Methods and reagents for enrichment of nucleic acid material for sequencing applications and other nucleic acid material interrogations

Abstract

To provide methods for improving cost efficiency, recovery efficiency, and other performance metrics as well as overall process speed for Duplex Sequencing and other sequencing applications for achieving high accuracy sequencing reads.SOLUTION: The present technology relates generally to methods and compositions for targeted nucleic acid sequence enrichment, as well as uses of such enrichment for error-corrected nucleic acid sequencing applications and other nucleic acid sequence interrogations. In some embodiments, provided methods provide non-amplification based targeted enrichment strategies compatible with use of molecular barcodes for error correction. Other embodiments provide methods for non-amplification based targeted enrichment strategies compatible with direct digital sequencing (DDS) and other sequencing strategies (e.g., single molecule sequencing modalities and interrogations) that do not use molecular barcoding.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 643,738, filed March 15, 2018, the disclosure of which is incorporated by reference in its entirety. [Background technology]

[0002] In massively parallel sequencing (MPS, sometimes known as next-generation DNA sequencing, or NGS), various approaches have been developed at the protocol development, chemistry / biology, and data processing levels to mitigate the impact of PCR-based errors. Additionally, techniques that can resolve PCR duplicate detection resulting from individual DNA fragments before or during amplification based on unique random shear points or through exogenous tagging (i.e., using molecular barcodes, also known as molecular tags, unique molecular identifiers [UMIs], and single molecule identifiers [SMIs]) are commonly used. This approach has been used to improve the counting accuracy of DNA and RNA templates. Because all amplicons derived from a single starting molecule can be unambiguously identified, any variations in the sequence of identically tagged sequencing reads can be used to correct for base errors that occur during PCR or sequencing. For example, Kinde et al. (Proc Natl Acad Sci USA 108, 9530-9535, 2011) introduced SafeSeqS, which uses single-strand molecular barcoding to reduce the sequencing error rate by grouping PCR copies that share sequencing barcodes and form consensus. However, the incorporation of single-strand molecular barcodes cannot completely eliminate PCR artifacts that arise during the first round of amplification and are carried on derivative copies as "jackpot" events.

[0003] Methods for more accurate genotyping of single nucleotide polymorphism (SNP) loci, short tandem repeat (STR) loci, and many other forms of mutations and genetic variants are desirable for a variety of applications in medicine, forensics, genetic toxicology, and other scientific industries. However, the challenge remains: how to most efficiently generate sequence information from as many related copies of the genetic material being sequenced as possible with the highest reliability and at a reasonable cost. Various consensus sequencing methods (both molecular barcode-based and non-molecular barcode-based) have been used to help better identify variants in mixtures for error correction (for a detailed discussion, see J. Salk et al., "Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations," Nature Reviews Genetics, 2018), but have come with various compromises in performance. We previously described duplex sequencing, an ultra-high-precision sequencing method based on genotyping and comparing independent sequenced strands of double-stranded nucleic acid molecules for the purpose of error correction. Aspects of the technology set forth herein describe methods for improving the cost efficiency, recovery efficiency and other performance criteria, as well as the overall processing speed of double-stranded sequencing and other sequencing applications to achieve highly accurate sequencing reads. Summary of the Invention

[0004] The present technology generally relates to methods for the enrichment of target nucleic acid sequences and the use of such enrichment for error-corrected nucleic acid sequencing applications and other nucleic acid material interrogation. In some embodiments, highly accurate, error-corrected, massively parallel sequencing of nucleic acid material is possible using the enriched target nucleic acid material from a sample. In some embodiments, the enriched target nucleic acid material is double-stranded, and one or more methods for uniquely labeling strands of double-stranded nucleic acid complexes can be used in a manner that not only informatively associates each strand with its complementary strand, but also allows it to be distinguished from its complementary strand after sequencing of each strand or the amplification products derived therefrom, and this information can be further used for error correction of the determined sequence. Some aspects of the present technology provide methods and compositions for improving the cost, conversion of sequenced molecules, and time efficiency of creating labeled molecules for targeted, ultra-high-precision sequencing. In some embodiments, the provided methods and compositions enable accurate analysis of very small amounts of nucleic acid material (e.g., from small clinical samples, or blood collected from crime scenes, or free-floating DNA in samples). In some embodiments, the methods and compositions provided allow for the detection of mutations in a sample of nucleic acid material that are present at a frequency of less than 1 in 100 cells or molecules (e.g., less than 1 in 1000 cells or molecules, less than 1 in 10,000 cells or molecules, less than 1 in 100,000 cells or molecules).

[0005] Aspects of the present technology are directed to a method for concentrating target nucleic acid material, the method comprising: providing the nucleic acid material; and cutting the nucleic acid material with one or more targeting endonucleases such that a predetermined length of the target region is separated from the remainder of the nucleic acid material. The method may further comprise enzymatically destroying non-target nucleic acid material, releasing the predetermined length of the target region from the targeting endonucleases, and analyzing the cleaved target region.

[0006] A further aspect of the present technology is directed to a method for concentrating a target nucleic acid material, the method comprising: providing the nucleic acid material; cleaving the nucleic acid material with one or more target endonucleases such that a target region of a predetermined length is separated from the remainder of the nucleic acid material, wherein at least one target endonuclease comprises a capture label; capturing the target region of a predetermined length using an extraction moiety configured to bind to the capture label; releasing the target region of a predetermined length from the target endonuclease; and analyzing the cleaved target region.

[0007] A further aspect of the present technology is directed to a method for concentrating a target nucleic acid material, the method comprising: providing the nucleic acid material; binding a catalytically inactive CRISPR-associated (Cas) enzyme to a target region of the nucleic acid material; enzymatically treating the nucleic acid material with one or more nucleic acid digestive enzymes such that non-target nucleic acid material is destroyed and the target region is protected from the digestive enzymes by the bound catalytically inactive Cas enzyme; releasing the target region from the catalytically inactive Cas enzyme; and analyzing the target region.

[0008] Another aspect of the present technology is directed to a method for concentrating a target nucleic acid material, the method comprising: providing the nucleic acid material; providing a pair of catalytically active target endonucleases and at least one catalytically inactive target endonuclease comprising a capture label, wherein the catalytically inactive target endonuclease is directed to bind to a target region of the nucleic acid material and the pair of catalytically active target endonucleases are directed to bind to the target region on either side of the catalytically inactive target endonuclease; cleaving the nucleic acid material with the pair of catalytically active target endonucleases such that the target region is separated from the remainder of the nucleic acid material; capturing the target region with an extraction moiety configured to bind to the capture label; releasing the target region from the target endonuclease; and analyzing the cleaved target region.

[0009] Further aspects include methods for concentrating target nucleic acid material from a sample comprising a plurality of nucleic acid fragments, the method comprising: providing one or more catalytically inactive CRISPR-associated (Cas) enzymes having capture labels to a sample comprising target nucleic acid fragments and non-target nucleic acid fragments, wherein the one or more catalytically inactive Cas enzymes are configured to bind to the target nucleic acid fragments; providing a surface comprising extraction moieties configured to bind to the capture labels; and separating the target nucleic acid fragments from the non-target nucleic acid fragments by capturing the target nucleic acid fragments via binding of the capture labels by the extraction moieties.

[0010] Various embodiments provide a method for concentrating a target double-stranded nucleic acid material, the method including providing the nucleic acid material, cleaving the nucleic acid material with one or more target endonucleases to create double-stranded target nucleic acid fragments comprising 5' sticky ends having a predetermined 5' nucleotide sequence and / or 3' sticky ends having a predetermined 3' nucleotide sequence, and separating the double-stranded target nucleic acid molecules from the remainder of the nucleic acid material via at least one of the 5' sticky ends and the 3' sticky ends.

[0011] A further embodiment provides a kit for enriching a target nucleic acid material, the kit comprising a nucleic acid library, the nucleic acid library comprising the nucleic acid material and a plurality of catalytically inactive Cas enzymes, the Cas enzymes comprising tags having sequence codes, and the plurality of Cas enzymes binding to a plurality of site-specific target regions along the nucleic acid material. The kit further comprises a plurality of probes, each probe comprising an oligonucleotide sequence comprising a complement to a corresponding sequence code and a capture label. The kit may also comprise a lookup table cataloging the relationship between the site-specific target regions, wherein the sequence codes are associated with the site-specific target regions and the probes comprise complements to the corresponding sequence codes.

[0012] In some embodiments, the error-corrected sequence reads are used to identify or identify cancer, cancer risk, cancer mutation, cancer metabolic state, mutator gene phenotype, carcinogen exposure, toxin exposure, chronic inflammatory exposure, age, neurodegenerative disease, pathogen, drug resistance variant, fetal molecule, forensically relevant molecule, immunologically relevant molecule, mutated T cell receptor, mutated B cell receptor, mutated immunoglobulin locus, catabolic site in the genome, hypermutable site in the genome, low frequency variant, subclonal variant, minor molecular population, contaminant source, nucleic acid synthesis error, enzymatic modification error, chemical modification error, gene editing error, gene therapy error, part of nucleic acid information reservoir, microbial polymorphism, viral polymorphism, organ transplant, organ transplant rejection, cancer recurrence, residual cancer after treatment, pre-neoplastic state, dysplastic state, microchimerism state, stem cell transplant state, cell therapy state, nucleic acid label affixed to another molecule, or combinations thereof in the organism or subject from which the double-stranded target nucleic acid molecule is derived. In some embodiments, the error-corrected sequence reads are used to identify carcinogenic compounds or exposures. In some embodiments, the error-corrected sequence reads are used to identify mutagenic compounds or exposures. In some embodiments, the nucleic acid material is derived from a forensic sample and the error-corrected sequence reads are used in forensic analysis.

[0013] In some embodiments, the single molecule identifier sequence comprises an endogenous shear point or an endogenous sequence that can be positionally associated with the shear point. In some embodiments, the single molecule identifier sequence is at least one of a degenerate or semi-degenerate barcode sequence, one or more nucleic acid fragment ends of a nucleic acid substance, or a combination thereof that uniquely labels a double-stranded nucleic acid molecule. In some embodiments, the adapter and / or adapter sequence comprises at least one nucleotide position that is at least partially non-complementary or contains at least one non-standard base. In some embodiments, the adapter comprises a single "U-shaped" oligonucleotide sequence formed by about five or more self-complementary nucleotides.

[0014] According to various embodiments, any of a variety of nucleic acid agents may be used. In some embodiments, the nucleic acid agent may include at least one modification to a polynucleotide in the canonical sugar-phosphate backbone. In some embodiments, the nucleic acid agent may include at least one modification in any base within the nucleic acid agent. For example, and not by way of limitation, in some embodiments, the nucleic acid agent is or includes at least one of double-stranded DNA, double-stranded RNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA).

[0015] In some embodiments, the provided method further comprises ligating an adapter molecule to the double-stranded nucleic acid molecule. In some embodiments, the ligating step comprises ligating the double-stranded nucleic acid material to at least one double-stranded degenerated barcode sequence to form a double-stranded nucleic acid molecule barcode complex, wherein the double-stranded degenerated barcode sequence comprises a single molecule identifier sequence on each strand. In some embodiments, the double-stranded nucleic acid molecule is a double-stranded DNA molecule or a double-stranded RNA molecule. In some embodiments, the double-stranded nucleic acid molecule comprises at least one modified nucleotide or non-nucleotide molecule.

[0016] In some embodiments, the ligating comprises the activity of at least one ligase. In some embodiments, the at least one ligase is selected from a DNA ligase and an RNA ligase. In some embodiments, the ligating comprises ligase activity at a ligation domain associated with an adapter molecule. In some embodiments, the ligating comprises ligase activity at a ligation domain associated with an adapter molecule and a ligatable end of the nucleic acid molecule. In some embodiments, the ligation domain and the ligatable end of the double-stranded nucleic acid molecule are compatible (e.g., have complementary single-stranded regions). In some embodiments, the ligation domain is a nucleotide sequence derived from one or more degenerate or semi-degenerate nucleotides or is associated with one or more degenerate or semi-degenerate nucleotides. In some embodiments, the ligation domain is a nucleotide sequence derived from one or more non-degenerate nucleotides. In some embodiments, the ligation domain contains one or more modified nucleotides. In some embodiments, the ligation domain and / or ligatable end comprises a T overhang, an A overhang, a CG overhang, a blunt end, a recombination sequence, an endonuclease cleavage site overhang, a restriction digest overhang, or another ligatable region. In some embodiments, at least one strand of the ligation domain is phosphorylated. In some embodiments, the ligation domain comprises an endonuclease cleavage sequence or a portion thereof.

[0017] In some embodiments, the endonuclease cleavage sequence is cleaved by an endonuclease (e.g., a tunable endonuclease, a restriction endonuclease) to yield a blunt end or an overhang with a ligatable region. In some embodiments, the ligatable end of a double-stranded nucleic acid molecule comprises an endonuclease cleavage sequence or a portion thereof. In some embodiments, the endonuclease (e.g., a programmable / targeted endonuclease, a restriction endonuclease) yields an overhang that includes a "sticky end" or single-stranded overhang region having a known nucleotide length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides) and sequence.

