5, or 4 nucleotides long. In some embodiments, the barcodes associated with some nucleic acids are of a different length than the barcodes associated with other nucleic acids. Generally, the barcodes are long enough to allow for the identification of the sample based on the barcode to which the sample associates, and contain sufficiently distinct sequences. In some embodiments, the barcode and the sample source to which it associates can be precisely identified after a mutation, insertion, or deletion of one or more nucleotides in the barcode sequence, e.g., a mutation, insertion, or deletion. In some embodiments, each barcode in a plurality of barcodes differs from any other barcode in the plurality of barcodes at two or more nucleotide positions, e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, one or more adapters include at least one of the plurality of barcode sequences. In some embodiments, the method of the technique further includes identifying a sample or source from which a target nucleic acid is obtained based on the barcode sequence to which the target nucleic acid binds. In some embodiments, the method of the technique further includes identifying the target nucleic acid based on the barcode sequence to which the target nucleic acid binds. Some embodiments of the method further include identifying a source or sample of a target nucleotide sequence by determining the barcode nucleotide sequence.Some embodiments of the method further include molecular counting applications (e.g., digital barcode counting and / or binning) to determine the expression level or copy number status of a desired target. Generally, the barcode may include a nucleic acid sequence that, when bound to the target nucleic acid, serves as an identifier for the sample from which the target polynucleotide was obtained.

[0047] As used herein, the terms “primer” or “extension primer” refer to oligonucleotides, whether naturally occurring or synthetically produced, that can act as starting points for nucleic acid synthesis when placed under appropriate conditions, for example, in a suitable buffer (where “buffer” includes appropriate pH, ionic strength, cofactors, etc.) and at a suitable temperature, in the presence of nucleotide triphosphates and a polymerase enzyme (e.g., a thermostable polymerase enzyme). In some embodiments, primers may be single-stranded for maximum amplification efficiency, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands and then used to prepare the extension product. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer must be long enough to initiate the synthesis of the extension product. The exact length of the primer depends on many factors, including temperature, the source of the primer, the polymerase enzyme (e.g., whether it is thermostable or not), and the method used.

[0048] Nucleic acids may contain nonspecific sequences. As used herein, the term “nonspecific sequence” refers to a nucleic acid sequence containing a set of residues that are not designed to be complementary to any other nucleic acid sequence, or are only partially complementary to it. For example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that do not function as a repressor nucleic acid when in contact with a cell or organism.

[0049] The term "complementary" as used herein refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides that can form a base pair with a complementary nucleotide or sequence of nucleotides. As described herein and as is commonly known in the art, the complementary (compatible) nucleotide of adenosine is thymidine, and the complementary (compatible) nucleotide of guanosine is cytosine. Thus, a complementary may include a sequence of nucleotides that form a base pair with the corresponding complementary nucleotide of a second nucleic acid sequence. The nucleotides of the complementary may be partially or completely compatible with the nucleotides of the second nucleic acid sequence. If the nucleotides of the complementary may be completely compatible with each nucleotide of the second nucleic acid sequence, the complementary will form a base pair with each nucleotide of the second nucleic acid sequence. If the nucleotides of the complementary may be partially compatible with the nucleotides of the second nucleic acid sequence, only a portion of the nucleotides of the complementary will form a base pair with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence contains nucleotides complementary to the coding sequence and thus forms a complement of the coding sequence. Further examples of complementary sequences are sense sequences and antisense sequences, where the sense sequence contains nucleotides complementary to the antisense sequence and thus forms a complement of the antisense sequence.

[0050] As described herein, sequence complementarity may be partial (only a portion of the nucleic acid fits according to base pairing) or complete (all nucleic acids fit according to base pairing). Thus, two complementary sequences may have a given percentage of identical nucleotides (i.e., about 60% identity across a given region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity).

[0051] As used herein, the terms “library,” “RNA library,” “DNA library,” or “library of DNA molecules” are used according to their plain and ordinary meanings and refer to a collection or group of nucleic acid fragments of comparable size having known adapter sequences (e.g., known adapters attached to the 5' and 3' ends of each fragment). In some embodiments, a library comprises multiple nucleic acid fragments, each containing one or more adapter sequences. In some embodiments, a library comprises a circular nucleic acid template. Libraries are typically constructed from input RNA, DNA, or cDNA and processed by fragmentation, size selection, end repair, adapter ligation, amplification, and purification. Alternative, non-amplified (i.e., non-PCR) methods for constructing a library of molecules involve shearing an input polynucleotide, size selection, and ligating adapters. Libraries may correspond to a single sample or a single origin. Multiple libraries, each having its own unique adapter sequences, may be pooled and sequenced in the same sequencing run using the methods described herein.

[0052] As used herein, the terms “extension” or “elongation” are used in their plain and common sense meanings and refer to the synthesis of a new polynucleotide chain (e.g., “extended chain”) complementary to the template chain by polymerase, by adding a free nucleotide (e.g., dNTP) from a reaction mixture complementary to the template in the 5'-to-3' direction, for example, by condensing the 5' phosphate group of the dNTP with the 3' hydroxyl group at the end of the nascent (extended) DNA chain.

[0053] As used herein, terms such as “sequencing,” “determining a sequence,” and “determining a nucleotide sequence” include determining partial or complete sequence information, including the identification, ordering, or positioning of nucleotides contained in the polynucleotide being sequenced, and including physical processes for generating such sequence information. That is, the term includes sequence comparison, consensus sequence determination, contig construction, fingerprinting, and similar levels of information regarding the target polynucleotide, as well as the explicit identification and ordering of nucleotides in the target polynucleotide. The term also includes the identification, ordering, and positioning of one, two, or three of the four types of nucleotides in the target polynucleotide. In some embodiments, the sequencing process described herein includes contacting a template and annealed primers with a suitable polymerase under conditions suitable for polymerase elongation and / or sequencing.

[0054] As used herein, the term “sequencing read” is used in its plain and ordinary sense and refers to the estimated sequence (or nucleotide base probability) of nucleotide bases corresponding to all or part of a single polynucleotide fragment. Sequencing reads may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotide bases.

[0055] The term "gene" refers to a segment of DNA involved in protein production, including the regions before and after the coding region (leader and trailer), as well as the intervening sequences (introns) between individual coding segments (exons). Leaders, trailers, and introns contain regulatory elements necessary for gene transcription and translation. Furthermore, a "protein gene product" is the protein expressed from a particular gene.

[0056] As used herein, the terms “hybridize” or “specifically hybridize” refer to the process by which two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Hybridization is typically, and preferably, carried out using oligonucleotides. The terms “annealing” and “hybridization” are used interchangeably to mean the formation of a stable double helix. The tendency for hybridization between nucleic acids depends on the temperature and ionic strength of their environments, the length of the nucleic acids, and the degree of complementarity. The effects of these parameters on hybridization are described, for example, in Sambrook J., Fritsch EF, Maniatis T., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York (1989). Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired level of complementarity stably hybridize, but sequences with lower levels of complementarity do not. When used herein, the hybridization of the primer or DNA elongation product is possible by the formation of a phosphodiester bond with an available nucleotide or a nucleotide analog capable of forming a phosphodiester bond with it.

[0057] For any specific protein described herein, the designated protein includes either the naturally occurring form of the protein, a variant, or a homolog that maintains the protein transcription factor activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the naturally occurring protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a segment of 50, 100, 150, or 200 amino acids) compared to the naturally occurring form. In other embodiments, the protein is a protein identified by its NCBI sequence reference. In other embodiments, the protein is a protein identified by its NCBI sequence reference, its homolog, or a functional fragment.

[0058] As used herein, the term “library,” when used in relation to nucleic acids, is intended to mean a collection of nucleic acids having different chemical compositions (e.g., different sequences, different lengths, etc.). Typically, nucleic acids in a library are different species that share common characteristics or features in some genus or class, but otherwise differ in some respect. For example, a library may include nucleic acid species that differ in nucleotide sequences but are similar in having a sugar-phosphate backbone. Libraries can be constructed using techniques known in the art. Examples of nucleic acids exemplified herein include nucleic acids obtained from any source, including, for example, the digestion of a genome (e.g., the human genome) or a mixture of genomes. In another example, nucleic acids may be obtained from metagenomic studies of a particular environment or ecosystem. The term also includes artificially constructed nucleic acid libraries, such as DNA libraries.

[0059] The terms “click chemistry” and “click reaction” are used interchangeably herein and are intended to be consistent with their use in the art. Generally, click chemistry reactions are fast (e.g., rapid to completion), simple, easily purified, and regiospecific. Examples of click chemistry, but not limited to, include copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-enhanced azide-alkyne cycloaddition (SPAAC), strain-enhanced alkyne-nitrone cycloaddition (SPANC), alkyne hydrothiolation, and alkene hydrothiolation, which are also known as copper-free click chemistry. Click chemistry using copper catalysts often involves unstable Cu(I) stabilizing ligands. Although not bound by any particular theory, ligands can stabilize Cu(I) ions or protect them from oxidation from reactive Cu(I) to Cu(II), and can also act as proton acceptors, reducing or eliminating the need for a base in the reaction. Click chemistry between polynucleotides can be facilitated in some embodiments by using a moiety that brings two reaction partners close enough to react.

[0060] As used herein, and unless otherwise specified, the terms "azide" or "azido" refer to N3, or -N=N+=N-, or -N--N+°N.