[0018] In some embodiments, the identifier sequence is or comprises a single molecule identifier (SMI) sequence. In some embodiments, the SMI sequence is an endogenous SMI sequence. In some embodiments, the endogenous SMI sequence is associated with a shear point. In some embodiments, the SMI sequence comprises at least one denatured or semi-denatured nucleic acid. In some embodiments, the SMI sequence is non-denatured. In some embodiments, the SMI sequence is a nucleotide sequence of one or more denatured or semi-denatured nucleotides. In some embodiments, the SMI sequence is a nucleotide sequence of one or more non-denatured nucleotides. In some embodiments, the SMI sequence comprises at least one modified nucleotide or non-nucleotide molecule. In some embodiments, the SMI sequence comprises a primer binding domain.

[0019] In some embodiments, the modified nucleotide or non-nucleotide molecule is 2-aminopurine, 2,6-diaminopurine (2-amino-dA), 5-bromo-dU, deoxyuridine, inverted dT, inverted dideoxy-T, dideoxy-C, 5-methyl-dC, deoxyinosine, Super T™, Super G™, locked nucleic acid, 5-nitroindole, 2'-O-methyl RNA bases, hydroxymethyl dC, iso-dG, iso-dC, fluoro C, fluoro U, fluoro A, fluoro G, 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 8-oxo-A, 8-oxo G, 5-hydroxymethyl-2'-deoxycytidine, 5'-methylisocytosine, tetrahydrofuran, iso-cytosine, iso-guanosine, uracil, methylated nucleotides, RNA nucleotides, ribose nucleotides, 8-oxo-G, BrdU, Loto dU, furan, fluorescent dyes, azido nucleotides, abasic nucleotides, 5-nitroindole nucleotides, and digoxigenin nucleotides.

[0020] In some embodiments, the cleavage site is or comprises a restriction endonuclease recognition sequence, hi some embodiments, the cleavage site is or comprises a user-directed recognition sequence for a targeting endonuclease (e.g., a CRISPR or CRISPR-like endonuclease) or other tunable endonuclease. In some embodiments, cleaving the nucleic acid material may include at least one of enzymatic digestion, enzymatic cleavage, enzymatic cleavage of one strand, enzymatic cleavage of both strands, incorporation of a modified nucleic acid followed by an enzymatic treatment causing cleavage of one or both strands, incorporation of a replication-blocking nucleotide, incorporation of a chain terminator, incorporation of a photocleavable linker, incorporation of uracil, incorporation of a ribose base, incorporation of an 8-oxo-guanine adduct, use of a restriction endonuclease, use of a ribonucleoprotein endonuclease (e.g., a Cas enzyme, e.g., Cas9 or CPF1), or other programmable endonucleases (e.g., homing endonucleases, zinc finger nucleases, TALENs, meganucleases (e.g., megaTAL nucleases), Argonaute nucleases, etc.), and any combination thereof.

[0021] In some embodiments, the capture label is or comprises at least one of an acrydite, azide, azide (NHS ester), digoxigenin (NHS ester), I-Linker, Amino modifier C6, Amino modifier C12, Amino modifier C6 dT, Unilink amino modifier, hexynyl, 5-octadiynyl dU, biotin, biotin (azide), biotin dT, biotin TEG, dual biotin, PC biotin, desthiobiotin TEG, thiol modifier C3, dithiol, thiol modifier C6 SS, and succinyl group.

[0022] In some embodiments, the extraction moiety is or includes at least one of an aminosilane, an epoxysilane, an isothiocyanate, an aminophenylsilane, an aminopropylsilane, a mercaptosilane, an aldehyde, an epoxide, a phosphonate, streptavidin, avidin, a hapten that recognizes an antibody, a specific nucleic acid sequence, magnetically attractable particles (Dynabeads), and a photosensitive resin.

[0023] In some embodiments, the provided methods further include amplifying the nucleic acid material by using primers specific to the adapter sequence and / or by using primers specific to the non-adapter portion of the nucleic acid product. It is contemplated that any of a variety of methods for amplifying the nucleic acid material may be used according to various embodiments. For example, in some embodiments, at least one amplification step includes polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), isothermal amplification, polony amplification in an emulsion, bridge amplification on a surface, on a bead or on a surface within a hydrogel, and any combination thereof. In some embodiments, amplifying the nucleic acid material includes using single-stranded oligonucleotides at least partially complementary to regions of the first and second adapter sequences (e.g., at least partially complementary to the adapter sequences on the 5' and / or 3' ends of each strand of the nucleic acid material). In some embodiments, amplifying the nucleic acid material includes using single-stranded oligonucleotides at least partially complementary to regions of the genomic sequence of interest and single-stranded oligonucleotides at least partially complementary to regions of the adapter sequence.

[0024] In some embodiments, amplifying the nucleic acid material comprises generating a plurality of amplicons derived from the first strand and a plurality of amplicons derived from the second strand.

[0025] In some embodiments, the provided methods further include cleaving the nucleic acid material with one or more target endonucleases to form target nucleic acid fragments of substantially known lengths, and isolating the target nucleic acid fragments based on the substantially known lengths. In some embodiments, the provided methods further include ligating adapters (e.g., adapter sequences) to the target nucleic acids (e.g., target nucleic acid fragments) of substantially known lengths (e.g., after the size enrichment step).

[0026] In some embodiments, the nucleic acid material may be or may include one or more target nucleic acid fragments. In some embodiments, the one or more target nucleic acid fragments each include a genomic sequence of interest from one or more locations in the genome. In some embodiments, the one or more target nucleic acid fragments include a target sequence from a substantially known region in the nucleic acid material. In some embodiments, isolating the target nucleic acid fragments based on their substantially known lengths includes concentrating the target nucleic acid fragments by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI bead purification.

[0027] In some embodiments, provided methods further include cleaving the double-stranded nucleic acid material with one or more target endonucleases to form double-stranded target nucleic acid fragments containing one or both ends with a substantially known length and / or single-stranded overhang sequence. In some embodiments, provided methods further include isolating the double-stranded target nucleic acid fragments based on the substantially known length and / or single-stranded overhang sequence. In some embodiments, provided methods further include ligating an adapter (e.g., an adapter sequence) to the double-stranded target nucleic acid (e.g., the target nucleic acid fragment) having a substantially known length and / or single-stranded overhang sequence. In some embodiments, the double-stranded target nucleic acid may have a ligatable end that is substantially uniquely compatible with (e.g., complementary to) the ligation domain of a ligation-selected adapter molecule such that one or more target nucleic acid fragments containing a target sequence from a substantially known region in the nucleic acid material can be selectively enriched by amplification using primers specific for the adapter sequence associated with the ligation-selected adapter(s).

[0028] According to various embodiments, some provided methods may be useful for sequencing any of a variety of suboptimal (e.g., damaged or degraded) samples of nucleic acid material, e.g., in some embodiments, at least a portion of the nucleic acid material is damaged.In some embodiments, the damage is oxidation, alkylation, deamination, methylation, hydrolysis, hydroxylation, nicking, intrastrand crosslinks, interstrand crosslinks, blunt-end breakage, sticky-end double-strand breakage, phosphorylation, dephosphorylation, sumoylation, glycosylation, deglycosylation, putrescinylation, carboxylation, halogenation, formylation, single-strand gaps, damage from heat, damage from desiccation, damage from UV exposure, damage from gamma radiation, damage from x-rays, damage from ionizing radiation, damage from non-ionizing radiation, damage from heavy particle radiation, damage from nuclear decay, damage from beta radiation, damage from alpha radiation, damage from neutron radiation, damage from proton radiation, damage from cosmic radiation, damage from high pH, ​​damage from low pH, damage from reactive oxidative species, damage from free radicals, damage from peroxides, damage from hypochlorite, damage from tissue fixation such as formalin or formaldehyde, damage from reactive iron, damage from low ionic conditions, damage from high ... damage from unbuffered conditions, damage from nucleases, damage from environmental exposure, damage from fire, damage from mechanical stress, damage from enzymatic degradation, damage from microorganisms, damage from mechanical shear in preparation, damage from enzymatic fragmentation in preparation, damage occurring spontaneously in vivo, damage occurring during nucleic acid extraction, damage occurring during sequencing library preparation, damage introduced by polymerases, damage introduced during nucleic acid repair, damage occurring during tailing of nucleic acid ends, damage occurring during nucleic acid ligation, damage occurring during sequencing, damage occurring from mechanical handling of DNA, damage occurring during passage through a nanopore, damage occurring as part of aging in an organism, damage occurring as a result of chemical exposure of an individual, damage caused by mutagens, damage caused by carcinogens, damage caused by clastogens, damage resulting from inflammatory damage in vivo due to oxygen exposure, damage resulting from one or more strand breaks, and any combination thereof.

[0029] It is contemplated that the nucleic acid material may be derived from a variety of sources. For example, in some embodiments, the nucleic acid material (e.g., comprising one or more double-stranded nucleic acid molecules) is provided from a sample derived from a human subject, an animal, a plant, a fungus, a virus, a bacterium, a protozoan, or any other living organism. In other embodiments, the sample comprises at least partially artificially synthesized nucleic acid material. In some embodiments, the sample is a body tissue, a biopsy, a skin sample, blood, serum, plasma, sweat, saliva, cerebrospinal fluid, mucus, uterine washing, vaginal swab, pap smear, nasal swab, oral swab, tissue scraping, hair, fingerprint, urine, stool, vitreous fluid, peritoneal washing, sputum, bronchial lavage, oral wash, pleural lavage, gastric lavage, gastric juice, bile, pancreatic duct lavage, bile duct lavage, common bile duct lavage, gallbladder fluid, synovial fluid, infected wound, non-infected wound, archaeological sample, forensic sample, water sample, tissue sample, food sample, bioreactor sample, plant sample, bacterial sample, protozoan sample, fungal sample, animal sample, viral sample, multi-organism sample, nail polish, semen, prostatic fluid, The nucleic acid material may be or include vaginal fluid, vaginal swab, fallopian tube washing, cell-free nucleic acid, intracellular nucleic acid, metagenomics sample, wash or swab of an implanted foreign body, nasal wash, intestinal fluid, epithelial brushing, epithelial wash, tissue biopsy, autopsy sample, necrosis sample, organ sample, human identification sample, non-human identification sample, artificially created nucleic acid sample, synthetic gene sample, deposited or preserved nucleic acid sample, tumor tissue, fetal sample, organ transplant sample, microbial culture sample, nuclear DNA sample, mitochondrial DNA sample, chloroplast DNA sample, apicoplast DNA sample, organelle sample, and any combination thereof. In some embodiments, the nucleic acid material is derived from two or more sources.

[0030] As described herein, in some embodiments, it is advantageous to treat nucleic acid material to improve the efficiency, accuracy, and / or speed of the sequencing process. In some embodiments, the nucleic acid material comprises nucleic acid molecules of substantially uniform length and / or substantially known length. In some embodiments, the substantially uniform length and / or substantially known length is from about 1 to about 1,000,000 bases. ) For example, in some embodiments, the substantially uniform length and / or the substantially known length may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, or 50,000 bases in length. In some embodiments, the substantially uniform and / or substantially known lengths may be up to 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 bases. As a specific, non-limiting example, in some embodiments, the substantially uniform and / or substantially known lengths are from about 100 to about 500 bases. In some embodiments, the methods described herein include providing nucleic acid molecules having one or more lengths and / or substantially known lengths by targeting concentrated nucleic acid material. In some embodiments, the nucleic acid material is cleaved by one or more targeting endonucleases into nucleic acid molecules of substantially uniform length and / or substantially known length. In some embodiments, the targeting endonucleases include at least one modification.

[0031] In some embodiments, the nucleic acid agent comprises a nucleic acid molecule having a length within one or more substantially known size ranges, which may be from 1 to about 1,000,000 bases, from about 10 to about 10,000 bases, from about 100 to about 1,000 bases, from about 100 to about 600 bases, from about 100 to about 500 bases, or some combination thereof.

[0032] In some embodiments, the targeting endonuclease is or includes at least one of the following restriction endonucleases (i.e., restriction enzymes) that cleave DNA at or near a recognition site (e.g., EcoRI, BamHI, XbaI, HindIII, AluI, AvaII, BsaJI, BstNI, DsaV, Fnu4HI, HaeIII, MaeIII, N1aIV, NSiI, MspJI, FspEI, NaeI, Bsu36I, NotI, HinF1, Sau3AI, PvuII, SmaI, HgaI, AluI, EcoRV, etc.). A list of several restriction endonucleases is available in both printed and computer-readable form and is provided by many commercial suppliers (e.g., New England Biolabs, Ipswich, MA). One of skill in the art will understand that any restriction endonuclease may be used in accordance with various embodiments of the present technology. In other embodiments, the targeting endonuclease is or includes at least one of a ribonucleoprotein complex, such as a CRISPR-associated (Cas) enzyme / guide RNA complex (e.g., Cas9 or Cpf1) or a Cas9-like enzyme. In other embodiments, the targeting endonuclease is or includes a homing endonuclease, a zinc finger nuclease, a TALEN and / or a meganuclease (e.g., a megaTAL nuclease), an Argonaute nuclease, or a combination thereof. In some embodiments, the targeting endonuclease includes Cas9 or CPF1, or a derivative thereof. In some embodiments, two or more targeting endonucleases (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) may be used. In some embodiments, a targeting endonuclease may be used to cleave two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) potential target regions of a nucleic acid material. In some embodiments, when two or more target regions of a nucleic acid material are present, each target region may be of the same (or substantially the same) length.In some embodiments, when there are two or more target regions of a nucleic acid material, at least two of the target regions having known lengths are different in length (e.g., a first target region having a length of 100 bp and a second target region having a length of 1,000 bp).

[0033] In some embodiments, at least one amplifying step includes at least one primer and / or adapter sequence that is or includes at least one non-standard nucleotide. By way of further example, in some embodiments, at least one adapter sequence is or includes at least one non-standard nucleotide. In some embodiments, the non-standard nucleotide is selected from uracil, methylated nucleotides, RNA nucleotides, ribose nucleotides, 8-oxo-guanine, biotinylated nucleotides, desthiobiotin nucleotides, thiol-modified nucleotides, acrydite-modified nucleotides, iso-dC, isodG, 2'-O-methyl nucleotides, inosine nucleotide-locked nucleic acids, peptide nucleic acids, 5-methyl dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotides, abasic nucleotides, 5-nitroindole nucleotides, adenylated nucleotides, azido nucleotides, digoxigenin nucleotides, I-linkers, 5' hexynyl-modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non-photocleavable spacers, click chemistry compatible modified nucleotides, fluorescent dyes, biotin, furan, BrdU, fluoro-dU, roto-dU, and any combination thereof.

[0034] According to some embodiments, various analytical steps may be used to enhance one or more of the accuracy, speed, and efficiency of the provided process. For example, in some embodiments, sequencing each of the first and second nucleic acid strands of the double-stranded nucleic acid molecule includes comparing the sequences of multiple strands derived from the first nucleic acid strand to determine a consensus sequence of the first strand, and comparing the sequences of multiple strands derived from the second nucleic acid strand to determine a consensus sequence of the second strand. In some embodiments, comparing the sequence of the first nucleic acid strand with the sequence of the second nucleic acid strand includes comparing the consensus sequence of the first strand and the consensus sequence of the second strand to provide an error-corrected consensus sequence. In other embodiments, the error-corrected sequence of the double-stranded target nucleic acid molecule can be determined by comparing a single sequence read from the first nucleic acid strand with a single sequence read from the second nucleic acid strand.

[0035] One aspect provided by some embodiments is the ability to generate high-quality sequencing information from very small amounts of nucleic acid material. In some embodiments, the provided methods and compositions may be used with starting amounts of nucleic acid material of up to about 1 picogram (pg), 10 pg, 100 pg, 1 nanogram (ng), 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng. In some embodiments, the provided methods and compositions may be used with input amounts of nucleic acid material of up to 1 molar copy or genome equivalent, 10 molar copies or genome equivalent, 100 molar copies or genome equivalent, 1,000 molar copies or genome equivalent, 10,000 molar copies or genome equivalent, 100,000 molar copies or genome equivalent, or 1,000,000 molar copies or genome equivalent. For example, in some embodiments, up to 1,000 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 10 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 pg of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 pg of nucleic acid material is initially provided for a particular sequencing process.

[0036] As used in this application, the terms "about" and "approximately" are used as equivalent terms. Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numbers used in this application, whether with or without about / approximately, are meant to encompass any normal variations understood by one of ordinary skill in the relevant art.

[0037] In various embodiments, concentration of nucleic acid material (including concentration of nucleic acid material in a region(s) of interest) is provided at a faster rate (e.g., in fewer steps), at a lower cost (e.g., utilizing fewer reagents), and to obtain more desired data. Various aspects of the present technology have many applications in preclinical and clinical testing and diagnostics, as well as other applications.

[0038] Specific details of several embodiments of the present technology are described below with reference to Figures 1-22C. While many of the embodiments are described herein with reference to double-stranded sequencing, other sequencing modes capable of generating error-corrected sequence reads and providing sequence information in addition to those described herein are within the scope of the present technology. Additionally, other nucleic acid interrogations are contemplated that would benefit from the nucleic acid enrichment methods and reagents described herein. Furthermore, other embodiments of the present technology may have different configurations, components, or procedures than those described herein. Thus, those skilled in the art will understand that the present technology may include other embodiments having additional elements and may include other embodiments that do not include some of the features shown and described below with reference to Figures 1-22C. [Brief explanation of the drawings]

[0039] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. [Figure 1] 1 is a graph plotting the relationship between nucleic acid insert size and family size obtained after amplification according to one embodiment of the present technology. [Figure 2A] 2A and 2B are schematic diagrams showing sequencing data generated for different nucleic acid insert sizes, according to embodiments of the present technology. [Figure 2B] Same as above. [Figure 3]

[0023] Figure 1 is a schematic diagram showing method steps for generating sized target fragments using CRISPR / Cas9, according to one embodiment of the present technology. Panel A shows gRNA-promoted binding of Cas9 at the target DNA site. Directed cleavage of Cas9 releases blunt-ended double-stranded target DNA fragments of known length, as shown in panel B. Panel C shows further processing steps for positive enrichment / selection of target DNA fragments via size selection. Optionally, enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing), as shown in panel D. [Figure 4] Schematic diagram showing steps of a method for generating target nucleic acid fragments of known / selected lengths using a CRISPR / Cas9 variant, according to one embodiment of the present technology. Using a CRISPR / Cas9 ribonucleoprotein complex engineered to remain bound to DNA in the appropriate state, panel A shows gRNA-facilitated binding of the variant Cas9 to a target DNA site. After cleavage, Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, while panel B shows treating the sample with an exonuclease to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. After negative / enrichment selection of target DNA fragments via exonuclease destruction of all non-target DNA, Cas9 dissociates from the DNA, releasing blunt-ended, double-stranded target DNA fragments of known length, as shown in panel C. Panel D shows optional further processing steps for positive enrichment / selection of target DNA fragments via size selection. Optionally, as shown in panel E, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing). [Figure 5]

[0023] Figure 1 is a schematic diagram showing steps of a method for generating target nucleic acid fragments of known / selected lengths using a CRISPR / Cas9 variant, according to another embodiment of the present technology. Panel A shows the use of a CRISPR / Cas9 ribonucleoprotein complex engineered to remain bound to DNA in the appropriate state, where the ribonucleoprotein complex contains a capture label. Binding of the variant Cas9 ribonucleoprotein complex containing the capture label, facilitated by a guide RNA (gRNA), is followed by cleavage of the double-stranded target DNA. After cleavage, Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, while panel B shows the treatment of the sample with an exonuclease to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. After negative / enrichment selection of target DNA fragments via exonuclease destruction of all non-target DNA, Cas9 remains bound, while panel C shows a positive enrichment / selection process for target nucleic acid capture, involving the stepwise addition of functionalized surfaces capable of binding capture labels associated with ribonucleoprotein complexes, as they remain bound to the target nucleic acid. After the affinity-based enrichment step, Cas9 dissociates from the DNA, releasing blunt-ended, double-stranded target DNA fragments of known length, as shown in panel D. Panel E shows an optional further processing step for positive enrichment / selection of target DNA fragments via size selection. Optionally, as shown in panel F, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing). [Figure 6]Figure 1 is a schematic diagram showing steps in a method for generating target nucleic acid fragments of known / selected lengths using a catalytically inactive variant of Cas9, according to one embodiment of the present technology. Using a catalytically inactive Cas9 ribonucleoprotein complex designed to target and bind to double-stranded DNA, panel A shows binding of the variant Cas9 to the target DNA site, facilitated by a gRNA. After binding, panel B shows treating the sample with an exonuclease to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. The catalytically inactive variant of Cas9 does not cleave the target DNA, but provides exonuclease resistance, such that exonuclease activity cleaves each nucleotide base until blocked by the bound Cas9 complex. After negative / enriched selection of target DNA fragments via exonuclease destruction of all non-target DNA, the catalytically inactive Cas9 dissociates from the DNA, releasing a double-stranded target DNA fragment of known length, as shown in panel C. Panel D shows optional further processing steps for positive enrichment / selection of target DNA fragments via size selection. Optionally, as shown in panel E, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing). [Figure 7]

[0023] Figure 1 is a schematic diagram showing steps in a method for generating sized target fragments using a catalytically inactive variant of Cas9, according to another embodiment of the present technology. Panel A shows the use of a catalytically inactive variant of Cas9 in a ribonucleoprotein complex engineered to remain bound to DNA in the appropriate state, where the ribonucleoprotein complex contains a capture label. Guide RNA (gRNA)-promoted binding of the catalytically inactive variant Cas9 ribonucleoprotein complex with the capture label is followed by the addition of an exonuclease to the sample to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. The catalytically inactive variant of Cas9 does not cleave the target DNA but provides exonuclease resistance, such that exonuclease activity cleaves each nucleotide base until blocked by the bound Cas9 complex. After negative / enrichment selection of target DNA fragments via exonuclease destruction of all non-target DNA, catalytically inactive Cas9 remains bound, while panel C shows a positive enrichment / selection process for target nucleic acid capture, involving the stepwise addition of functionalized surfaces capable of binding capture labels associated with ribonucleoprotein complexes, which remain bound to the target nucleic acid. After the affinity-based enrichment step, as shown in panel D, Cas9 dissociates from the DNA, releasing double-stranded target DNA fragments of known length. Panel E shows an optional further processing step for positive enrichment / selection of target DNA fragments via size selection. Optionally, as shown in panel F, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing). [Figure 8]

[0023] Figure 1 is a schematic diagram showing a target nucleic acid enrichment scheme using both catalytically active and catalytically inactive Cas9 according to another embodiment of the present technology. Both the catalytically active Cas9 ribonucleoprotein complex and the catalytically inactive Cas9 ribonucleoprotein complex can target a desired sequence in a sample. The catalytically active Cas9 ribonucleoprotein complex is directed to a region adjacent to the target DNA region and used to cleave the target double-stranded DNA, releasing blunt-ended double-stranded target DNA fragments of known length. One or more catalytically inactive ribonucleoprotein complexes bearing a capture label are directed to the target sequence region between two site-selected cleavage sites. After cleaving the target DNA and releasing the DNA fragments, the addition of a functionalized surface capable of binding the capture label associated with the catalytically inactive ribonucleoprotein complex can facilitate positive enrichment / selection of the target fragments. [Figure 9A] 9A and 9B are schematic diagrams of steps in a method for positive enrichment / selection of target nucleic acid fragments using a catalytically inactive variant of a Cas9 ribonucleoprotein complex bearing a capture label, according to one embodiment of the present technology. Fragmented double-stranded DNA fragments in a sample (e.g., mechanically sheared, acoustically fragmented, cell-free DNA, etc.) can be positively enriched / selected via target-directed binding by the catalytically inactive Cas9 ribonucleoprotein complex in solution (FIG. 9A). Stepwise addition of functionalized surfaces capable of binding capture labels associated with the ribonucleoprotein complex, which remain bound to the target nucleic acid, facilitates pull-down (e.g., affinity purification) of desired double-stranded DNA fragments while discarding non-target fragments (FIG. 9B). [Figure 9B] Same as above. [Figure 10]

[0023] Figure 1 is a schematic diagram showing steps of a method for positive enrichment / selection of target nucleic acid fragments using a catalytically inactive variant of a Cas9 ribonucleoprotein complex bearing a capture label, according to one embodiment of the present technology. Panel A shows multiple fragmented double-stranded DNA fragments of various sizes in a sample, including molecule 2, which is too small to be reliably enriched by size-selection or affinity-based methods. Panel B shows further elongation of such DNA fragments by ligating adapters to the 5' and 3' ends of the molecules in the sample. Panel C shows the positive enrichment / selection step of molecule 2 via target-directed binding by a catalytically inactive Cas9 ribonucleoprotein complex bearing a capture label in solution, followed by affinity purification by a pull-down method. [Figure 11] Schematic diagrams showing steps in a method for enriching target nucleic acid material using a negative enrichment scheme (Panel A) and a positive enrichment scheme (Panel B) according to one embodiment of the present technology. Panel A shows ligation of hairpin adapters to the 5' and 3' ends of double-stranded target DNA molecules to generate adapter-nucleic acid complexes without exposed ends. The adapter-nucleic acid complexes are treated with exonuclease in a negative enrichment / selection scheme to remove nucleic acid material fragments and adapters with unprotected 5' and 3' ends (e.g., adapter-nucleic acid complexes without four ligated phosphodiester bonds, unligated DNA, single-stranded nucleic acid material, free adapters, etc.), as shown on the right side of Panel B. Exonuclease-resistant adapter-nucleic acid complexes can be further enriched by size selection or target sequence (e.g., CRISPR / Cas9 pulldown) (Panel B, left). The desired adapter-target nucleic acid complexes can be further processed by amplification and / or sequencing. [Figure 12] FIG. 10 shows an embodiment in which a hairpin adaptor with a capture label is ligated to a target double-stranded DNA for affinity-based enrichment, according to another embodiment of the present technology. [Figure 13]FIG. 1 is a schematic diagram showing the steps of a method for positive enrichment of adaptor-target nucleic acid complexes using hairpin adaptors (Panel A), followed by rolling circle amplification (Panels B and C) and an amplicon generation step (Panel D) to generate amplicons of first and second strands of double-stranded nucleic acid fragments in substantially equal ratios, according to one embodiment of the present technology. [Figure 14]