[0061] As used herein, “label” refers to a chemical or biochemical portion useful for labeling nucleic acids. Examples of “labels” include fluorescent agents, chemiluminescent agents, affinity agents, blocking groups, chromogenic agents, quenchers, radioactive nucleotides, enzymes, substrates, cofactors, inhibitors, nanoparticles, magnetic particles, and other portions known in the art. Labels can produce a measurable signal and may be covalently or acovalently bound to oligonucleotides or nucleotides. In some examples, a portion of the oligonucleotide of an oligonucleotide or oligonucleotide-linked nucleotide disclosed herein may function as a label.

[0062] As used herein, the term “tagged adapter-targeted polynucleotide” refers to an adapter-targeted polynucleotide to which one or more tags are attached.

[0063] As used herein, the term “tag” refers to an adapter—a biomolecule capable of binding to a target polynucleotide. In some embodiments, the tag contains a polynucleotide sequence complementary to a region of the target polynucleotide. In some embodiments, the tag can bind to a complementary region of the target polynucleotide. In some embodiments, the tag contains half of a homozygous binding pair.

[0064] As used herein, the term “solid support” refers to an insoluble, chemically inert object having a solid surface. Examples of solid supports provided herein, but not limited to, include beads, columns, chips, wells, arrays, microfluidic channels, resins, particles, microparticles, or nanoparticles. In some embodiments, the surface of the solid support may be smooth, porous, or granular. In some embodiments, the solid support comprises half of a congener pair. In some embodiments, the surface of the solid support comprises half of a congener pair.

[0065] As used herein, the term “homozygous pair” refers to a pair of biomolecules that are non-covalently bound to one another. In several embodiments, a homozygous pair includes a capture portion and a binding portion. In some embodiments, a solid support includes the capture portion. In some embodiments, a tag includes the binding portion. In several embodiments, the non-covalent binding of a homozygous pair forms a homozygous capture complex. In some embodiments, the non-covalent binding of a homozygous pair forms a pull-down complex. In several embodiments, a homozygous pair includes binding pairs of streptavidin and biotin, maltose and maltose-binding protein, glutathione and glutathione S-transferase, chitin and chitin-binding protein, an aptamer and its antigen, SpyCatcher and SpyTag, or an antibody and its antigen. In several embodiments, a homozygous pair includes a binding pair of streptavidin and biotin. In several embodiments, a homozygous pair includes a binding pair of maltose and maltose-binding protein. In several embodiments, the homogeneous pair includes a binding pair of glutathione and glutathione S-transferase. In several embodiments, the homogeneous pair includes a binding pair of chitin and chitin-binding protein. In several embodiments, the homogeneous pair includes a binding pair of an aptamer and its antigen. In several embodiments, the homogeneous pair includes a binding pair of a SpyCatcher and a SpyTag. In several embodiments, the homogeneous pair includes a binding pair of an antibody and its antigen.

[0066] As used herein, the term “pull-down complex” refers to a complex formed when a binding portion hybridizes with its corresponding capture portion, thereby binding a tagged adapter-target polynucleotide to a solid support. In some embodiments, the pull-down complex comprises a tagged adapter-target polynucleotide and a solid support.

[0067] As used herein, the term “sequencing adapter” refers to a synthetic oligonucleotide or synthetic polynucleotide that is adjacent to one or both sides of the polynucleotide sequence to be sequenced. In some embodiments, the sequencing adapter includes a flow cell binding sequence. In some embodiments, the sequencing adapter includes a primer binding site for a sequencing device. In some embodiments, the sequencing adapter includes an index region or a barcode region. In some embodiments, the sequencing adapter includes a motor protein binding site.

[0068] As used herein, the term “motor protein binding site” refers to an oligonucleotide sequence that enables the binding of a motor protein. In some embodiments, the motor protein is a polypeptide that unwinds a double-stranded polynucleotide for sequencing of a single-stranded polynucleotide. In some embodiments, the motor protein contains helicase activity.

[0069] As used herein, the term “flow cell binding sequence” refers to an oligonucleotide sequence complementary to an oligonucleotide bound to a sequencing flow cell. In some embodiments, the flow cell binding sequence binds the polynucleotide sequence of interest to the flow cell.

[0070] As used herein, the term “primer binding site for sequencing apparatus” refers to an oligonucleotide sequence that enables the binding of a sequencing primer. In some embodiments, the sequencing primer is a synthetic oligonucleotide that recruits a polymerase to bind and extend the oligonucleotide.

[0071] As used herein, the terms “index region” or “barcode region” refer to regions of a sequencing adapter that enable the identification of individual sequencing adapters. In some embodiments, one or more sequencing adapters include one or more index regions that enable multiplexing.

[0072] As used herein, the term “sequencing complex” refers to a complex formed by the ligation of one or more sequencing adapters to an adapter-target polynucleotide. In some embodiments, the sequencing complex comprises one or more sequencing adapters and an adapter-target polynucleotide.

[0073] As used herein, the term “sequencing” refers to the process of determining the sequence of a biopolymer. In some embodiments, the biopolymer is a polynucleotide. In some embodiments, sequencing does not include bisulfite sequencing. In some embodiments, sequencing does not include whole-genome sequencing.

[0074] As used herein, the term “bisulfite sequencing” refers to sequencing methods used to determine the methylation patterns of polynucleotides. In several embodiments, bisulfite sequencing includes bisulfite conversion. In several embodiments, bisulfite conversion includes treating the polynucleotide with a bisulfite before sequencing. In several embodiments, bisulfite treatment introduces specific changes to the sequence of the polynucleotide. In several embodiments, bisulfite treatment converts cytosine residues to uracil. In several embodiments, bisulfite treatment does not convert 5-methylcytosine residues to uracil. In several embodiments, bisulfite sequencing includes reduced representative region bisulfite sequencing (RRBS).

[0075] As used herein, the terms “Reduced Representative Region Bisulfite Sequencing” or “RRBS” refer to sequencing techniques that analyze whole-genome methylation patterns at the single-nucleotide level. In several embodiments, RRBS includes restriction enzyme and bisulfite sequencing to enrich genomic regions with high CpG content. In several embodiments, RRBS does not target any promoter regions.

[0076] As used herein, the terms “Reduced Methylation Sequencing” or “RRMS” refer to sequencing techniques that analyze whole-genome methylation patterns without bisulfite conversion. In several embodiments, RRMS includes adaptive sampling to enrich the region of interest. In several embodiments, adaptive sampling excludes off-target regions during sequencing.

[0077] As used herein, the terms “whole-genome sequencing” or “WGS” refer to sequencing techniques used to determine the entire or nearly entire nucleotide sequence of an organism’s genome.

[0078] As used herein, the terms “cell-free DNA” or “cfDNA” refer to freely circulating (e.g., not contained within cells) DNA fragments found in biological samples.

[0079] A "biological sample" or "sample" refers to a substance obtained from or originating from a subject or patient. Examples of biological samples include tissue sections obtained for histological purposes, such as biopsy and autopsy specimens, and frozen sections. Such samples include body fluids, such as blood and blood fractions or blood products (e.g., serum, plasma, platelets, red blood cells), sputum, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, semen, synovial fluid, joint tissue, synovial tissue, synovial cells, fibroblast-like synovial cells, macrophage-like synovial cells, immune cells, hematopoietic cells, fibroblasts, macrophages, and T cells. Biological samples are typically obtained from eukaryotes, such as mammals, such as primates, such as chimpanzees or humans, cattle, horses, goats, pigs, dogs, cats, rodents, such as guinea pigs, rats, mice, rabbits, or birds, reptiles, or fish. In several embodiments, the biological sample is obtained from vertebrates. In several embodiments, the biological sample is obtained from invertebrates. In several embodiments, the biological sample is obtained from bacteria. In several embodiments, the biological sample is obtained from viruses. In several embodiments, the biological sample is obtained from plants. In several embodiments, the biological sample is obtained from archaea.

[0080] As used herein, the term “barcode” refers to a known nucleic acid sequence that enables the identification of a certain characteristic of the polynucleotide to which the barcode associates. In some embodiments, the characteristic of the polynucleotide to be identified is the sample or source from which the polynucleotide is obtained. In some embodiments, the barcode is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides long, or more. In some embodiments, the barcode is less than 10, 9, 8, 7, 6, 5, or 4 nucleotides long. In some embodiments, the barcode associated with some polynucleotides is of a different length from the barcode associated with other polynucleotides. Generally, the barcodes are long enough to enable the identification of a sample based on the barcode to which the sample associates and contain sufficiently different sequences. In some embodiments, each barcode in a group of barcodes differs from any other barcode in the group of barcodes at two or more nucleotide positions, e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, one or more adapters include at least one of a plurality of barcode sequences. In some embodiments, the method of this technique further includes identifying a polynucleotide based on the barcode sequence to which the polynucleotide is bound. Generally, the barcode may include an oligonucleotide sequence that, when bound to a polynucleotide, serves as an identifier for the sample from which the polynucleotide was obtained.

[0081] method Methods for sequencing target polynucleotides are provided herein. In some embodiments, the target polynucleotides are enriched without amplification. In some embodiments, sequencing includes detecting or identifying base modifications in the target polynucleotides.

[0082] In some embodiments, the method includes a first step of ligating an adapter to a target polynucleotide. In some embodiments, the first step includes ligating one or more adapters to a target polynucleotide to form an adapter-target polynucleotide. In some embodiments, one or more adapters include a primer binding site.

[0083] In some embodiments, the method includes a second step of hybridizing a tag to a target polynucleotide. In some embodiments, the second step includes hybridizing one or more tags to a target polynucleotide to form a tagged adapter-target polynucleotide. In some embodiments, one or more tags include a polynucleotide sequence complementary to the target polynucleotide. In some embodiments, one or more tags include half of a congenerate pair. In some embodiments, one or more tags include a binding moiety.