[0023] Figure 1 is a schematic diagram showing steps of a method for using CRISPR / Cpf1 to generate target nucleic acid fragments of known / selected lengths with distinct 5' and 3' ligatable ends containing single-stranded overhang regions of known nucleotide length and sequence, according to one embodiment of the present technology. Panel A shows gRNA-promoted binding of Cpf1 at the target DNA site. Cpf1-directed cleavage generates a sticky break, providing a 4 (as shown) or 5-nucleotide overhang (e.g., "sticky end"). Site-directed Cpf1 cleavage adjacent to the target DNA sequence creates double-stranded target DNA fragments of known length (which may be enriched, for example, by size selection) with sticky end 1 at the 5' end of the fragment and sticky end 2 at the 3' end (Panel B). Panel B further shows the attachment of adaptor 1 to the 5' end of the fragment and adaptor 2 to the 3' end, where adaptor 1 and adaptor 2 contain overhang sequences at least partially complementary to sticky ends 1 and 2 of the fragment, respectively. [Figure 15] 14A-14C are schematic diagrams illustrating the steps of a method for affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments produced by the method of FIG. 14 ), according to one embodiment of the present technology. Panel A shows the stepwise addition of a functionalized surface capable of binding sticky ends that associate with cleaved target DNA fragments in solution. Upon binding to the functionalized surface, affinity interactions facilitate the pull-down (e.g., affinity purification) of desired double-stranded DNA fragments while discarding non-target fragments, as shown in panel B. [Figure 16]14A-14C are schematic diagrams illustrating steps in a method for affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments produced by the method of FIG. 14) according to another embodiment of the present technology. Panel A shows the stepwise addition of oligonucleotides bearing capture labels that have nucleotide sequences at least partially complementary to the portions of the sticky ends that associate with cleaved target DNA fragments in solution. As shown in panel B, the further addition of a functionalized surface capable of binding the capture label facilitates pull-down (e.g., affinity purification) of desired double-stranded DNA fragments while discarding non-target fragments. [Figure 17-1]

[0023] Figure 1 is a schematic diagram showing steps of a method for enriching target fragments of nucleic acid material of known length with distinct 5' and 3' ligatable ends containing long single-stranded overhang regions of known nucleotide length and sequence using Cas9 nickase, according to one embodiment of the present technology. Panel A shows gRNA-targeted binding of paired Cas9 nickase at the target DNA region. A double-stranded break is introduced by the use of paired nickases to excise the target DNA region; when paired Cas9 nickases are used, long overhangs (sticky ends 1 and 2) are generated for each cleaved end instead of blunt ends, as shown in panel B. Panel C shows the stepwise addition of a functionalized surface capable of binding the long sticky ends (e.g., sticky end 1) associated with the cleaved target DNA fragments in solution. Upon binding to the functionalized surface, the affinity interaction facilitates the pull-down (e.g., affinity purification) of the desired double-stranded DNA fragments while discarding non-target fragments, as shown in panel D. Panel E shows a variation of the positive enrichment step, involving the addition of an oligonucleotide bearing a capture label that has a nucleotide sequence at least partially complementary to a portion of the long sticky end (e.g., sticky end 1) that associates with the cleaved target DNA fragments in solution. Panel F shows the annealing of a second oligo strand that is at least partially complementary to a portion of the oligonucleotide bearing the capture label. Enzymatic extension of the second oligo strand and ligation to the template DNA fragment generates an adaptor-target DNA complex. A further step may include the introduction of a functionalized surface (not shown) capable of binding the capture label to facilitate the pull-down (e.g., affinity purification) of the desired adaptor-double-stranded DNA complex while discarding non-target fragments. [Figure 17-2] Same as above. [Figure 17-3] Same as above. [Figure 18]

[0023] Figure 1 is a schematic diagram showing a target nucleic acid enrichment scheme using catalytically inactive Cas9 according to another embodiment of the present technology. Catalytically inactive Cas9 ribonucleoprotein complexes can target desired sequences in a sample. One or more catalytically inactive ribonucleoprotein complexes carrying one or more capture labels direct other protein complex structures to the target DNA region. When this protein complex structure covers the target DNA region, it provides exonuclease resistance. After treatment with an exonuclease or a combination of an endonuclease and an exonuclease, affinity purification of the protein complex (e.g., via capture labels bound to a functionalized surface, antibody pull-down, etc.), the target nucleic acid fragments can be released from the binding of the ribonucleotide complexes. [Figure 19] Figures 19A and 19B are conceptual diagrams of prepared DNA libraries and reagents that can be used as tools for selectively interrogating DNA regions of interest, according to one embodiment of the present technology. Uniquely tagged catalytically inactive Cas9s target multiple (e.g., repeated) regions of isolated / unfragmented genomic DNA (or other large fragments of DNA) (Figure 19A). Each catalytically inactive Cas9 ribonucleoprotein contains a known oligonucleotide tag with a known sequence (e.g., a coding sequence) and binds to a predesigned region of the genome. When using a DNA library, users can stepwise add one or more probes containing the complement of the coding sequence (e.g., an anti-coding sequence) corresponding to the region of the genome of interest. Genomic DNA can be fragmented into various sizes using fragmentation methods (e.g., restriction enzyme digestion, mechanical shearing, etc.). The probes contain capture labels immobilized or incorporated into the probes (Figure 19B). The addition of functionalized surfaces capable of binding capture labels may be added for affinity purification and positive enrichment of desired genomic regions for interrogation. [Figure 20-1]

[0043] Figures 1A-1C show steps in a method for affinity-based enrichment and sequencing of target DNA fragments for use with direct digital sequencing methods, according to one embodiment of the present technology. Panel A shows the attachment of selected adapters to target DNA fragments containing sticky end(s) (e.g., target DNA fragments generated by the methods of Figures 14 or 17). Panel A further shows the attachment of adapter 1 to the 5' end of the fragment and adapter 2 to the 3' end, where adapters 1 and 2 contain overhanging sequences at least partially complementary to sticky ends 1 and 2 of the fragment, respectively. Adapter 1 has a Y-shape and includes 5' and 3' single-stranded arms bearing different labels (A and B) with distinct properties. Adapter 2 is a hairpin-shaped adapter. Panel B shows steps in a direct digital sequencing method in which label A is configured to bind to a functionalized surface. Label B provides a physical property (e.g., charge, magnetic property, etc.) that causes electroextension of the DNA fragments after denaturation of the first and second strands of the double-stranded adapter-DNA complex occurs by application of an electric or magnetic field. The first and second strands remain tethered by the hairpin adapter, such that sequence information from the enriched / targeted strand provides double-stranded sequence information for error correction and other nucleic acid interrogations (e.g., assessment of DNA damage, etc.). [Figure 20-2] Same as above. [Figure 21-1]Figure 1 shows steps of a method for affinity-based enrichment for sequencing of target DNA fragments using a direct digital sequencing method according to another embodiment of the present technology. Panel A shows affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments produced by the methods of Figure 14 or Figure 17). As shown, hairpin adapters are attached to the 3' ends of the double-stranded DNA fragments in a sequence-dependent manner. The target DNA molecule(s) may be flowed over a functionalized surface capable of binding sticky ends associated with cleaved target DNA fragments (e.g., with attached oligonucleotides). In addition, a second oligonucleotide strand containing label B and at least partially complementary to a portion of the attached oligonucleotide is added to the solution. Annealing and ligation of the adapter / DNA fragment components provides a surface-bound adapter-target double-stranded DNA complex suitable for direct digital sequencing (Panel B). Application of an electric or magnetic field and electroextension of the adapter-DNA complex for the sequencing step can be performed, for example, as described in Figure 20. [Figure 21-2] Same as above. [Figure 22A] 1 shows a nucleic acid adapter molecule for use with some embodiments of the present technology, and a double-stranded adapter nucleic acid molecule complex resulting from ligation of the adapter molecule to a double-stranded nucleic acid fragment in accordance with embodiments of the present technology. [Figure 22B-C] 22B and 22C are schematic diagrams of various double-stranded sequencing method steps according to one embodiment of the present technology.

[0040] definition In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0041] In this application, unless otherwise clear from the context, the term "a" may be understood to mean "at least one." As used in this application, the term "or" may be understood to mean "and / or." As used in this application, the terms "comprising" and "including" may be understood to encompass the listed element or step, whether stated by itself or with one or more additional elements or steps. When ranges are presented herein, their ends are inclusive. As used in this application, the term "comprise" and variations of this term, such as "comprising" and "comprises," are not intended to exclude other additives, elements, integers, or steps.

[0042] About: The term "about," when used herein in reference to a value, refers to a similar value in terms of the referenced value. Generally, one of ordinary skill in the art familiar with the context will understand the relevant degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or an even smaller range of the referenced value.

[0043] Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with the reference substance, e.g., shares a core or consensus structure, but also differs in certain distinct ways. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by the performance of a synthetic process that is substantially similar (e.g., shares multiple steps) to the synthetic process that produces the reference substance. In some embodiments, an analog is produced, or can be produced, by the performance of a synthetic process that is different from that used to produce the reference substance.

[0044] Biological sample: As used herein, the term "biological sample" or "sample" typically refers to a sample obtained from or derived from a biological source of interest (e.g., a tissue or organism or cell culture), as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In other embodiments, the source of interest includes a microorganism, such as a bacterium, a virus, a protozoan, or a fungus. In further embodiments, the source of interest may be a synthetic tissue, organism, cell culture, nucleic acid, or other material. In yet further embodiments, the source of interest may be a plant-based organism. In yet another embodiment, the sample may be an environmental sample, such as, for example, a water sample, a soil sample, an archaeological sample, or other sample collected from a non-living source. In other embodiments, the sample may be a multi-organism sample (e.g., a mixed biological sample). In some embodiments, the biological sample is or includes a biological tissue or biological fluid. In some embodiments, the biological sample may be or include bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing bodily fluid, free-floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, feces, lymphatic fluid, gynecological fluid, skin swab, vaginal swab, Pap smear, oral swab, nasal swab, stain or washing such as ductal washing or bronchopulmonary lavage, vaginal fluid, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, fetal tissue or fluid, surgical specimen, feces, other bodily fluid, secretion, and / or excretion, and / or cells therefrom, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells from the individual from whom the sample is obtained. In certain embodiments, the biological sample is a liquid biopsy obtained from a subject. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means.For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), e.g., by filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components, etc.

[0045] Capture Label: As used herein, the term "capture label" (which may also be referred to as a "capture tag," "capture moiety," "affinity label," "affinity tag," "epitope tag," "tag," "prey" moiety, or chemical group, among other names) refers to a moiety that can be incorporated into or onto a target molecule or substrate for purification purposes. In some embodiments, the capture label is selected from the group including a small molecule, a nucleic acid, a peptide, or any uniquely bindable moiety. In some embodiments, the capture label is immobilized at the 5' end of the nucleic acid molecule. In some embodiments, the capture label is immobilized at the 3' end of the nucleic acid molecule. In some embodiments, the capture label is attached to a nucleotide in the internal sequence of the nucleic acid molecule, rather than at either end. In some embodiments, the capture label is the sequence of nucleotides in the nucleic acid molecule. In some embodiments, the capture label is selected from the group consisting of biotin, biotin-deoxythymidine dT, biotin-NHS, biotin-TEG, decio-biotin-NHS, digoxigenin-NHS, DNP-TEG, thiol, among others. In some embodiments, capture labels include, but are not limited to, biotin, avidin, streptavidin, haptens recognized by antibodies, specific nucleic acid sequences, and magnetically attractable particles. In some embodiments, chemical modifications of nucleic acid molecules (such as Acridite™ modification, adenylation, azide modification, alkyne modification, I-Linker™ modification, etc.) can function as capture labels.

[0046] Cleavage site: Also called a "cleavage site" and a "nick site," this is a bond or bond pair between nucleotides in a nucleic acid molecule. In the case of a double-stranded nucleic acid molecule (e.g., double-stranded DNA), the cleavage site may involve bonds (typically phosphodiester bonds) that are immediately adjacent to each other in the double-stranded molecule, such that a "blunt" end is formed after cleavage. The cleavage site may also involve two nucleotide bonds on each single strand of the pair that are not immediately opposite each other, such that upon cleavage, a "sticky end" is left, thereby leaving a region of single-stranded nucleotides at the end of the molecule. The cleavage site can be defined by a specific nucleotide sequence that can be recognized by an enzyme (e.g., a restriction enzyme) or another endonuclease with sequence recognition capabilities, such as CRISPER / Cas9. The cleavage site may be within the recognition sequence of such an enzyme (i.e., a type 1 restriction enzyme) or may be adjacent to these enzymes by a defined nucleotide interval (i.e., a type 2 restriction enzyme). The cleavage site can also be defined by the location of a modified nucleotide that can be recognized by a specific nuclease. For example, abasic sites can be recognized and cleaved by endonuclease VII and the enzyme FPG. Uracil systems can be recognized and rendered abasic by the enzyme UDG. Ribose-containing nucleotides in other DNA sequences can be recognized and cleaved by RNAse H2 when annealed to complementary DNA sequences.

[0047] Determining: Many methodologies described herein include a "determining" step. Those skilled in the art will understand, upon reading this specification, that such "determining" can be utilized or accomplished through the use of any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, determining involves the physical manipulation of the sample. In some embodiments, determining involves the review and / or manipulation of data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0048] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) the creation of an RNA template from a DNA sequence (e.g., by transcription), (2) the processing of the RNA transcript (e.g., by splicing, editing, 5' capping and / or 3' end formation), (3) the translation of the RNA into a polypeptide or protein, and / or (4) the post-translational modification of the polypeptide or protein.

[0049] Extracting moiety: As used herein, the term "extracting moiety" (which may also be referred to as a "binding partner," "affinity partner," "bait" moiety, or chemical group, among other names) refers to an isolatable moiety or any type of molecule that allows for affinity separation of a nucleic acid bearing a capture label from a nucleic acid lacking the capture label. In some embodiments, the extracting moiety is selected from the group including a small molecule, a nucleic acid, a peptide, an antibody, or any uniquely binding moiety. The extracting moiety may be linked or linkable to a solid phase or other surface to form a functionalized surface. In some embodiments, the extracting moiety is a sequence of nucleotides linked to a surface (e.g., a solid surface, a bead, a magnetic particle, etc.). In some embodiments, the extracting moiety is selected from the group including avidin, streptavidin, an antibody, a polyhistidine tag, a FLAG tag, or any chemical modification of a surface for attachment chemistry. Non-limiting examples of these latter include azide and alkyne groups that can form 1,2,3-triazole bonds via "click" technology, or thiol-, azide-, and terminal alkynes, thiol-modified surfaces that can react covalently with acrydite-modified oligonucleotides, and aldehyde- and ketone-modified surfaces that can react to bind to I-Linker™-labeled oligonucleotides.

[0050] Functionalized Surface: As used herein, the term "functionalized surface" refers to a solid surface, bead, or other fixed surface capable of binding or immobilizing a capture label. In some embodiments, the functionalized surface comprises an extraction moiety capable of binding a capture label. In some embodiments, the extraction moiety is directly linked to a surface. In some embodiments, a chemical modification of the surface serves as the extraction moiety. In some embodiments, the functionalized surface may include controlled pore glass (CPG), magnetic porous glass (MPG), among other glass or non-glass surfaces. Chemical functionalization may involve ketone, aldehyde, thiol, azide, and alkyne modifications, among others. In some embodiments, the functionalized surface and oligonucleotides used in adapter synthesis are linked using one or more of the following groups of immobilization chemistries: amide, alkylamine, thiourea, diazo, and hydrazine bonds, among other surface chemistries. In some embodiments, the functionalized surface and oligonucleotides used in adapter synthesis are ligated using one or more of a group of reagents including EDAC, NHS, sodium periodate, glutaraldehyde, pyridyl disulfide, nitric acid, biotin, among other ligation reagents.

[0051] gRNA: As used herein, "gRNA" or "guide RNA" refers to a short RNA molecule that includes a scaffold sequence suitable for binding a targeting endonuclease (such as a Cas enzyme (e.g., Cas9 or Cpfl) or another ribonucleoprotein with similar properties) to a substantially target-specific sequence that facilitates cleavage of a specific region of DNA or RNA.

[0052] Nucleic acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone and are considered within the scope of the present technology. Alternatively, or in addition, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose, or locked nucleic acids) compared to sugars commonly present in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid may be a non-protein-coding RNA product, such as a microRNA, ribosomal RNA, or CRISPER / Cas9 guide RNA. In some embodiments, the nucleic acid serves a regulatory purpose in a genome. In some embodiments, the nucleic acid does not originate from a genome. In some embodiments, the nucleic acid comprises an intergenic sequence. In some embodiments, the nucleic acid is derived from an extrachromosomal element or a non-nuclear genome (such as mitochondria or chloroplasts). In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.In some embodiments, the nucleic acids are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 residues in length, or longer. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a polypeptide or includes at least one element that is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity. In some embodiments, the nucleic acid performs a mechanical function, for example, in a ribonucleic acid protein complex or transfer RNA. In some embodiments, the nucleic acid functions as an aptamer. In some embodiments, the nucleic acid may be used for data storage. In some embodiments, the nucleic acid may be chemically synthesized in vitro.

[0053] Reference: As used herein, describes a standard or control relative to which a comparison is made. For example, in some embodiments, an agent, animal, individual, collection, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, collection, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify reliance on and / or comparison to a particular possible reference or control.

[0054] Single Molecular Identifier (SMI): As used herein, the term "single molecular identifier" or "SMI" (which may also be referred to as a "tag," "barcode," "molecular barcode," "unique molecular identifier" or "UMI") refers to any substance (e.g., a nucleotide sequence, nucleic acid molecular feature) capable of distinguishing an individual molecule within a larger, heterogeneous collection of molecules. In some embodiments, the SMI may be or include an exogenously applied SMI. In some embodiments, the exogenously applied SMI may be or include a degenerate or semi-degenerate sequence. In some embodiments, a substantially degenerate SMI may be known as a random unique molecular identifier (R-UMI). In some embodiments, the SMI may include a code (e.g., a nucleic acid sequence) from within a pool of known codes. In some embodiments, a predetermined SMI code is known as a defined unique molecular identifier (D-UMI). In some embodiments, the SMI may be or include an endogenous SMI. In some embodiments, endogenous SMI may be or include information related to specific shear points of a target sequence or features associated with the ends of individual molecules comprising the target sequence. In some embodiments, SMI may be related to sequence variations in nucleic acid molecules caused by random or semi-random damage, chemical, enzymatic, or other modifications to the nucleic acid molecule. In some embodiments, the modification may be deamination of methylcytosine. In some embodiments, the modification may involve the site of a nucleic acid nick. In some embodiments, SMI may include both exogenous and endogenous elements. In some embodiments, SMI may include physically adjacent SMI elements. In some embodiments, SMI elements may be spatially distinct within the molecule. In some embodiments, SMI may be non-nucleic acid. In some embodiments, SMI may include two or more different types of SMI information.Various embodiments of SMIs are further disclosed in International Patent Publication No. WO2017 / 100441, which is incorporated herein by reference in its entirety.

[0055] Strand Defining Element (SDE): As used herein, the term "strand defining element" or "SDE" refers to any material that allows for the identification of a particular strand of double-stranded nucleic acid material and therefore its differentiation from other / complementary strands (e.g., any material that allows the amplification products of each of two single-stranded nucleic acids obtained from a target double-stranded nucleic acid to be substantially distinguishable from one another after sequencing or other nucleic acid interrogation). In some embodiments, an SDE may be or include one or more segments of substantially non-complementary sequence in an adapter sequence. In certain embodiments, the segment of substantially non-complementary sequence in an adapter sequence may be provided by an adapter molecule comprising a Y-shape or "loop" shape. In other embodiments, the segment of substantially non-complementary sequence in an adapter sequence may form an unpaired "bubble" in the middle of adjacent complementary sequences in the adapter sequence. In other embodiments, an SDE may incorporate a nucleic acid modification. In some embodiments, an SDE may include a physical separation into a reaction compartment physically separate from the paired strands. In some embodiments, an SDE may include a chemical modification. In some embodiments, the SDE may comprise a modified nucleic acid. In some embodiments, the SDE may be associated with sequence variations in a nucleic acid molecule caused by random or semi-random damage, chemical modification, enzymatic modification, or other modification to the nucleic acid molecule. In some embodiments, the modification may be deamination of methylcytosine. In some embodiments, the modification may involve the formation of a nucleic acid nick. Various embodiments of SDEs are further disclosed in International Patent Publication No. WO2017 / 100441, the entire contents of which are incorporated herein by reference.

[0056] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including, in some embodiments, prenatal human forms). In some embodiments, the subject is afflicted with a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is someone who possesses one or more characteristics characteristic of susceptibility to, or risk for, a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or after diagnosis and / or treatment is to be performed.

[0057] Substantially: As used herein, the term "substantially" refers to a qualitative state indicating the total or nearly total extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go completely to completion and / or move toward complete completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potentially never-completeness inherent in many biological and chemical phenomena. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present technology generally relates to methods for concentrating nucleic acid material for sequencing applications and other nucleic acid material interrogation, as well as related reagents for use in such methods. Some embodiments of the present technology are directed to enriching one or more regions of interest within nucleic acid material for sequencing applications, such as double-stranded sequencing and other sequencing applications, to achieve highly accurate sequencing reads. For example, various embodiments of the present technology include selectively enriching nucleic acid material (e.g., genomic DNA material) for a region of interest and performing double-stranded sequencing to provide error-corrected sequence reads of the enriched nucleic acid material. Further examples of the present technology are directed to methods of performing double-stranded sequencing or other sequencing methods (e.g., single-consensus sequencing, Hyb&Seq™ sequencing, nanopore sequencing, etc.) on nucleic acid material enriched for a region of interest. In various embodiments, enrichment of nucleic acid material (including enrichment of nucleic acid material in a region(s) of interest) is provided at a faster rate (e.g., with fewer steps), at a reduced cost (e.g., utilizing fewer reagents), and to obtain more desired data. Various aspects of the present technology have many applications in preclinical and clinical testing and diagnostics, as well as other applications.

[0059] Double-stranded sequencing (DS) is a method for generating error-corrected nucleic acid sequences from double-stranded nucleic acid molecules.In a specific embodiment of this technology, DS can be used to independently sequence both strands of individual nucleic acid molecules in such a way that derivative sequence reads can not only be recognized as coming from the same double-stranded nucleic acid parent molecule during massively parallel sequencing, but also be distinguished from each other as distinct entities after sequencing.The sequence reads obtained from each strand are then compared for the purpose of obtaining the error-corrected sequence of the original double-stranded nucleic acid molecule, known as double-stranded consensus sequence.The DS process allows to determine whether one or both strands of the original double-stranded nucleic acid molecule appear in the generated sequencing data used to form double-stranded consensus sequence.

[0060] Standard next-generation sequencing has an error rate of approximately 1 / 100 to 1 / 1000. If fewer than 1 / 100 to 1 / 1000 of molecules contain a sequence variant, its presence is obscured by the background error rate of the sequencing process. DS, on the other hand, can accurately detect extremely low-frequency variants due to the high degree of error correction it provides. The high degree of error correction provided by DS's strand comparison technology reduces sequencing errors of double-stranded nucleic acid molecules by several orders of magnitude compared to standard next-generation sequencing methods. This error reduction improves sequencing accuracy for almost all types of sequences, but may be particularly well-suited for biochemically demanding sequences that are well known in the art to be error-prone, or when the molecular population being sequenced is heterogeneous (i.e., a small subset of molecules contains a sequence variant that the rest of the molecules do not possess). A non-limiting example of such a type of sequence is a homopolymer or other microsatellite / short tandem repeat. Another non-limiting example of an error-prone sequence that could benefit from DS error correction is a molecule damaged by, for example, heat, radiation, mechanical stress, or various chemical exposures that generate error-prone chemical adducts during copying by one or more nucleotide polymerases, and also generate single-stranded DNA at the ends of the molecule or as nicks and gaps. In forensic applications where highly damaged DNA (oxidation, deamination, etc.) occurs due to the fixation process (i.e., FFPE in clinical pathology), or ancient DNA, or where the material has been exposed to harsh chemicals or environments, double-stranded sequencing is particularly useful for reducing the high error levels that result from such damage.

[0061] In a further embodiment, DS can also be used to accurately detect minor sequence variants within a collection of double-stranded nucleic acid molecules. A non-limiting example of this application is the detection of a minority of cancer-derived DNA molecules among a large number of non-mutated molecules from non-cancerous tissues within a subject. DS is also well suited for accurate genotyping of difficult-to-sequence regions of the genome (homopolymers, microsatellites, guanine quadruplets, etc.), where the error rate of standard sequencing is particularly high. Another non-limiting application for rare variant detection by DS is the early detection of DNA damage resulting from exposure to genotoxic agents. A further non-limiting application of DS is the detection of mutations generated by genotoxic or non-genotoxic carcinogens by focusing on gene clones that emerge with driver mutations. Yet another non-limiting application for accurate detection of minor sequence variants is the creation of mutagenic signatures associated with genotoxic agents. Further non-limiting examples of the usefulness of DS can be found in Salk et al., Nature Reviews Genetics 2018, PMID 29576615, which is incorporated herein by reference in its entirety.

[0062] Various embodiments relating to the enrichment of nucleic acid material for sequencing applications and other nucleic acid material interrogation are useful in single-molecule sequencing and direct digital sequencing methods. In some embodiments, techniques using single-molecule hybridization with barcoded probes may be used to characterize and / or quantify genomic regions. Generally, such techniques use molecular "barcodes" and single-molecule imaging to detect and count specific nucleic acid targets in a single reaction without amplification. Typically, each color-coded barcode binds to a probe specific to a single target corresponding to a genomic region of interest. When mixed with a control, a multiplexed code set is formed. In some embodiments, two probes are used to hybridize each individual target nucleic acid. In a specific configuration, a reporter probe carries a signal, and a capture probe allows the complex to be immobilized for data collection. After hybridization, excess probe is removed, and the immobilized probe / target complex may be analyzed by a digital analyzer for data collection. The color codes are counted and tabulated for each target molecule (e.g., genomic region of interest). Suitable digital analyzers include the nCounter® Analysis System (NanoString™ Technologies; Seattle, WA). Methods and reagents, including molecular "barcodes," and equipment suitable for NanoString™ technology are further described, for example, in U.S. Patent Publication Nos. 2010 / 0112710, 2010 / 0047924, and 2010 / 0015607, the contents of each of which are incorporated herein by reference in their entireties.

[0063] Direct digital sequencing (DDS) technology includes methods for simultaneously capturing and directly sequencing DNA and RNA for various research, diagnostic, and other applications, providing highly accurate single-molecule sequencing. DDS provides both short and long sequencing reads without library creation or amplification steps, and is described, for example, in International Patent Publication No. WO2016 / 081740, incorporated herein by reference. Generally, direct sequencing of nucleic acid targets is achieved by hybridizing fluorescent molecular barcodes to natural nucleic acid targets. As further described in U.S. Patent No. 7,919,237 and available from NanoString™ Technologies, Inc. (Seattle, WA), oligomers, which are extensions of target nucleotide sequences, are extended by an electrical extension technique that spatially separates monomers, each of which is connected to a unique label. Thus, the pattern of labeled monomers can be used to identify the barcode on the oligomer tag.