[0084] In some embodiments, the method includes a third step, which involves the formation of a pull-down complex. In some embodiments, the formation of the pull-down complex involves the binding of a congeneral pair. In some embodiments, the formation of the pull-down complex involves the binding of the binding portion to the capture portion. In some embodiments, the pull-down complex includes a tagged adapter-target polynucleotide bound to a solid support. In some embodiments, the solid support includes half of the congeneral pair. In some embodiments, the solid support includes the capture portion.

[0085] In some embodiments, the method includes a fourth step of pulling down the target polynucleotide. In some embodiments, the fourth step includes isolating the pull-down complex. In some embodiments, the isolation of the pull-down complex enriches the target polynucleotide. In some embodiments, the isolation of the pull-down complex may include exposing the pull-down complex to a magnet. In some embodiments, the isolation of the pull-down complex may include exposing the pull-down complex to a magnet for about 1 minute. In some embodiments, the isolation of the pull-down complex may include one or more exposures to a magnet. In some embodiments, the isolation of the pull-down complex may include one or more exposures of the pull-down complex to a magnet, where each exposure is about 1 minute. In some embodiments, the isolation of the pull-down complex may include two exposures of the pull-down complex to a magnet, where each exposure is about 1 minute. In some embodiments, the isolation of the pull-down complex may include three exposures of the pull-down complex to a magnet, where each exposure is about 1 minute. In several embodiments, isolation of the pull-down complex may include incubation of the pull-down complex with one or more wash buffers. In several embodiments, isolation of the pull-down complex may include incubation of the pull-down complex with one wash buffer. In several embodiments, isolation of the pull-down complex may include incubation of the pull-down complex with two wash buffers. In several embodiments, the pull-down complex is incubated with the wash buffer for 5 minutes. In several embodiments, the pull-down complex is incubated with one or more wash buffers, where each incubation lasts for 5 minutes. In several embodiments, the pull-down complex is incubated with the wash buffer at 68°C. In several embodiments, the pull-down complex is incubated with the wash buffer at 48°C.

[0086] In some embodiments, the method optionally includes a fifth step for eluting the target polynucleotide from the pull-down complex. In some embodiments, the optional fifth step includes eluting the adapter-target polynucleotide from the pull-down complex. In some embodiments, the elution of the adapter-target polynucleotide from the pull-down complex may include incubation for about 5 minutes. In some embodiments, the elution of the adapter-target polynucleotide from the pull-down complex may include incubation at room temperature (RT).

[0087] In some embodiments, the method includes a sixth step for second-chain synthesis. In some embodiments, the sixth step includes synthesizing a complementary adapter-target polynucleotide. In some embodiments, the pull-down complex or eluted adapter-target polynucleotide is brought into contact with polymerase under conditions that promote the production of the complementary adapter-target polynucleotide. In some embodiments, the complementary adapter-target polynucleotide includes a 3' protecting group. In some embodiments, the 3' protecting group prevents sequencing of the synthesized complementary adapter-target polynucleotide. In some embodiments, the synthesized complementary adapter-target polynucleotide includes a 5' protecting group. In some embodiments, the 5' protecting group prevents sequencing of the synthesized complementary adapter-target polynucleotide.

[0088] In some embodiments, the method includes a seventh step for forming a sequencing complex. In some embodiments, the seventh step includes ligating a sequencing adapter to an adapter-target polynucleotide to form a sequencing complex. In some embodiments, a 3' protecting group prevents ligation of the sequencing adapter to its complementary adapter-target polynucleotide. In some embodiments, a 5' protecting group prevents ligation of the sequencing adapter to its complementary adapter-target polynucleotide. In some embodiments, a primer without a 5' is used to prevent ligation of the sequencing adapter to its complementary adapter-target polynucleotide.

[0089] In some embodiments, the method includes an eighth step, which involves sequencing the sequencing complex. In some embodiments, the complementary adapter-target polynucleotide is not sequenced.

[0090] In another embodiment, a method for detecting base modifications in a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) producing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting base modifications in a target polynucleotide, comprising: (d) constructing a body; (e) isolating a pull-down complex; (f) optionally eluting an adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the construction of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide, thereby constructing a sequencing complex; and (h) sequencing the sequencing complex, which does not involve sequencing the complementary adapter-target polynucleotide but does involve sequencing the target polynucleotide sequence.

[0091] In another embodiment, a method for sequencing a target polynucleotide, comprising: (a) contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; and (b) contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, wherein one or more tags include a target (c) Constructing a tagged adapter-target polynucleotide complementary to one or more regions of a polynucleotide; (d) Constructing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support; (e) Optionally, eluting the adapter-target polynucleotide from the pull-down complex; (f) Constructing the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the construction of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group. (g) Constructing a sequencing complex by contacting an adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote ligation of the sequencing adapter to the adapter-target polynucleotide; and (h) Sequencing the sequencing complex, which includes sequencing the target polynucleotide sequence but does not include sequencing the complementary adapter-target polynucleotide, thereby sequencing the target polynucleotide, wherein the target polynucleotide optionally includes one or more base modifications.

[0092] In one embodiment, a method for quantifying base modifications in a target polynucleotide is provided, comprising: (a) preparing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) preparing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) preparing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for quantifying base modifications in a target polynucleotide, comprising: (d) preparing a pull-down complex; (e) optionally eluting an adapter-target polynucleotide from the pull-down complex; (f) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the preparation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) preparing a sequencing complex by contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide; and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence.

[0093] In another embodiment, a method for detecting multiple base modifications in multiple target polynucleotides, comprising: (a) producing multiple adapter-target polynucleotides by contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, wherein one or more adapters include a primer binding site; (b) producing multiple tagged adapter-target polynucleotides by contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; and (c) producing multiple pull-down complexes by contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting multiple base modifications in multiple target polynucleotides, comprising: (d) isolating multiple pull-down complexes; (e) optionally eluting multiple adapter-target polynucleotides from the multiple pull-down complexes; (f) contacting the multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to the multiple adapter-target polynucleotides to create multiple sequencing complexes; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing the multiple complementary adapter-target polynucleotides but does involve sequencing the multiple target polynucleotide sequences.

[0094] In another embodiment, a method for sequencing multiple target polynucleotides, comprising: (a) contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, thereby producing multiple adapter-target polynucleotides, wherein one or more adapters include a primer binding site; (b) contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, thereby producing multiple tagged adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; (c) contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support, thereby producing multiple pull-down complexes; and (d) isolating the multiple pull-down complexes. A method is provided for sequencing multiple target polynucleotides, comprising: (e) optionally eluting multiple adapter-target polynucleotides from multiple pull-down complexes; (f) contacting multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to multiple adapter-target polynucleotides to create multiple sequencing complexes; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing multiple complementary adapter-target polynucleotides but does involve sequencing multiple target polynucleotide sequences.

[0095] In another embodiment, a method for detecting multiple base modifications in a target polynucleotide, comprising: (a) preparing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein one or more adapters include a primer binding site; (b) preparing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of one or more tags to the adapter-target polynucleotide, wherein one or more tags are complementary to one or more regions of the target polynucleotide; and (c) preparing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support. A method is provided for detecting multiple base modifications in a target polynucleotide, comprising: (d) constructing a body; (e) isolating a pull-down complex; (f) optionally eluting an adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the construction of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide, thereby constructing a sequencing complex; and (h) sequencing the sequencing complex, which does not involve sequencing the complementary adapter-target polynucleotide but does involve sequencing the target polynucleotide sequence.

[0096] In another embodiment, a method for detecting base modifications in multiple target polynucleotides, comprising: (a) contacting multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of one or more adapters to multiple target polynucleotides, thereby producing multiple adapter-target polynucleotides, wherein one or more adapters include a primer binding site; (b) contacting multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of one or more tags to multiple adapter-target polynucleotides, thereby producing multiple tagged adapter-target polynucleotides, wherein one or more tags are complementary to one or more regions of the multiple target polynucleotides; and (c) contacting multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of one or more tags to the solid support, thereby producing multiple pull-down complexes. A method is provided for detecting base modifications in the multiple target polynucleotides, comprising: (d) isolating multiple pull-down complexes; (e) optionally eluting multiple adapter-target polynucleotides from the multiple pull-down complexes; (f) contacting the multiple pull-down complexes or multiple eluted adapter-target polynucleotides with a first polymerase under conditions that promote the creation of multiple complementary adapter-target polynucleotides, wherein each complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group; (g) contacting the multiple adapter-target polynucleotides with multiple sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the multiple sequencing adapters to the multiple adapter-target polynucleotides; and (h) sequencing the multiple sequencing complexes, which does not involve sequencing the multiple complementary adapter-target polynucleotides but does involve sequencing the multiple target polynucleotide sequences.

[0097] In several embodiments, conditions that facilitate the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production include the presence of a first ligase, one or more adapters, and the target polynucleotide in a suitable buffer and at a suitable temperature. In several embodiments, the buffer includes suitable components (e.g., pH, ionic strength, cofactors, etc.) that facilitate the ligation of one or more adapters to the target polynucleotide. In several embodiments, conditions that facilitate the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include one or more adapters. In several embodiments, conditions that facilitate the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include one or more barcodes. In several embodiments, conditions that facilitate the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include a ligation enhancer. In several embodiments, conditions that facilitate the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include a first ligase. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include T4 DNA ligase. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include polyethylene glycol (PEG). In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include adenosine triphosphate (ATP). In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at room temperature (RT).In several embodiments, conditions to facilitate the ligation of one or more adapters to the target polynucleotide for creating the adapter-target polynucleotide may include incubation for about 15 minutes.