[0064] In addition, various embodiments relating to the enrichment of nucleic acid materials are useful in other forms of characterization and / or quantification of nucleic acid materials, and are known in the art. For example, characterization of nucleic acid materials to determine the presence or absence of genomic mutations, DNA variants, quantification of DNA or RNA copy numbers, and other applications can benefit from selective enrichment of target nucleic acid materials as provided herein. Some exemplary methodologies include, but are not limited to, single molecule sequencing (e.g., single molecule real-time sequencing, nanopore sequencing, high-throughput sequencing or next-generation sequencing (NGS)), digital PCR, bridge PCR, emulsion PCR, and semiconductor sequencing, among others. Those skilled in the art will recognize other nucleic acid interrogation methods and techniques that can be appropriately used to examine and / or benefit from enriched nucleic acid materials.

[0065] Methods incorporating DS and other sequencing modalities may involve ligating one or more sequencing adapters to a target double-stranded nucleic acid molecule to generate a double-stranded target nucleic acid complex. Such adapter molecules may contain one or more of a variety of features suitable for MPS platforms, such as a sequencing primer recognition site, an amplification primer recognition site, a barcode (e.g., a single molecule identifier (SMI) sequence), an index sequence, a single-stranded portion, a double-stranded portion, or a strand-discriminating element or feature. The use of highly pure sequencing adapters for DS or any next-generation sequencing technology is important for obtaining high-quality, reproducible data and maximizing a sample's sequence yield (i.e., the relative proportion of input molecules that are converted into independent sequence reads). This is particularly important for DS, due to the need to successfully recover both strands of the original double-stranded molecule.

[0066] Regarding the efficiency of the DS process or other high-precision sequencing methods, this specification further describes two types of efficiency: conversion efficiency and workflow efficiency.For the purpose of discussing DS efficiency, conversion efficiency can be defined as the fraction of unique nucleic acid molecules input into a sequencing library preparation reaction that produces at least one double-stranded consensus sequence read.Workflow efficiency can be related to the amount of time required to perform these steps to generate a double-stranded sequencing library and / or perform targeted enrichment for the sequence of interest, the relative number of steps, and / or the relative inefficiency of the procurement costs of reagents / materials.

[0067] In some cases, limitations in either or both conversion efficiency and workflow efficiency may limit the usefulness of high-precision DS for some applications that would otherwise be very well suited. For example, low conversion efficiency may result in a situation where the copy number of the target double-stranded nucleic acid is limited, which may result in less than desired sequence information being generated. Non-limiting examples of this concept include DNA from circulating tumor cells or cell-free DNA from tumors, or prenatal infants that have shed DNA in body fluids such as plasma and mixed with excess DNA from other tissues. DS typically has an accuracy that can separate one mutant molecule among more than 100,000 unmutated molecules. However, for example, if there are only 10,000 available molecules in a sample, even if the ideal efficiency of converting them into double-stranded consensus sequence reads is 100%, the lowest measurable mutation frequency will be 1 / (10,000*100%) = 1 / 10,000. For clinical diagnostics, it may be important to have maximum sensitivity for detecting low-level signals of cancer- or treatment-related mutations, so a relatively low conversion efficiency may be undesirable in this context. Similarly, in forensic applications, there is often very little DNA available for testing. When only nanogram or picogram amounts can be recovered from crime scenes or natural disaster sites, or when DNA from multiple individuals is mixed, it may be important to have maximum conversion efficiency in terms of being able to detect the presence of DNA from all individuals in the mixture.

[0068] In some cases, workflow inefficiencies can similarly pose challenges for certain nucleic acid interrogation applications. One non-limiting example of this is clinical microbiology testing. Sometimes, it is desirable to rapidly detect the nature of a microbial or polymicrobial bloodstream infection, where one or more infectious organisms, e.g., some organisms, are resistant to a particular antibiotic based on unique genetic variants they possess. However, the time required to culture and empirically determine the antibiotic sensitivity of the infectious organisms is much longer than the time required to make a therapeutic decision about the antibiotic to be used for treatment. DNA sequencing of DNA from blood (or other infected tissues or bodily fluids) has the potential to be even more rapid, and DS, among other high-precision sequencing methods, can highly accurately detect, for example, minor therapeutically important variants in infectious populations based on DNA signatures. Because workflow turnaround time to data generation can be critical for determining treatment options (e.g., as in the example used herein), applications that increase the speed at which data output is achieved may also be desirable.

[0069] Further disclosed herein are methods and compositions for target nucleic acid sequence enrichment for various nucleic acid material interrogation applications. In particular, some aspects of the present technology are directed to methods and compositions for target nucleic acid material enrichment and the use of such enrichment for error-corrected nucleic acid sequencing applications, which offer improvements in cost, conversion of molecules to be sequenced, and time efficiency of creating labeled molecules for targeted ultra-high precision sequencing.

[0070] I. SELECTED EMBODIMENTS OF METHODS AND REAGENTS FOR THE CONCENTRATION OF NUCLEIC In some embodiments, provided methods provide targeted enrichment strategies that are compatible with the use of molecular barcodes for error correction. Other embodiments provide methods for non-amplification-based targeted enrichment strategies that are compatible with DDS and other sequencing strategies that do not use molecular barcoding (e.g., single-molecule sequencing modes and interrogation).

[0071] In some embodiments, it is advantageous to treat nucleic acid material to improve the efficiency, accuracy, and / or speed of the sequencing process. According to a further aspect of the present technology, for example, the efficiency of DS can be increased by fragmenting the target nucleic acid. Typically, fragmentation of nucleic acids (e.g., genomes, mitochondria, plasmids, etc.) is achieved by either physical shearing (e.g., sonication) or relatively non-sequence-specific enzymatic techniques that utilize enzyme cocktails to cleave DNA phosphodiester bonds. The result of any of the above methods is a sample in which intact nucleic acid material (e.g., genomic DNA (gDNA)) has been reduced to a mixture of randomly or semi-randomly sized nucleic acid fragments. While effective, these techniques can create nucleic acid fragments of various sizes, leading to amplification bias (e.g., short fragments tend to PCR amplify more efficiently than long fragments and may more easily be clustered during polony formation) and uneven sequencing depth. For example, Figure 1 is a graph plotting the relationship between nucleic acid insert size and family size obtained after amplification of a collection of DNA molecules tagged with diverse molecular barcodes during library preparation. As shown in Figure 1, short fragments tend to amplify preferentially, so that on average, many copies of each of these short fragments are generated and sequenced, providing uneven levels of sequencing depth for these regions.

[0072] Furthermore, when using long fragments, the portion of DNA between the limits of a sequencing read (or between the ends of a paired-end sequencing read) cannot be interrogated if it extends beyond the maximum read length of the sequencing platform, resulting in "dark" fragments despite being successfully ligated, amplified, and captured (Figure 2A). Similarly, when using short fragments, overlapping reads provide redundant information when covering the same sequence in the center of the molecule from both reads when using paired-end sequencing, making it cost-inefficient (Figure 2B). Random or semi-random nucleic acid fragmentation can also result in unpredictable breakpoints within the target molecule, resulting in fragments that may not be complementary or may have reduced complementarity to the bait strand for hybrid capture, thereby reducing target capture efficiency. Random or semi-random fragmentation can destroy the sequence of interest and / or result in very small or very large fragments that are lost during other stages of library preparation, reducing data yield and efficiency.

[0073] Another problem with many random fragmentation methods (especially mechanical or acoustic methods) is that they induce damage beyond double-strand breaks, potentially rendering some double-stranded DNA no longer double-stranded. For example, mechanical shearing can generate 3' or 5' overhangs at the ends of molecules and single-stranded nicks or gaps in the middle of the molecule. Artificially re-double-stranding these single-stranded portions using, for example, a cocktail of "end repair" enzymes, can result in artificial error sources (e.g., "false double-stranded molecules" as described herein). In many embodiments, it is optimal to maximize the amount of double-stranded nucleic acid of interest that remains in its natural double-stranded form during handling. In addition, the high energy involved in many random or semi-random mechanical fragmentation methods increases the amount of DNA damage (e.g., oxidation, deamination, or other adduct formation that can be mutagenic or inhibitory during amplification or sequencing, introducing artificial base calls or signal reductions). Some random or semi-random enzymatic fragmentation methods may similarly leave mutagenic or blocking "scars" at partial cleavage sites.

[0074] In addition, for DS processing, both strands of the original target nucleic acid molecule must be successfully ligated. For example, in embodiments in which adapters are ligated to both the 5' and 3' ends of the molecule, four phosphodiester bonds must be successfully formed. If one of these bonds is not formed, it will be impossible to amplify and sequence both strands of the molecule. As mentioned above, for example, failure to form the necessary bonds can occur for several reasons, including, inter alia, damage to the ends of the target double-stranded nucleic acid molecule, incomplete end repair or tail repair of the library fragments, incomplete synthesis or damaged adapter molecules, and contamination of the ligation or subsequent reaction with, for example, undesirable enzymatic activity (e.g., exonuclease activity that can destroy the ligatable ends of the adapter or library fragments, or denaturation of the ligation enzyme that renders its catalytic activity inefficient in multiple dimensions). Damage to the ends of library fragments may be particularly common with high-energy sonication or other mechanical DNA fragmentation.

[0075] In addition to successful adapter ligation, both the first and second strands of the adapter-target nucleic acid complex must be amplifiable to achieve double-stranded sequence accuracy. For example, if a specific strand of the target nucleic acid molecule is nicked or damaged in a way that the polymerase cannot cross, amplification of this specific strand will not occur and double-stranded consensus sequence reads will not be generated. Non-limiting examples of uncrossable damage can be introduced by ultrasonic DNA fragmentation, high temperature or long-term enzymatic processes, or single-strand nicking activity during library preparation.

[0076] Therefore, DS, among other applications, would benefit from improved efficiency by utilizing one or more methods for concentrating target nucleic acids in a sample, including concentrating target nucleic acid material prior to the amplification step. Regardless of the underlying method, detection of rare nucleic acid variants requires screening a large number of molecules. However, the more molecules (i.e., genome equivalents) that are simultaneously prepared in a library, the lower the relative efficiency of the process.

[0077] Various aspects of the present technology provide methods, reagents, nucleic acid libraries, and kits for enriching nucleic acid material for sequencing applications and other nucleic acid interrogation. Further aspects of the present technology provide multiple solutions for improving both the conversion efficiency and workflow efficiency of DS and other sequencing modalities to overcome most of the limitations listed above.

[0078] Some embodiments of the present technology are directed to methods for enriching a region(s) of interest using a clustered regularly interspaced short palindromic repeats (CRISPR) programmable endonuclease system. In other embodiments, CRISPER-like or other programmable endonucleases, such as zinc finger nucleases, TALEN nucleases, or other sequence-specific endonucleases, such as homing endonucleases or simple restriction nucleases, or derivatives thereof, can be used alone or in combination as part of the disclosed technology.

[0079] In particular, CRISPR / Cas9 (or other programmable or non-programmable endonucleases, or combinations thereof) can be used to selectively cleave a nucleic acid backbone at one or more defined or semi-defined regions, functionally cleaving one or more sequence regions of interest and functionally cleaving from this region longer nucleic acid molecules designed so that the cleaved target region(s) are one or more predetermined lengths or substantially predetermined lengths, thereby enabling enrichment of one or more nucleic acid target regions of interest via size selection prior to library preparation for sequencing applications such as DS. In other embodiments, CRISPR / Cas9 (or other programmable or non-programmable endonucleases, or combinations thereof) can be used to selectively cleave one or more sequence regions of interest, designed so that the cleaved target region(s) have substantially predetermined lengths and overhanging sequences. These programmable endonucleases can be used alone or in combination with other forms of target nucleases, such as restriction endonucleases, or other enzymatic or non-enzymatic methods for cleaving nucleic acids.

[0080] In some embodiments, the provided methods may include providing a nucleic acid material, cleaving the nucleic acid material with a targeting endonuclease (e.g., a ribonucleic acid protein complex) such that one or more target regions of substantially predetermined lengths are separated or enriched from the remainder of the nucleic acid material, and analyzing the cleaved target regions. In other embodiments, the one or more cleaved regions may be negatively enriched (i.e., depleted) from the remainder of the nucleic acid material and not analyzed. In some embodiments, the provided methods may further include ligating at least one SMI and / or adapter sequence to at least one of the 5' or 3' ends of the cleaved target regions of predetermined lengths. In some embodiments, analyzing may be or include quantification and / or sequencing.

[0081] In some embodiments, quantification may be or include spectrophotometry, real-time PCR, and / or fluorescence-based quantification (e.g., using fluorescent dye tagging). In some embodiments, sequencing may be or include Sanger sequencing, shotgun sequencing, bridge PCR, nanopore sequencing, single-molecule real-time sequencing, ion torrent sequencing, pyrosequencing, digital sequencing (e.g., digital barcode-based sequencing), sequencing by ligation, polony-based sequencing, current-based sequencing (e.g., tunneling current), mass spectrometry sequencing, microfluidics-based sequencing, Illumina sequencing, next-generation sequencing, massively parallel, and any combination thereof.

[0082] In some embodiments, the targeting endonuclease is or includes at least one of a CRISPR-associated (Cas) enzyme (e.g., Cas9 or Cpfl) or other ribonucleoprotein complex, a homing endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute nuclease, a megaTAL nuclease, a meganuclease, and / or a restriction endonuclease. In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting endonucleases may be used. In some embodiments, the targeting nuclease may be used to cleave two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) potential target regions of a predetermined length. In some embodiments where two or more target regions of a predetermined length are present, each target region may be of the same (or substantially the same) length. In some embodiments where there are two or more target regions of a predetermined length, at least two of the target regions of a predetermined length are different in length (e.g., a first target region having a length of 100 bp and a second target region having a length of 1,000 bp).

[0083] The present disclosure provides, inter alia, methods and reagents for affinity-based enrichment of target nucleic acid material. In some embodiments, including such methods, one or more capture labels or moieties may be used to enrich / select desired target nucleic acid material from a sample containing genomic material, non-target nucleic acid material, contaminating nucleic acid material, nucleic acid material from a mixed sample, cfDNA material, etc. For example, some embodiments include the use of one or more capture labels / moieties for positive enrichment / selection of desired target nucleic acid material (e.g., a fragment containing a target sequence or genomic region of interest, a target genomic region of interest in unfragmented genomic DNA). In other embodiments, the capture labels may be used for negative enrichment / selection to remove or reduce the amount of undesired genomic material.

[0084] For example, in some embodiments involving positive enrichment, the adapter oligonucleotide may have a capture label that is or includes an immobilized chemical moiety (e.g., biotin) that can be used to isolate or separate the desired adapter-nucleic acid complex via capture in one or more subsequent purification steps, e.g., via an extracting moiety (e.g., streptavidin) bound to a functionalized surface (e.g., paramagnetic beads or other forms of beads). In some embodiments involving negative enrichment, the capture label that is or includes an immobilized chemical moiety (e.g., biotin) may be used to purify away or separate undesired genomic material (e.g., non-target nucleic acid fragments, etc.) ligated to or attached to the adapter (or other probe containing the capture label), e.g., via capture in one or more subsequent purification steps, e.g., via an extracting moiety (e.g., streptavidin) bound to a functionalized surface (e.g., paramagnetic beads or other forms of beads).

[0085] Size-based enrichment of nucleic acid material In some embodiments, the provided methods and compositions utilize targeting endonucleases (e.g., ribonucleoprotein complexes (CRISPR-associated endonucleases (e.g., Cas9, Cpfl), homing endonucleases, zinc finger nucleases, TALENs, Argonaute nucleases, meganucleases, restriction endonucleases and / or meganucleases (e.g., megaTAL nucleases, etc.), or combinations thereof) or other technologies (e.g., one or more restriction enzymes) that can cleave nucleic acid material to cleave a target sequence of interest into optimal fragment sizes for sequencing. In some embodiments, the targeting endonuclease has the ability to specifically and selectively cleave a precise sequence region of interest. For example, a programmable endonuclease (e.g., CRISPR-associated (C) nuclease) that generates fragments of predetermined, substantially uniform sizes is utilized. By preselecting the cleavage site using a (as) enzyme / guide RNA complex, bias and the presence of uninformative reads can be dramatically reduced. Furthermore, due to the size difference between the cleaved fragments and the remaining uncleaved DNA, a size selection step (described further below) can be performed to remove large off-target regions, thereby pre-concentrating the sample before any further processing steps. The need for end-repair steps may also be reduced or eliminated, thereby saving time, reducing the risk of false double-strand problems, and in some cases reducing or eliminating the need for computational trimming of data near the ends of the molecules, thereby improving efficiency. Thus, an additional advantage of targeted enzymatic fragmentation is the potential to reduce nicks or nucleic acid adducts or other forms of damage caused by mechanical fragmentation methods.

[0086] A method called CRISPR-DS allows for extremely high label enrichment (potentially reducing the need for a subsequent hybrid capture step), significantly reducing time and cost, as well as increasing conversion efficiency. Figure 3 is a schematic diagram illustrating method steps for generating sized target fragments using CRISPR / Cas9, according to various embodiments of the present technology. For example, CRISPR / Cas9 may be used to cleave at one or more specific sites (e.g., protospacer adjacent motif or "PAM" sites) in a target sequence (Figure 3, panel A) through binding facilitated by the Cas9 gRNA. Cas9-directed cleavage releases blunt-ended, double-stranded target DNA fragments of known length, as shown in panel B. Panel C of Figure 3 illustrates further processing steps for positive enrichment / selection of target DNA fragments via size selection. One method for isolating the cleaved target portion involves using SPRI / Ampure beads and magnetic purification to remove high-molecular-weight DNA while retaining short fragments of interest. In other embodiments, cleaved segments of a predetermined length can be separated from undesired DNA fragments and other high-molecular-weight genomic DNA (if applicable) using various size selection methods, including, but not limited to, electrophoresis, gel purification, liquid chromatography, size exclusion purification, and / or filtration purification methods. After size selection, the CRISPR-DS method may include steps consistent with those of the DS method, including A-tailing (which leaves blunt ends after CRISPR / Cas9 cleavage), adapter (e.g., DS adapter) ligation, double-strand amplification, optional capture steps, and amplification (e.g., PCR), before sequencing each strand and creating a double-stranded consensus sequence. In addition to improving workflow efficiency, CRISPR-based size selection / target enrichment provides optimal fragment lengths for highly efficient amplification and sequencing. Aspects of CRISPR-DS are disclosed in International Patent Publication No. WO / 2018 / 175997, the entire contents of which are incorporated herein by reference.

[0087] In certain embodiments, CRISPR-DS solves several common problems associated with NGS, including, for example, inefficient target enrichment (which can be optimized by CRISPR-based size selection), sequencing errors (which can be removed using DS methodology to create error-corrected double-stranded consensus sequences), and uneven fragment sizes (which are mitigated by pre-designed CRISPR / Cas9 fragmentation). As will be appreciated by those skilled in the art, as described herein, CRISPR-DS can have applications for sensitive identification of mutations in situations where samples are limited to DNA (e.g., particularly in forensic and early cancer detection applications).

[0088] In vitro digestion of DNA material with Cas9 nuclease relies on the formation of a ribonucleoprotein complex that recognizes and cleaves predetermined sites (e.g., PAM sites, panel A of Figure 3). This complex is formed using a guide RNA ("gRNA," e.g., crRNA + tracrRNA) and Cas9. For multiple cleavages, the gRNA can be complexed by pooling all crRNAs and then complexing with the tracrRNA, or by complexing the crRNA and tracrRNA separately and then pooling them. In some embodiments, the second option may be preferred to eliminate competition between crRNAs. Other CRISPER systems using different Cas proteins may rely on different or no PAM motif sequences, or may rely on other forms of nucleic acid sequences to direct nuclease delivery to the target nucleic acid region.

[0089] In some embodiments, the nucleic acid agent comprises nucleic acid molecules of substantially uniform length, which in some embodiments is from about 1 to about 1,000,000 bases. For example, in some embodiments, the substantially uniform length may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, or 50,000 bases in length. In some embodiments, the substantially uniform length may be up to 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 bases. As a specific, non-limiting example, in some embodiments, the substantially uniform length is about 100 to about 500 bases. In some embodiments, a size selection step (e.g., as described herein) may be performed before any particular amplification step. In some embodiments, a size selection step (e.g., as described herein) may be performed after any particular amplification step. In some embodiments, a size selection step (e.g., as described herein) may be followed by additional steps, such as a digestion step and / or another size selection step. In some embodiments, size selection may be performed before or after the adapter ligation step. In some embodiments, size selection may be performed simultaneously with the cleaving step. In some embodiments, size selection may be performed after the cleaving step.

[0090] In addition to the use of targeting endonuclease(s), any other application-appropriate method(s) for achieving substantially uniform length nucleic acid molecules may be used. By way of non-limiting example, such methods may be or include the use of one or more of agarose or other gels, gel electrophoresis, affinity columns, HPLC, PAGE, filtration, gel filtration, exchange chromatography, SPRI / Ampure-type beads, or any other suitable method recognized by those of skill in the art.

[0091] In some embodiments, processing nucleic acid material to generate nucleic acid molecules of substantially uniform length (or mass) may be used to recover one or more desired target regions (e.g., target sequences of interest) from a sample. In some embodiments, processing nucleic acid material to generate nucleic acid molecules of substantially uniform length (or mass) may be used to exclude certain portions of a sample (e.g., nucleic acid material from undesired species or undesired subjects of the same species). In some embodiments, nucleic acid material may be present in a variety of sizes (e.g., not substantially uniform length or mass).

[0092] In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) targeting endonucleases or other methods for providing nucleic acid molecules of substantially uniform length may be used. In some embodiments, a targeting nuclease may be used to cleave two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) potential target regions of a nucleic acid material. In some embodiments where two or more target regions of a nucleic acid material are present, each target region may be of the same (or substantially the same) length. In some embodiments where two or more target regions of a nucleic acid material are present, at least two of the target regions having known lengths are different in length (e.g., a first target region having a length of 100 bp and a second target region having a length of 1,000 bp).

[0093] In some embodiments, multiple targeting endonucleases (e.g., programmable endonucleases) may be used in combination to fragment multiple regions of a target nucleic acid of interest. In some embodiments, one or more programmable targeting endonucleases may be used in combination with other targeting nucleases. In some embodiments, one or more targeting endonucleases may be used in combination with random or semi-random nucleases. In some embodiments, one or more targeting endonucleases may be used in combination with other random or semi-random methods of nucleic acid fragmentation, such as mechanical or acoustic shearing. In some embodiments, it may be advantageous to perform cleavage in sequential steps with one or more intervening size selection steps. In some embodiments where targeted fragmentation is used in combination with random or semi-random fragmentation, the random or semi-random nature of the latter may be useful for purposes of unique molecular identifier (UMI) sequences. In some embodiments where targeted fragmentation is used in combination with random or semi-random fragmentation, the random or semi-random nature of the latter may be useful to facilitate sequencing of regions of the nucleic acid that are not easily cleaved in a targeted manner, such as one or more long or highly repetitive regions that have significant similarity to other regions in one or more genomes that may otherwise be difficult to enrich by conventional methods of hybrid capture.

[0094] Targeted Endonuclease Targeting endonucleases (e.g., CRISPR-associated ribonucleoprotein complexes, e.g., Cas9 or Cpfl, homing nucleases, zinc finger nucleases, TALENs, megaTAL nucleases, Argonaute nucleases, and / or derivatives thereof) can be used to selectively cleave and excise target portions of nucleic acid material for the purpose of enriching such target portions for sequencing applications. In some embodiments, the targeting endonuclease may be modified (e.g., possessing amino acid substitutions) to provide, for example, improved thermostability, salt tolerance, and / or pH tolerance, or improved specificity or alternative PAM site recognition, or higher binding affinity. In other embodiments, the targeting endonuclease may be biotinylated, fused to streptavidin, and / or incorporate other affinity-based (e.g., bait / prey) technologies. In certain embodiments, the targeting endonuclease may have altered recognition site specificity (e.g., SpCas9 variants with altered PAM site specificity). In other embodiments, the targeting endonuclease may be catalytically inactive so that cleavage does not occur upon binding to the target portion of the nucleic acid material. In some embodiments, the targeting endonuclease is modified (e.g., a nickase variant) to cleave a single strand of the target portion of the nucleic acid material, thereby generating a nick in the nucleic acid material. CRISPR-based targeting endonucleases are further described herein, providing further detailed, non-limiting examples of the use of targeting endonucleases. Note that the nomenclature surrounding such targeting endonucleases is still in flux. For purposes herein, the term "CRISPR-based" is used to generally refer to endonucleases that contain a nucleic acid sequence that can be modified to redefine the nucleic acid sequence to be cleaved. Cas9 and CPF1 are examples of such targeting endonucleases currently in use, but many more are likely to exist in different locations in nature, and the availability of different varieties of such targeted, easily tunable nucleases is expected to grow rapidly in the coming years.For example, Cas12a, Cas13, CasX, etc. are contemplated for use in various embodiments. Similarly, multiple engineered variants of these enzymes are becoming available to improve or modify their properties. The use of substantially functionally similar targeting endonucleases not expressly described herein or yet to be discovered to achieve similar purposes as those disclosed herein is expressly contemplated herein.

[0095] Restriction endonucleases It is specifically contemplated that any of a variety of restriction endonucleases (i.e., enzymes) may be used to provide nucleic acid material of substantially uniform length and / or to cleave target regions of nucleic acid material. Generally, restriction enzymes are typically produced by particular bacteria / other prokaryotes and cleave at, near, or between specific sequences in a given segment of DNA.

[0096] It will be apparent to one skilled in the art that a restriction enzyme may be selected to cleave at a specific site, or alternatively, at a site engineered to create a restriction site for cleavage. In some embodiments, the restriction enzyme is a synthetic enzyme. In some embodiments, the restriction enzyme is not a synthetic enzyme. In some embodiments, the restriction enzymes used herein are modified to introduce one or more changes into their own genome. In some embodiments, the restriction enzyme generates a double-stranded break between defined sequences in a given portion of DNA.