[0098] In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 17°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 18°C ​​to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 19°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 20°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 21°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 22°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 23°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 24°C to approximately 37°C.In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 25°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 26°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 27°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 28°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 29°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at about 30°C to about 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at about 31°C to about 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at about 32°C to about 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at about 33°C to about 37°C.In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 34°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 35°C to approximately 37°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 36°C to approximately 37°C.

[0099] In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 36°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 35°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 34°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 33°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 32°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 31°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 30°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 29°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 28°C.In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 27°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 26°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 25°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 24°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 23°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 22°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 21°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 20°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 19°C.In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 16°C to approximately 18°C. In several embodiments, conditions that promote the ligation of one or more adapters to the target polynucleotide for adapter-target polynucleotide production may include incubation at approximately 17°C to approximately 36°C.

[0100] In several embodiments, conditions that facilitate the hybridization of one or more tags to an adapter-target polynucleotide for producing a tagged adapter-target polynucleotide include the presence of one or more tags and the adapter-target polynucleotide in a suitable buffer and at a suitable temperature. In several embodiments, the buffer includes suitable components (e.g., pH, ionic strength, cofactors, etc.) that facilitate the hybridization of one or more tags to an adapter-target polynucleotide. In several embodiments, conditions that facilitate the hybridization of one or more tags to an adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include a hybridization enhancer. In several embodiments, conditions that facilitate the hybridization of one or more tags to an adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include a synthetic polynucleotide. In several embodiments, the synthetic polynucleotide prevents nonspecific binding. In several embodiments, conditions that facilitate the hybridization of one or more tags to an adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include a nonspecific synthetic polynucleotide. In several embodiments, the nonspecific synthetic polynucleotide prevents nonspecific binding. In several embodiments, the conditions that promote hybridization of one or more tags to the adapter-target polynucleotide for the production of the tagged adapter-target polynucleotide may include DNA from a different species than the target polynucleotide. In several embodiments, the conditions that promote hybridization of one or more tags to the adapter-target polynucleotide for the production of the tagged adapter-target polynucleotide may include salmon sperm DNA. In several embodiments, salmon sperm DNA prevents nonspecific binding. In several embodiments, the conditions that promote hybridization of one or more tags to the adapter-target polynucleotide for the production of the tagged adapter-target polynucleotide may include incubation at approximately 70°C.In several embodiments, conditions that facilitate the hybridization of one or more tags to the adapter-target polynucleotide for the production of the tagged adapter-target polynucleotide may include incubation for about 20 hours.

[0101] In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 80°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 55°C to about 80°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 60°C to about 80°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 65°C to about 80°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 70°C to about 80°C. In several embodiments, conditions that facilitate the hybridization of one or more tags to the adapter-target polynucleotide for producing the tagged adapter-target polynucleotide may include incubation at approximately 75°C to approximately 80°C.

[0102] In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 75°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 70°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 65°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 60°C. In several embodiments, conditions that promote the hybridization of one or more tags to the adapter-target polynucleotide for producing a tagged adapter-target polynucleotide may include incubation at about 50°C to about 55°C.

[0103] In several embodiments, conditions that facilitate the hybridization of one or more tags to a solid support for forming a pull-down complex include the presence of one or more tags bound to an adapter-target polynucleotide and a solid support in a suitable buffer and at a suitable temperature. In several embodiments, the buffer includes suitable components (e.g., pH, ionic strength, cofactors, etc.) that facilitate the hybridization of one or more tags to a solid support. In several embodiments, conditions that facilitate the hybridization of one or more tags to a solid support for forming a pull-down complex may include incubation at about 68°C. In several embodiments, conditions that facilitate the hybridization of one or more tags to a solid support for forming a pull-down complex may include incubation for about 5 minutes.

[0104] In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides include the presence of nucleotide triphosphates and polymerase enzymes (e.g., thermostable polymerase enzymes) in a suitable buffer and at a suitable temperature. In several embodiments, the buffer includes suitable components that facilitate nucleic acid synthesis (e.g., pH, ionic strength, cofactors, etc.). In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include standard polymerase chain reaction conditions. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include incubation at approximately 60°C. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include incubation for approximately 15 minutes. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include polyethylene glycol (PEG). In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include adenosine triphosphate (ATP). In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include Bst3.0 DNA polymerase. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include BST buffer. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include elution primers. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include one or more free nucleotides. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include one or more deoxynucleotide triphosphates. In several embodiments, conditions that promote the synthesis of complementary adapter-target polynucleotides may include MgSO4.

[0105] In several embodiments, conditions that facilitate the ligation of a sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex include the presence of a second ligase, a sequencing adapter, and an adapter-target polynucleotide in a suitable buffer and at a suitable temperature. In several embodiments, the buffer includes suitable components (e.g., pH, ionic strength, cofactors, etc.) that facilitate the ligation of the sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex. In several embodiments, conditions that facilitate the ligation of a sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex may include incubation for about 15 minutes. In several embodiments, conditions that facilitate the ligation of a sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex may include incubation at room temperature (RT). In several embodiments, conditions that facilitate the ligation of a sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex may include a ligation adapter (LA). In several embodiments, conditions that facilitate the ligation of a sequencing adapter to an adapter-target polynucleotide for creating a sequencing complex may include a ligation buffer (LNB). In several embodiments, conditions that promote ligation of a sequencing adapter to an adapter-target polynucleotide for constructing a sequencing complex may include a Quick ligase. In several embodiments, conditions that promote ligation of a sequencing adapter to an adapter-target polynucleotide for constructing a sequencing complex may include a Quick T4 ligase.

[0106] In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 17°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 18°C ​​to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 19°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 20°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 21°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 22°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 23°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 24°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 25°C to approximately 37°C.In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 26°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 27°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 28°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 29°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 30°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 31°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 32°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 33°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 34°C to approximately 37°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 35°C to approximately 37°C.In several embodiments, conditions that facilitate the ligation of the sequencing adapter to the adapter-target polynucleotide for creating the sequencing complex may include incubation at approximately 36°C to approximately 37°C.

[0107] In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 36°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 35°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 34°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 33°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 32°C. In several embodiments, conditions that promote the ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 31°C. In several embodiments, conditions that promote the ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 30°C. In several embodiments, conditions that promote the ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 29°C. In several embodiments, conditions that promote the ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 28°C. In several embodiments, conditions that promote the ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 27°C.In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 26°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 25°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 24°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 23°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 22°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 21°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 20°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 19°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 18°C. In several embodiments, conditions that promote ligation of the sequencing adapter to the target polynucleotide for creating the sequencing complex may include incubation at approximately 16°C to approximately 17°C.

[0108] In some embodiments, the method further includes (i) analyzing the sequenced sequencing complex to detect base modifications.

[0109] In some embodiments, the method further includes repeating steps (a) to (i) at least once using one or more additional adapters.

[0110] In some embodiments, the target polynucleotide is isolated from the biological sample before step (a). In several embodiments, the biological sample is of human origin.

[0111] In some embodiments, the target polynucleotide includes genomic DNA (gDNA) or cell-free DNA (cfDNA).

[0112] In some embodiments, the target polynucleotide is fragmented before step (a). In some embodiments, fragmentation includes mechanical fragmentation or enzymatic fragmentation. In some embodiments, fragmentation includes mechanical fragmentation. In some embodiments, mechanical fragmentation includes focused ultrasonic shearing, hydrodynamic shearing, or spraying. In some embodiments, mechanical fragmentation includes focused ultrasonic shearing. In some embodiments, mechanical fragmentation includes hydrodynamic shearing. In some embodiments, mechanical fragmentation includes spraying. In some embodiments, fragmentation includes enzymatic fragmentation. In some embodiments, enzymatic fragmentation includes a transposase, restriction enzyme, or nonspecific nicking enzyme. In some embodiments, enzymatic fragmentation includes a transposase. In some embodiments, enzymatic fragmentation includes a restriction enzyme. In some embodiments, enzymatic fragmentation includes a nonspecific nicking enzyme.

[0113] In some embodiments, the target polynucleotide fragment is approximately 75 base pairs (bp) to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 100 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 125 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 150 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 175 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 200 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 300 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 400 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 500 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 600 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 700 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 800 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 900 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 1,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 2,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 3,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 4,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 5,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 6,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 7,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 8,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 9,000 bp to approximately 50,000 bp.In some embodiments, the target polynucleotide fragment is approximately 10,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 15,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 20,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 25,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 30,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 35,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 40,000 bp to approximately 50,000 bp. In some embodiments, the target polynucleotide fragment is approximately 45,000 bp to approximately 50,000 bp.

[0114] In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 45,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 40,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 35,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 30,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 25,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 20,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 15,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 10,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 9,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 8,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 7,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 6,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 5,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 4,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 3,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 2,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 1,000 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 900 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 800 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 700 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 600 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to 500 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to 400 bp.In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 300 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 200 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 175 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 150 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 125 bp. In some embodiments, the target polynucleotide fragment is approximately 75 bp to approximately 100 bp.

[0115] In some embodiments, the target polynucleotide fragment is 75 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 100 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 125 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 150 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 175 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 200 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 300 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 400 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 500 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 600 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 700 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 800 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 900 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 1,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 2,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 3,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 4,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 5,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 6,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 7,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 8,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 9,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 10,000 bp to 50,000 bp.In some embodiments, the target polynucleotide fragment is 15,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 20,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 25,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 30,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 35,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 40,000 bp to 50,000 bp. In some embodiments, the target polynucleotide fragment is 45,000 bp to 50,000 bp.

[0116] In some embodiments, the target polynucleotide fragment is 75 bp to 45,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 40,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 35,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 30,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 25,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 20,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 15,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 10,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 9,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 8,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 7,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 6,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 5,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 4,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 3,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 2,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 1,000 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 900 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 800 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 700 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 600 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 500 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 400 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 300 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 200 bp.In some embodiments, the target polynucleotide fragment is 75 bp to 175 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 150 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 125 bp. In some embodiments, the target polynucleotide fragment is 75 bp to 100 bp.

[0117] In some embodiments, one or more adapters further include a first barcode.

[0118] In some embodiments, one or more additional adapters further include one or more additional barcodes.

[0119] In some embodiments, one or more tags comprise half of a congenerate pair. In some embodiments, one or more tags comprise one or more binding moieties. In some embodiments, one or more tags comprise streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody. In some embodiments, one or more tags comprise streptavidin. In some embodiments, one or more tags comprise biotin. In some embodiments, one or more tags comprise maltose. In some embodiments, one or more tags comprise maltose-binding protein. In some embodiments, one or more tags comprise glutathione. In some embodiments, one or more tags comprise glutathione S-transferase. In some embodiments, one or more tags comprise chitin. In some embodiments, one or more tags comprise chitin-binding protein. In some embodiments, one or more tags comprise aptamers. In some embodiments, one or more tags comprise antigens. In some embodiments, one or more tags comprise SpyCatcher. In some embodiments, one or more tags include SpyTag. In some embodiments, one or more tags include antibodies.

[0120] In some embodiments, one or more tags target the same region of the target polynucleotide.

[0121] In some embodiments, one or more tags target different regions of the target polynucleotide.

[0122] In some embodiments, the solid support comprises half of a congenerate pair. In some embodiments, the solid support comprises a capture portion. In some embodiments, the solid support comprises streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody. In some embodiments, the solid support comprises streptavidin. In some embodiments, the solid support comprises biotin. In some embodiments, the solid support comprises maltose. In some embodiments, the solid support comprises maltose-binding protein. In some embodiments, the solid support comprises glutathione. In some embodiments, the solid support comprises glutathione S-transferase. In some embodiments, the solid support comprises chitin. In some embodiments, the solid support comprises chitin-binding protein. In some embodiments, the solid support comprises an aptamer. In some embodiments, the solid support comprises an antigen. In some embodiments, the solid support comprises a SpyCatcher. In some embodiments, the solid support comprises a SpyTag. In some embodiments, the solid support contains an antibody.

[0123] In some embodiments, the solid support is beads or a column. In some embodiments, the solid support is beads. In some embodiments, the solid support is a column. In some embodiments, the beads are paramagnetic.

[0124] In some embodiments, the elution of step (e) includes separating the adapter-target polynucleotide from one or more tags, thereby separating the adapter-target polynucleotide from the solid support. In some embodiments, the separation of the elution of step (e) includes denaturing one or more tags. In some embodiments, the denaturation of one or more tags dissociates one or more tags, thereby separating the adapter-target polynucleotide from the solid support. In some embodiments, the separation of the elution of step (e) includes a chain-displacing polymerase that removes one or more tags from the adapter-target polynucleotide, thereby separating the adapter-target polynucleotide from the solid support. In some embodiments, the separation of the elution step (e) includes a polymerase having exonuclease activity that digests one or more tags, thereby separating the adapter-target polynucleotide from the solid support.

[0125] In some embodiments, the elution of step (e) includes NaOH, heat, a chain-substituted polymerase, or a polymerase having exonuclease activity. In some embodiments, the elution step of (e) includes a base. In some embodiments, the elution of step (e) may include incubation with a base for about 5 minutes. In some embodiments, the elution of step (e) may include incubation with a base at room temperature (RT). In some embodiments, the elution of step (e) includes NaOH. In some embodiments, the elution of step (e) may include incubation with NaOH for about 5 minutes. In some embodiments, the elution of step (e) may include incubation with NaOH at room temperature (RT). In some embodiments, the elution of step (e) includes heat. In some embodiments, the elution of step (e) includes a chain-substituted polymerase. In some embodiments, the elution of step (e) includes a polymerase having exonuclease activity.

[0126] In some embodiments, the 3' protecting group prevents sequencing of the complementary adapter-target polynucleotide. In some embodiments, the 3' protecting group includes a nucleotide overhang. In some embodiments, the nucleotide overhang has at least 1, 2, or 3 nucleotides. In some embodiments, the nucleotide overhang has at least 1 nucleotide. In some embodiments, the nucleotide overhang has at least 2 nucleotides. In some embodiments, the nucleotide overhang has at least 3 nucleotides.

[0127] In some embodiments, the 5' protecting group prevents sequencing of the complementary adapter-target polynucleotide. In some embodiments, the 5' protecting group includes an aldehyde. In some embodiments, the 5' protecting group includes an amine. In some embodiments, the 5' protecting group includes a thiol.

[0128] In some embodiments, the primer does not contain a 5' phosphate group.

[0129] In some embodiments, the sequencing adapter includes a second barcode.

[0130] In some embodiments, the base modification includes 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), N6-methyladenine (6mA), 7-methylguanine (m7G), 2'-O-methylation (2'-O-methyl), or bromodeoxyuridine (BrdU). In some embodiments, the base modification includes 5-methylcytosine (5mC). In some embodiments, the base modification includes 5-hydroxymethylcytosine (5hmC). In some embodiments, the base modification includes N6-methyladenine (6mA). In some embodiments, the base modification includes 7-methylguanine (m7G). In some embodiments, the base modification includes 2'-O-methylation (2'-O-methyl). In some embodiments, the base modification includes bromodeoxyuridine (BrdU).

[0131] In some embodiments, the multiple base modifications include one or more of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), N6-methyladenine (6mA), 7-methylguanine (m7G), 2'-O-methylation (2'-O-methyl), or bromodeoxyuridine (BrdU). In some embodiments, the multiple base modifications include 5-methylcytosine (5mC). In some embodiments, the multiple base modifications include 5-hydroxymethylcytosine (5hmC). In some embodiments, the multiple base modifications include N6-methyladenine (6mA). In some embodiments, the multiple base modifications include 7-methylguanine (m7G). In some embodiments, the multiple base modifications include 2'-O-methylation (2'-O-methyl). In some embodiments, the multiple base modifications include bromodeoxyuridine (BrdU).

[0132] In some embodiments, the method does not involve bisulfite conversion or amplification of the target polynucleotide.

[0133] In some embodiments, the method does not include reduced representative region bisulfite sequencing (RRBS) or reduced representative region methylation sequencing (RRMS).

[0134] In some embodiments, the first or second ligase is T4 DNA ligase, Quick ligase, T3 ligase, T7 ligase, or Tag ligase.

[0135] In some embodiments, the first ligase is T4 DNA ligase, Quick ligase, T3 ligase, T7 ligase, or Tag ligase.

[0136] In some embodiments, the second ligase is T4 DNA ligase, Quick ligase, T3 ligase, T7 ligase, or Tag ligase.

[0137] In some embodiments, the first polymerase is Taq polymerase, BST polymerase, sulfolobus polymerase, therminator polymerase, Klenow polymerase, or deep vent polymerase.

[0138] In some embodiments, sequencing is performed in a sequencing device capable of identifying modified bases.

[0139] In some embodiments, base modifications in the target polynucleotide are retained until sequencing.

[0140] In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 400 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 500 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 600 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 700 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 800 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 900 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1000 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1100 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1200 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1300 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1400 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1500 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 1600 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by approximately 1700 to 2400 times compared to whole-genome sequencing.In some embodiments, the method enriches base modifications in the target polynucleotide by about 1800 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by about 1900 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by about 2000 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by about 2100 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by about 2200 to about 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by about 2300 to about 2400 times compared to whole-genome sequencing.

[0141] In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 2300 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 2200 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 2100 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 2000 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1900 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1800 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1700 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1600 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1500 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1300 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1200 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1100 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 1000 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by approximately 300 to 900 times compared to whole-genome sequencing.In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 800 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 700 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 600 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 500 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by about 300 to about 400 times compared to whole-genome sequencing.

[0142] In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 400 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 500 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 600 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 700 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 800 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 900 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1000 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1100 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1200 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1300 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1400 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1500 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1600 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 1700 to 2400 times compared to whole-genome sequencing.In some embodiments, the method enriches base modifications in the target polynucleotide by 1800 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by 1900 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by 2000 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by 2100 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by 2200 to 2400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in the target polynucleotide by 2300 to 2400 times compared to whole-genome sequencing.

[0143] In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 2300 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 2200 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 2100 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 2000 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1900 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1800 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1700 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1600 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1500 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1400 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1300 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1200 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1100 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 1000 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by a factor of 300 to 900 compared to whole-genome sequencing.In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 800 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 700 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 600 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 500 times compared to whole-genome sequencing. In some embodiments, the method enriches base modifications in target polynucleotides by 300 to 400 times compared to whole-genome sequencing. [Examples]

[0144] Those skilled in the art will understand that the examples described herein are for illustrative purposes only and that this disclosure is not limited by such examples.

[0145] Example 1. The inventors have created a novel method for isolating specific regions of a genome using oligohybridization pulldown to enable direct identification of modified bases from the pulldown product. A key innovation in this method is the post-hybridization preparation of a second strand configured to allow adapter ligation to only one end. This limits sequencing to the native strand, which is the only strand with potential modifications. Therefore, the inventors' method efficiently utilizes the maximum number of sequencing reads.

[0146] The inventors reduced this method to a practice using a pool of polynucleotides targeting 1,235 2kb regions of the genome that can be used to assess sperm quality using methylation patterns. The protocol used is attached. Next, CpG loci in each region were evaluated and their methylation fractions were quantified. The methylation fractions ranged from 0 (completely unmethylated) to 1 (completely methylated) (Figure 2A) and correlated with typical results using the MethylationEpic array, a common method for identifying methylated cytosines (Figure 2B). This finding supports the inventors' claim that only the native strand was sequenced. Since the synthetic strand is never entirely methylated, it is impossible to have CpGs that are entirely methylated (methylation fraction 1) if both the native and synthetic strands were sequenced. However, the inventors found more than 800 entirely methylated sites. To more comprehensively confirm that only the native strand was sequenced, the inventors also searched for reverse complement sequencing reads of the Nanopore ligation sequencing kit (SQK-LSK114) adapter. The presence of the adapter indicates that both ends of the enriched DNA were ligated, resulting in sequencing of both the native and synthetic strands. The inventors identified forward adapter sequences in over 85,000 sequences, but no reverse complement sequences. In summary, these results demonstrate that the inventors' method can sequence only the native strand, identify the full range of base modification states, yield results consistent with other methods, and provide superior data for the potential to achieve significantly longer reads.

[0147] Example 2. Materials and Methods Sample preparation and barcoding. Extracted DNA (1.5 ug) was fragmented, repaired, and end-treated using an Ultra II FS DNA module (NEB). The DNA was then purified using 0.8x ratio Ampure XP beads (Beckman Coulter) according to the manufacturer's instructions. The DNA fragment (750 ng) was mixed with 100 pmol of an adapter containing a 3'T overhang (a 20 base pair sequence used for sample identification) and a 30 base pair primer binding site, and ligated at room temperature for 15 minutes using Blunt / TA Master Mix (NEB). The reaction was then stopped using 0.5 M EDTA.

[0148] Oligohybridization. Twelve barcoded samples were mixed and purified using 0.5x Ampure XP beads according to the manufacturer's recommendations, and eluted in 12 µl of buffer containing blocker and blocker solution. In a separate tube, 20 µl of hybridization mix was mixed with 100 ng of oligoprobe library, heated at 95°C for 2 minutes, and then placed on ice. The eluted DNA fragments were then heated at 95°C for 5 minutes, and both mixtures were allowed to cool to room temperature for 5 minutes. Both mixtures were combined, and 30 µl of hybridization enhancer was added on top of the reaction. The complete mix was then incubated at 70°C for 20 hours.

[0149] The following day, the hybridization reaction was transferred to streptavidin-conjugated beads and incubated at 68°C for 5 minutes. After four washes, the DNA was eluted from the beads in 0.1N NaOH. The reaction was neutralized using 4 µl of 1M Tris, pH 5.2.

[0150] Second strand synthesis. Enriched DNA was mixed with 10 pmol of primer configured to bind to the 3' adapter and prevent ligation to its ends. The second strand was synthesized using BST3.0, which can eliminate any oligoprobes still bound to the DNA and result in a 3'A overhang. The reaction was incubated at 60°C for 15 minutes to synthesize the second strand.

[0151] Library preparation. Using double-stranded enriched DNA, nanopore sequencing libraries were constructed using a ligation sequencing kit (nanopore) with the following modifications: First, quantification was not performed because the amount of enriched DNA was below the detection level. Second, the adapter was diluted 1:10 in 1×LNB buffer before ligation. Finally, up to eight pulldowns were mixed into a single sequencing reaction.

[0152] Example 3. Prediction of infertility treatment success based on methylation. The examples described herein relate to a method for assessing the likelihood that a couple may require in vitro fertilization (IVF) instead of intrauterine insemination (IUI) or timed intercourse to conceive. This is based solely on targeted sequencing using the method described herein, which involves genetic and epigenetic evaluation of sperm. Our model generation yielded the following results in our test group (not involved in model generation): [Table 1]

[0153] Importantly, when the inventors tested this same model with known fertilizable sperm donors, 15 out of 16 were predicted to achieve pregnancy without IVF. The current model used by Inherent is constructed from array data and falls far short of the amount and resolution of information obtained using the method described herein. Their results for the same 14 samples were as follows: The prediction was low quality (failure without IVF) - 1 out of 2 individuals achieved pregnancy without IVF. The quality was predicted to be average (moderate chance of success without IVF) - 4 out of 8 women achieved pregnancy without IVF. High quality (high probability of pregnancy without IVF) was predicted - 1 out of 4 women achieved pregnancy without IVF.

[0154] Analysis of data generated using the methods described herein dramatically improves results compared to other tests currently available on the market, and the same techniques can be used to improve diagnostics in many fields.

[0155] The inventors partnered with a company that developed a methylation array-based method to predict whether artificial insemination would be effective for a particular patient. This assay investigated the methylation status of CpG in a defined 2 kbp region of sperm that had been previously identified as beneficial.

[0156] The inventors prepared an oligonucleotide pool that recognizes the same region and performed nanopore sequencing with Nanohyb enrichment and innate methylation determination. The samples consisted of DNA from 24 patients with known treatment outcomes who had previously undergone runs using array methods. Run statistics are shown in Table 1 below.

[0157] [Table 2]

[0158] The methylation patterns were consistent with previous results (Figure 3A). In addition, intra-region methylation patterns were even more consistent with the array (Figure 3B). When considering the average value over a 2kb region, the correlation between the array and sequencing improves. However, we also found that performance can be improved by investigating methylation across the entire read, which is not possible with the array.

[0159] Although the probe set was not designed to measure copy number variations (CNVs), the inventors decided to investigate whether the probe set could measure CNVs by measuring the average coverage of probes across chromosomes. Chromosomal copy numbers were determined by averaging the number of reads per region across the entire chromosome and then normalizing this to the average across all autosomes. The Y chromosome had one probe, the X chromosome had 28 probes, and autosomes ranged from 20 to 149 probes per chromosome. Despite the wide range of probe numbers, including only one on the Y chromosome, the copy number estimation was remarkably accurate and stable across repeated trials (Figure 4).

[0160] Example 4. Variant detection in genes that are difficult to sequence. Some key gene subsets are considered "difficult to sequence" due to their high GC content, long length, repetitive regions, and the presence of similar or pseudogenetic genes resulting from genomic duplication. One set of these genes harbors mutations that can cause polycystic kidney disease (PKD): PKD1, PKD2, and PKHD1. In addition, multiple distinct mutations across the entire gene are associated with the disease, and the nature of the mutations and the gene itself have a significant impact on the prognosis.

[0161] To demonstrate that Nanohyb can sequence these genes, we developed probe sets for these three genes and performed pulldowns in individuals with known mutations in PKD2. Run statistics are shown in Table 2 below.

[0162] [Table 3]

[0163] [Table 4] Example 5. Exemplary protocol for oligohybridization enrichment for natural chain sequencing. Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7

[0164] surface Table 6

[0165] Table 7

[0166] References

[0167] 1.Martin, S., Heavens, D., Lan, Y., Horsfield, S., Clark, MD, & Leggett, RM (2022). Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples. Genome Biology, 23(1),11.doi.org / 10.1186 / s13059-021-02582-x

[0168] 2. Kovaka, S., Fan, Y., Ni, B., Timp, W., & Schatz, M. C. (2021). Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED. Nature Biotechnology, 39(4), 431-441. doi.org / 10.1038 / s41587-020-0731-9

[0169] 3. Mariya, T., Kato, T., Sugimoto, T., Miyai, S., Inagaki, H., Ohye, T., Sugihara, E., Muramatsu, Y., Mizuno, S., & Kurahashi, H. (n.d.). Target enrichment nanopore sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes. Nanopore Website.

[0170] 4. McDonald, T. L., Zhou, W., Castro, C. P., Mumm, C., Switzenberg, J. A., Mills, R. E., & Boyle, A. P. (2021). Cas9 targeted enrichment of mobile elements using nanopore sequencing. Nature Communications, 12(1), 3586. doi.org / 10.1038 / s41467-021-23918-y

[0171] Embodiments Embodiment 1. A method for detecting base modifications in a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide; and (c) producing a tagged adapter-target polynucleotide by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of the one or more tags to the solid support. The method comprising: (d) preparing a complex; (e) isolating the pull-down complex; (f) optionally eluting the adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the preparation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group; (g) preparing a sequencing complex by contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide; and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence, thereby detecting the base modification in the target polynucleotide.

[0172] Embodiment 2. A method for sequencing a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide; (c) producing a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of the one or more tags to the solid support; and (d) The method comprising: (e) optionally eluting the adapter-target polynucleotide from the pull-down complex; (f) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex; and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence, thereby sequencing the target polynucleotide, wherein optionally the target polynucleotide contains one or more base modifications.

[0173] Embodiment 3. A method for quantifying base modifications in a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, or the adapter-target polynucleotide to the one or more tags, wherein the one or more tags are complementary to one or more regions of the target polynucleotide; and (c) producing the tagged adapter-target polynucleotide with a solid support, wherein the one or more tags are hybridized to the solid support. (d) Contacting under conditions that promote ligation to prepare a pull-down complex; (e) Isolating the pull-down complex; (f) Optionally eluting the adapter-target polynucleotide from the pull-down complex; (g) Contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the preparation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group; (g) Contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide to prepare a sequencing complex; (h) Sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence, thereby quantifying the base modification in the target polynucleotide.

[0174] Embodiment 4. A method for detecting multiple base modifications in multiple target polynucleotides, comprising: (a) producing multiple adapter-target polynucleotides by contacting the multiple target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the multiple target polynucleotides, wherein the one or more adapters include a primer binding site; (b) producing multiple tagged adapter-target polynucleotides by contacting the multiple adapter-target polynucleotides with one or more tags under conditions that promote hybridization of the one or more tags to the multiple adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the multiple target polynucleotides; and (c) contacting the multiple tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of the one or more tags to the solid support. (d) to produce a plurality of pull-down complexes; (e) optionally to elute the plurality of adapter-target polynucleotides from the plurality of pull-down complexes; (f) to contact the plurality of pull-down complexes or the plurality of eluted adapter-target polynucleotides with a first polymerase under conditions that promote the production of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group; (g) to produce a plurality of sequencing complexes by contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides; and (h) to sequence the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides but includes sequencing the plurality of target polynucleotide sequences.The method for detecting multiple base modifications in the multiple target polynucleotides.

[0175] Embodiment 5. A method for sequencing a plurality of target polynucleotides, comprising: (a) contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote the ligation of the one or more adapters to the plurality of target polynucleotides, wherein the one or more adapters include a primer binding site; (b) contacting the plurality of adapter-target polynucleotides with one or more tags under conditions that promote the hybridization of the one or more tags to the plurality of adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides; and (c) contacting the plurality of tagged adapter-target polynucleotides with a solid support under conditions that promote the hybridization of the one or more tags to the solid support, thereby forming a plurality of pull-down complexes. (d) preparing, (e) isolating the plurality of pull-down complexes, (f) optionally eluting the plurality of adapter-target polynucleotides from the plurality of pull-down complexes, (g) contacting the plurality of pull-down complexes or the plurality of eluted adapter-target polynucleotides with a first polymerase under conditions that promote the preparation of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group, (g) contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to prepare a plurality of sequencing complexes, (h) sequencing the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides but includes sequencing the plurality of target polynucleotide sequences, thereby sequencing the plurality of target polynucleotides.The method wherein, optionally, the plurality of target polynucleotides include one or more base modifications.

[0176] Embodiment 6. A method for detecting multiple base modifications in a target polynucleotide, comprising: (a) producing an adapter-target polynucleotide by contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site; (b) producing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide; and (c) producing a tagged adapter-target polynucleotide by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of the one or more tags to the solid support. The method comprising: (d) preparing a complex; (e) isolating the pull-down complex; (f) optionally eluting the adapter-target polynucleotide from the pull-down complex; (g) contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the preparation of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group; (g) contacting the adapter-target polynucleotide with a sequencing adapter in the presence of a second ligase under conditions that promote the ligation of the sequencing adapter to the adapter-target polynucleotide to prepare a sequencing complex; and (h) sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide but includes sequencing the target polynucleotide sequence, thereby detecting the plurality of base modifications in the target polynucleotide.

[0177] Embodiment 7. A method for detecting base modifications in a plurality of target polynucleotides, comprising: (a) contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the plurality of target polynucleotides, wherein the one or more adapters include a primer binding site; (b) contacting the plurality of adapter-target polynucleotides with one or more tags under conditions that promote hybridization of the one or more tags to the plurality of adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides; and (c) contacting the plurality of tagged adapter-target polynucleotides with a solid support under conditions that promote hybridization of the one or more tags to the solid support. (d) preparing a plurality of pull-down complexes; (e) optionally eluting the plurality of adapter-target polynucleotides from the plurality of pull-down complexes; (f) contacting the plurality of pull-down complexes or the plurality of eluted adapter-target polynucleotides with a first polymerase under conditions that promote the preparation of a plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group; (g) preparing a plurality of sequencing complexes by contacting the plurality of adapter-target polynucleotides with a plurality of sequencing adapters in the presence of a second ligase under conditions that promote the ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides; and (h) sequencing the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides but includes sequencing the plurality of target polynucleotide sequences.The method for detecting base modifications in the plurality of target polynucleotides.

[0178] Embodiment 8.(i) The method according to any one of the prior embodiments, further comprising analyzing the sequenced sequencing complex to detect the base modification.

[0179] Embodiment 9. The method according to any one of the prior embodiments, wherein the sequencing comprises identifying a base modification in the target polynucleotide.

[0180] Embodiment 10. The method according to any one of the prior embodiments, further comprising repeating steps (a) to (i) at least once using one or more additional adapters.

[0181] Embodiment 11. The method according to any one of the prior embodiments, wherein the target polynucleotide is isolated from the biological sample before step (a).

[0182] Embodiment 12. The method according to Embodiment 11, wherein the biological sample is of human origin.

[0183] Embodiment 13. The method according to any one of the prior embodiments, wherein the target polynucleotide comprises genomic DNA (gDNA) or cell-free DNA (cfDNA).

[0184] Embodiment 14. The method according to any one of the prior embodiments, wherein the target polynucleotide is fragmented before step (a).

[0185] Embodiment 15. The method according to any one of the prior embodiments, wherein the one or more adapters further include a first barcode.

[0186] Embodiment 16. The method according to Embodiment 15, wherein the one or more additional adapters further include one or more additional barcodes.

[0187] Embodiment 17. The method according to any one of the prior embodiments, wherein the one or more tags include streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody.

[0188] Embodiment 18. The method according to any one of the prior embodiments, wherein one or more tags target the same region of the target polynucleotide.

[0189] Embodiment 19. The method according to any one of Embodiments 1 to 17, wherein one or more tags target different regions of the target polynucleotide.

[0190] Embodiment 20. The method according to any one of the prior embodiments, wherein the solid support comprises streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody.

[0191] Embodiment 21. The method according to any one of the prior embodiments, wherein the solid support is beads or a column.

[0192] Embodiment 22. The method according to Embodiment 21, wherein the beads are paramagnetic.

[0193] Embodiment 23. The method according to any one of the prior embodiments, wherein the elution in step (e) comprises NaOH, heat, a chain-substituted polymerase, or a polymerase having exonuclease activity.

[0194] Embodiment 24. The method according to any one of the prior embodiments, wherein the 3' protecting group prevents sequencing of the complementary adapter-target polynucleotide.

[0195] Embodiment 25. The method according to any one of the prior embodiments, wherein the 3' protecting group includes a nucleotide overhang.

[0196] Embodiment 26. The method according to Embodiment 25, wherein the nucleotide overhang has at least 1 nucleotide, 2 nucleotides, or 3 nucleotides.

[0197] Embodiment 27. The method according to any one of the prior embodiments, wherein the primer does not contain a 5' phosphate group.

[0198] Embodiment 28. The method according to any one of the prior embodiments, wherein the sequence determination adapter includes a second barcode.

[0199] Embodiment 29. The method according to any one of the prior embodiments, wherein the base modification comprises 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), N6-methyladenine (6mA), 7-methylguanine (m7G), 2'-O-methylation (2'-O-methyl), or bromodeoxyuridine (BrdU).

[0200] Embodiment 30. The method according to any one of the prior embodiments, wherein neither bisulfite conversion nor amplification of the target polynucleotide is included.

[0201] Embodiment 31. A method according to any one of the prior embodiments, which does not include reduced representative region bisulfite sequencing (RRBS) or reduced representative region methylation sequencing (RRMS).

[0202] Embodiment 32. The method according to any one of the prior embodiments, wherein the first ligase or the second ligase is a T4 DNA ligase, a Quick ligase, a T3 ligase, a T7 ligase, or a tag ligase.

[0203] Embodiment 33. The method according to any one of the prior embodiments, wherein the first polymerase is Taq polymerase, BST polymerase, sulfolobus polymerase, therminator polymerase, Klenow polymerase, or deep vent polymerase.

[0204] Embodiment 34. The method according to any one of the prior embodiments, wherein the sequencing is performed in a sequencing device capable of identifying modified bases.

[0205] Embodiment 35. The method according to any one of the prior embodiments, wherein the base modification in the target polynucleotide is retained until sequencing.

[0206] Embodiment 36. The method according to any one of the prior embodiments, wherein the base modification in the target polynucleotide is enriched by a factor of about 300 to about 2400 compared to whole genome sequencing.

Claims

1. A method for detecting base modifications in a target polynucleotide, (a) Contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site, (b) Contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide to produce a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide. (c) To produce a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the pull-down complex, (e) Optionally, eluting the adapter-target polynucleotide from the pull-down complex, (f) Contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group, (g) Contacting the adapter-target polynucleotide with the sequencing adapter in the presence of a second ligase under conditions that promote ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex, (h) Sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide, but includes sequencing the target polynucleotide sequence. Includes, This allows detection of the base modification in the target polynucleotide. The aforementioned method.

2. A method for sequencing a target polynucleotide, (a) Contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site, (b) Contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide to produce a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide. (c) To produce a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the pull-down complex, (e) Optionally, eluting the adapter-target polynucleotide from the pull-down complex, (f) Contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group, (g) Contacting the adapter-target polynucleotide with the sequencing adapter in the presence of a second ligase under conditions that promote ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex, (h) Sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide, but includes sequencing the target polynucleotide sequence. Includes, This allows for sequencing of the target polynucleotide, and optionally, the target polynucleotide contains one or more base modifications. The aforementioned method.

3. A method for quantifying base modifications in a target polynucleotide, (a) Contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site, (b) Constructing a tagged adapter-target polynucleotide by contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide, or the adapter-target polynucleotide to the one or more tags, wherein the one or more tags are complementary to one or more regions of the target polynucleotide. (c) To produce a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the pull-down complex, (e) Optionally, eluting the adapter-target polynucleotide from the pull-down complex, (f) Contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group, (g) Contacting the adapter-target polynucleotide with the sequencing adapter in the presence of a second ligase under conditions that promote ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex, (h) Sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide, but includes sequencing the target polynucleotide sequence. Includes, This allows for the quantification of the base modification in the target polynucleotide. The aforementioned method.

4. A method for detecting multiple base modifications in multiple target polynucleotides, (a) To produce a plurality of adapter-target polynucleotides by contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the plurality of target polynucleotides, wherein the one or more adapters include a primer binding site. (b) Conveying the plurality of adapter-target polynucleotides to one or more tags under conditions that promote hybridization of the one or more tags to the plurality of adapter-target polynucleotides to produce a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides, (c) To produce a plurality of pull-down complexes by bringing the plurality of tagged adapter-target polynucleotides into contact with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the plurality of pull-down complexes, (e) Optionally, eluting the plurality of adapter-target polynucleotides from the plurality of pull-down complexes, (f) Contacting the plurality of pull-down complexes or the plurality of elution adapter-target polynucleotides with a first polymerase under conditions that promote the production of the plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group or a 5' protecting group, (g) Contacting the plurality of adapter-target polynucleotides with the plurality of sequencing adapters in the presence of a second ligase under conditions that promote ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to produce a plurality of sequencing complexes, (h) Sequencing the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides, but includes sequencing the plurality of target polynucleotide sequences. Includes, This allows for the detection of multiple base modifications in the multiple target polynucleotides. The aforementioned method.

5. A method for sequencing multiple target polynucleotides, (a) To produce a plurality of adapter-target polynucleotides by contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the plurality of target polynucleotides, wherein the one or more adapters include a primer binding site. (b) Conveying the plurality of adapter-target polynucleotides to one or more tags under conditions that promote hybridization of the one or more tags to the plurality of adapter-target polynucleotides to produce a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides, (c) To produce a plurality of pull-down complexes by bringing the plurality of tagged adapter-target polynucleotides into contact with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the plurality of pull-down complexes, (e) Optionally, eluting the plurality of adapter-target polynucleotides from the plurality of pull-down complexes, (f) Contacting the plurality of pull-down complexes or the plurality of elution adapter-target polynucleotides with a first polymerase under conditions that promote the production of the plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group or a 5' protecting group, (g) Contacting the plurality of adapter-target polynucleotides with the plurality of sequencing adapters in the presence of a second ligase under conditions that promote ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to produce a plurality of sequencing complexes, (h) Sequencing the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides, but includes sequencing the plurality of target polynucleotide sequences. Includes, This allows for sequencing of the plurality of target polynucleotides, and optionally, the plurality of target polynucleotides include one or more base modifications. The aforementioned method.

6. A method for detecting multiple base modifications in a target polynucleotide, (a) Contacting the target polynucleotide with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the target polynucleotide, wherein the one or more adapters include a primer binding site, (b) Contacting the adapter-target polynucleotide with one or more tags under conditions that promote hybridization of the one or more tags to the adapter-target polynucleotide to produce a tagged adapter-target polynucleotide, wherein the one or more tags are complementary to one or more regions of the target polynucleotide. (c) To produce a pull-down complex by contacting the tagged adapter-target polynucleotide with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the pull-down complex, (e) Optionally, eluting the adapter-target polynucleotide from the pull-down complex, (f) Contacting the pull-down complex or the eluted adapter-target polynucleotide with a first polymerase under conditions that promote the production of a complementary adapter-target polynucleotide, wherein the complementary adapter-target polynucleotide contains a 3' protecting group or a 5' protecting group, (g) Contacting the adapter-target polynucleotide with the sequencing adapter in the presence of a second ligase under conditions that promote ligation of the sequencing adapter to the adapter-target polynucleotide to produce a sequencing complex, (h) Sequencing the sequencing complex, which does not include sequencing the complementary adapter-target polynucleotide, but includes sequencing the target polynucleotide sequence. Includes, This allows for the detection of the multiple base modifications in the target polynucleotide. The aforementioned method.

7. A method for detecting base modifications in multiple target polynucleotides, (a) To produce a plurality of adapter-target polynucleotides by contacting the plurality of target polynucleotides with one or more adapters in the presence of a first ligase under conditions that promote ligation of the one or more adapters to the plurality of target polynucleotides, wherein the one or more adapters include a primer binding site. (b) Conveying the plurality of adapter-target polynucleotides to one or more tags under conditions that promote hybridization of the one or more tags to the plurality of adapter-target polynucleotides to produce a plurality of tagged adapter-target polynucleotides, wherein the one or more tags are complementary to one or more regions of the plurality of target polynucleotides, (c) To produce a plurality of pull-down complexes by bringing the plurality of tagged adapter-target polynucleotides into contact with a solid support under conditions that promote hybridization of one or more tags to the solid support, (d) Isolating the plurality of pull-down complexes, (e) Optionally, eluting the plurality of adapter-target polynucleotides from the plurality of pull-down complexes, (f) Contacting the plurality of pull-down complexes or the plurality of elution adapter-target polynucleotides with a first polymerase under conditions that promote the production of the plurality of complementary adapter-target polynucleotides, wherein each of the complementary adapter-target polynucleotides contains a 3' protecting group or a 5' protecting group, (g) Contacting the plurality of adapter-target polynucleotides with the plurality of sequencing adapters in the presence of a second ligase under conditions that promote ligation of the plurality of sequencing adapters to the plurality of adapter-target polynucleotides to produce a plurality of sequencing complexes, (h) Sequencing the plurality of sequencing complexes, which does not include sequencing the plurality of complementary adapter-target polynucleotides, but includes sequencing the plurality of target polynucleotide sequences. Includes, This allows for the detection of base modifications in the multiple target polynucleotides. The aforementioned method.

8. (i) Analyze the sequenced sequencing complex to detect the base modification. The method according to any one of the prior claims, further comprising:

9. The method according to any one of the prior claims, wherein the sequencing includes identifying a base modification in the target polynucleotide.

10. The method according to any one of the prior claims, further comprising repeating steps (a) to (i) at least once using one or more additional adapters.

11. The method according to any one of the prior claims, wherein the target polynucleotide is isolated from the biological sample before step (a).

12. The method according to claim 11, wherein the biological sample is of human origin.

13. The method according to any one of the prior claims, wherein the target polynucleotide comprises genomic DNA (gDNA) or cell-free DNA (cfDNA).

14. The method according to any one of the prior claims, wherein the target polynucleotide is fragmented before step (a).

15. The method according to any one of the prior claims, wherein the one or more adapters further include a first barcode.

16. The method according to claim 15, wherein the one or more additional adapters further include one or more additional barcodes.

17. The method according to any one of the prior claims, wherein the one or more tags include streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody.

18. The method according to any one of the prior claims, wherein one or more tags target the same region of the target polynucleotide.

19. The method according to any one of claims 1 to 17, wherein one or more tags target different regions of the target polynucleotide.

20. The method according to any one of the prior claims, wherein the solid support comprises streptavidin, biotin, maltose, maltose-binding protein, glutathione, glutathione S-transferase, chitin, chitin-binding protein, aptamer, antigen, SpyCatcher, SpyTag, or antibody.

21. The method according to any one of the prior claims, wherein the solid support is beads or columns.

22. The method according to claim 21, wherein the beads are paramagnetic.

23. The method according to any one of the prior claims, wherein the elution in step (e) comprises NaOH, heat, a chain-substituted polymerase, or a polymerase having exonuclease activity.

24. The method according to any one of the prior claims, wherein the 3' protecting group or the 5' protecting group prevents sequencing of the complementary adapter-target polynucleotide.

25. The method according to any one of the prior claims, wherein the 3' protecting group includes a nucleotide overhang.

26. The method according to claim 25, wherein the nucleotide overhang has at least one nucleotide, two nucleotides, or three nucleotides.

27. The method according to any one of the prior claims, wherein the primer does not contain a 5' phosphate group.

28. The method according to any one of the prior claims, wherein the sequence determination adapter includes a second barcode.

29. The method according to any one of the prior claims, wherein the base modification comprises 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), N6-methyladenine (6mA), 7-methylguanine (m7G), 2'-O-methylation (2'-O-methyl), or bromodeoxyuridine (BrdU).

30. The method according to any one of the prior claims, wherein neither bisulfite conversion nor amplification of the target polynucleotide is included.

31. The method according to any one of the prior claims, wherein the method does not include reduced representative region bisulfite sequencing (RRBS) or reduced representative region methylation sequencing (RRMS).

32. The method according to any one of the prior claims, wherein the first ligase or the second ligase is T4 DNA ligase, Quick ligase, T3 ligase, T7 ligase, or Tag ligase.

33. The method according to any one of the prior claims, wherein the first polymerase is Taq polymerase, BST polymerase, sulfolobus polymerase, therminator polymerase, Klenow polymerase, or deep vent polymerase.

34. The method according to any one of the prior claims, wherein the sequencing is performed in a sequencing apparatus capable of identifying modified bases.

35. The method according to any one of the prior claims, wherein the base modification in the target polynucleotide is maintained until sequencing.

36. The method according to any one of the prior claims, wherein the base modifications in the target polynucleotide are concentrated by a factor of about 300 to about 2400 compared to whole genome sequencing.

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