[0097] While any restriction enzyme (e.g., Type I, Type II, Type III and / or Type IV) may be used according to some embodiments, the following represents a non-limiting list of restriction enzymes that may be used: AluI, ApoI, AspHI, BamHI, BfaI, BsaI, CfrI, DdeI, DpnI, DraI, EcoRI, EcoRII, EcoRV, HaeII, HaeIII, HgaI, HindII, HindIII, HinFI, HPYCH4III, KpnI, MamI, MNL1, MseI, MstI, M stII, NcoI, NdeI, NotI, PacI, PstI, PvuI, PvuII, RcaI, RsaI, SacI, SacII, SalI, Sau3AI, ScaI, SmaI, SpeI, SphI, StuI, TaqI, XbaI, XhoI, XhoII, XmaI, XmaII, and any combination thereof. Extensive, but not exhaustive, lists of suitable restriction enzymes can be found in publicly available catalogs or on the Internet (e.g., available from New England Biolabs, Ipswich, MA, USA). Those skilled in the art will appreciate that various enzymes, ribozymes, or other nucleic acid-modifying enzymes that can be used alone or in combination to target phosphodiester backbone cleavage of nucleic acid molecules, which can achieve the same purpose, may not be included in the above list or may not yet be discovered. Various nucleic acid-modifying enzymes can recognize base modifications (e.g., CpG methylation) that can be used to target further modifications (e.g., to generate abasic sites) of adjacent nucleic acid sequences that can be cleaved (e.g., by an enzyme with lyase activity). Thus, substantial sequence specificity of cleavage can be achieved based on the recognition of DNA or RNA modifications, which can be used alone or in combination with a targeting endonuclease to achieve fragmentation of the target nucleic acid.

[0098] Methods for negative and positive enrichment / selection of nucleic acid material In some embodiments, provided methods and compositions utilize targeting endonucleases (e.g., ribonucleoprotein complexes (CRISPR-associated endonucleases, e.g., Cas9, Cpfl), homing endonucleases, zinc finger nucleases, TALENs, Argonaute nucleases, and / or meganucleases (e.g., megaTAL nucleases, etc., or combinations thereof), or other technologies capable of site-directed interaction with nucleic acid material to positively enrich for desired (target) nucleic acid molecules. Other embodiments provide methods and compositions for negatively enriching / selecting for desired nucleic acid molecules by removing undesired (e.g., off-target) nucleic acid material from a sample. Some embodiments described herein combine both positive and negative enrichment schemes. In some embodiments, provided methods may further include ligating at least one SMI and / or adapter sequence to at least one of the 5' or 3' ends of the enriched target region. In some embodiments, analyzing may be or include quantification and / or sequencing.

[0099] In some embodiments, negative enrichment / selection of target nucleic acid material can be facilitated by the removal or destruction of non-target or undesired nucleic acid material. Figure 4 is a schematic diagram illustrating method steps for generating target nucleic acid fragments of a substantially known / selected length using a CRISPR / Cas9 variant, according to one embodiment of the present technology. Using a CRISPR / Cas9 ribonucleoprotein complex, optionally engineered to have enhanced thermal stability and / or remain bound to dsDNA under appropriate conditions (e.g., enzyme replacement until removed), panel A shows gRNA-facilitated binding of the variant Cas9 to the target DNA site described above. In one embodiment, after cleavage, Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragment, but the sample may be treated with an exonuclease to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA (panel B). During exonuclease treatment, undesired or non-target DNA is destroyed by enzymatic activity, leaving only exonuclease-resistant target dsDNA fragments. As shown in Figure 4, the bound ribonucleoprotein complex can provide exonuclease protection. After negative / enrichment selection of target DNA fragments via exonuclease destruction of non-target DNA, Cas9 dissociates from the DNA, releasing blunt-ended, double-stranded target DNA fragments of known length, as shown in panel C. In some embodiments, the method may also incorporate a positive enrichment / selection scheme using such size selection (panel D). In some embodiments, enriching for fragments of the desired and / or predicted target size allows for further filtering out of genomic fragments that remain undigested and / or are protected by non-target Cas9 binding. Optionally, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing), as shown in panel E.For example, the blunt ends of the target fragments can be directly ligated to blunt-ended adapters. The embodiment of ligating adapters to cleaved double-stranded nucleic acid material may also include end-repair and 3'-dA tailing of the fragments, if required for a particular application. In other embodiments, further processing of the fragments to create suitable ligatable ends of the fragments may include or be any of a variety of forms or steps to form ligatable ends having "sticky" ends that include, for example, blunt ends, a-3' overhangs, 1 nucleotide 3' overhangs, 2 nucleotide 3' overhangs, 3 nucleotide 3' overhangs, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide 3' overhangs, 1 nucleotide 5' overhangs, 2 nucleotide 5' overhangs, 3 nucleotide 5' overhangs, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotide 5' overhangs, among others. The 5' base of the ligation site may be phosphorylated and the 3' base may have a hydroxyl group, or either may be dephosphorylated or dehydrated, alone or in combination, or may be further chemically modified to promote improved ligation of one strand, optionally preventing ligation of the other strand until a later time point.

[0100] In another embodiment, positive enrichment / selection of target nucleic acid material using CRISPR / Cas can be facilitated by affinity-based enrichment of the target nucleic acid material. Figure 5 is a schematic diagram showing steps of a method for generating target nucleic acid fragments of a substantially known / selected length using a CRISPR / Cas9 variant according to another embodiment of the present technology. Panel A shows the use of a CRISPR / Cas9 ribonucleoprotein complex, optionally further engineered to remain tightly bound to DNA under appropriate conditions (as described above), where the ribonucleoprotein complex contains a capture label (e.g., biotin). The capture label may be incorporated into the gRNA (e.g., crRNA, tracrRNA) or the Cas9 protein. Thus, the ribonucleoprotein complex provides an affinity label for the subsequent pull-down step.

[0101] After binding facilitated by the guide RNA (gRNA) of the variant Cas9 ribonucleoprotein complex in the presence of a capture label, cleavage of the double-stranded target DNA is performed. After cleavage, while Cas9 remains bound to the cleaved 5'- and 3'-ends of the target DNA fragment, the reaction mixture is contacted with a functionalized surface to which one or more extraction moieties are attached. The provided extraction moiety can bind to the capture label for immobilization and separation of molecules bearing the capture label (e.g., streptavidin beads when the capture label is biotin). In particular, the extraction moiety can be any member of a binding pair, such as biotin / streptavidin or hapten / antibody or complementary nucleic acid sequences (e.g., DNA / DNA pair, DNA / RNA pair, RNA / RNA pair, LNA / DNA pair, etc.). In an exemplary embodiment, the capture label attached to the CRISPR / Cas9 ribonucleic acid protein complex that binds to the (cleaved) target dsDNA fragment is captured by its binding partner (e.g., extraction moiety) attached to an isolatable moiety (e.g., a magnetically attractive particle or a large particle that can be precipitated by centrifugation). Thus, the capture label may be any type of molecule / moiety that allows affinity separation of nucleic acids associated with the capture label (e.g., bound by Cas9) from nucleic acids that are not associated with the capture label. One example of a capture label is biotin, which allows affinity separation by binding to streptavidin attached to or connectable to a solid phase or oligonucleotide, and then to a complementary oligonucleotide attached to or connectable to a solid phase. Undesired or non-target nucleic acid material may remain free in solution. Advantageously, the free / unbound nucleic acid material does not have a capture label or is not associated with a capture label, and can be effectively removed / separated from the desired target nucleic acid material. In a further embodiment, the functionalized surface (S) may be washed to remove any residual by-products or other contaminants.

[0102] Using the affinity-based enrichment scheme shown in Figure 5, unwanted or non-target nucleic acid material can be substantially reduced in amount. Collection of the desired / target nucleic acid fragments may be achieved in any manner suitable for the application. As a specific example, in some embodiments, collection of the desired nucleic acid material may be achieved through one or more of removal of the functionalized surface via size filtration, magnetic methods, charge methods, centrifugal density methods, or any other method, or collection of elution fractions when using column-based purification methods or similar methods, or any other purification practice commonly understood by those of skill in the art.

[0103] In some embodiments, an affinity-based positive enrichment step may be combined or used in combination with a negative enrichment step. For example, after cleavage, Cas9 remains bound to the cleaved 5' and 3' ends of the target DNA fragments (either before or after the affinity-based enrichment step), while the sample can be treated with an exonuclease to destroy any undesired nucleic acid material or contaminants in the sample. After the affinity-based enrichment step and, optionally, the negative exonuclease removal step shown in panels A and B, Cas9 dissociates from the DNA, releasing blunt-ended, double-stranded target DNA fragments of known length (panel D). Optionally, the enrichment step described above may be combined with the size-based enrichment step described above (panel E), and in some embodiments, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing) as described above (panel F).

[0104] Figure 6 is a schematic diagram showing steps in a method for negative enrichment / selection of target nucleic acid material according to another embodiment of the present technology. For example, enrichment of target double-stranded nucleic acid material can be facilitated by the removal or destruction of non-target or undesired nucleic acid material. Figure 6 illustrates an enrichment embodiment using catalytically inactive variants of Cas9 to create target nucleic acid fragments of a substantially known / selected length. Using a catalytically inactive Cas9 ribonucleoprotein complex engineered to target double-stranded DNA and selectively bind to double-stranded DNA, a gRNA promotes binding of a pair of catalytically inactive Cas9 variants to adjacent target DNA regions (Panel A). After binding, the sample may be treated with one or more exonucleases to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. The catalytically inactive variants of Cas9 do not cleave the target DNA but provide exonuclease resistance, such that exonuclease activity cleaves each nucleotide base until blocked by the bound Cas9 complex. Thus, exonuclease treatment destroys all non-target nucleic acid material in the sample with exposed ends, leaving behind fragments protected by catalytically inactive Cas9 pairs. In certain embodiments, a cocktail of endonucleases and exonucleases can be used to destroy undesired nucleic acid material. For example, an endonuclease (e.g., a site-specific restriction enzyme) can be used to generate multiple exposed 5' and 3' ends that allow exonuclease enzyme activity.

[0105] After negative / enrichment selection of target DNA fragments via exonuclease destruction of all non-target DNA (Panel B), catalytically inactive Cas9 dissociates from the DNA, thereby releasing double-stranded target DNA fragments of known length, as shown in Panel C. As described above, a further size selection step may be performed for further enrichment of the target double-stranded DNA fragments (Panel D). Optionally, the enriched DNA fragments can be polished, blunted, or tailed to create suitable ligatable ends, which may then be ligated to adapters for nucleic acid interrogation (e.g., sequencing) (Panel E).

[0106] In another embodiment, shown in Figure 7, both negative and positive enrichment schemes can be implemented using a catalytically inactive variant of Cas9. Panel A shows the use of a catalytically inactive variant of Cas9 in a ribonucleoprotein complex engineered to remain bound to DNA in the appropriate state, where the ribonucleoprotein complex contains a capture label (e.g., on a guide RNA or tethered to the Cas9 protein). Guide RNA (gRNA)-promoted binding of the catalytically inactive variant Cas9 ribonucleoprotein complex with the capture label is followed by the addition of an exonuclease to the sample to hydrolyze exposed phosphodiester bonds at the exposed 3' or 5' ends of the DNA. The catalytically inactive variant of Cas9 does not cleave the target DNA but provides exonuclease resistance such that exonuclease activity cleaves each nucleotide base until blocked by the bound Cas9 complex. After negative / enrichment selection of target DNA fragments via exonuclease destruction of all non-target DNA, catalytically inactive Cas9 remains bound, but remains bound to the target nucleic acid, allowing the stepwise addition of a functionalized surface (e.g., a functionalized surface to which one or more extraction moieties are attached) capable of binding a capture label that associates with the ribonucleoprotein complex to immobilize and / or separate the molecules bearing the capture label and / or associated with the capture label from the undesired nucleic acid material remaining in the sample (Panel B). In some embodiments, the provided methods allow for the removal of all or substantially all undesired nucleic acid material in a sample, or allow for a substantial reduction in its amount. Collection of the desired target nucleic acid material may be achieved in a manner suitable for any application. As specific examples, in some embodiments, collection of the desired target nucleic acid fragments may be achieved through one or more of removal of the functionalized surface via size filtration, magnetic methods, charge methods, centrifugal density methods, or any other method, or collection of elution fractions when using column-based purification methods or similar methods, or any other commonly understood purification practice.

[0107] After the affinity-based enrichment step, Cas9 dissociates from the DNA, releasing double-stranded target DNA fragments of known length, as shown in panel D. Panel E shows an optional further processing step for positive enrichment / selection of target DNA fragments via size selection. Optionally, as shown in panel F, the enriched DNA fragments may be ligated to adapters for nucleic acid interrogation (e.g., sequencing).

[0108] In some embodiments, a combination of catalytically active and catalytically inactive CRISPR / Cas complexes can be used to positively enrich fragments containing target double-stranded nucleic acid regions. Referring to Figure 8, catalytically active and inactive Cas9 ribonucleoprotein complexes can target desired nucleic acid regions (e.g., specific genomic loci) in a sample in a sequence-dependent manner. The catalytically active Cas9 ribonucleoprotein complex is directed to regions adjacent to the target DNA region and used to cleave the target double-stranded DNA, releasing blunt-ended double-stranded target DNA fragments of known length. One or more catalytically inactive ribonucleoprotein complexes bearing a capture label (e.g., biotin) are directed to the target sequence region between the two site-selected cleavage sites. After cleaving the target DNA and releasing the DNA fragments, the addition of a functionalized surface capable of binding the capture label associated with the catalytically inactive ribonucleoprotein complex can facilitate positive enrichment / selection of the target fragments. It will be appreciated that many other forms of target nucleic acid fragmentation, such as those described above, can be substituted for active Cas9 ribonucleoprotein complexes in the sample.

[0109] In some embodiments, the positive enrichment / selection step can also be performed to enrich target sequences from samples in which nucleic acid material has already been fragmented (e.g., mechanically sheared or from cell-free DNA samples (e.g., from liquid biopsies)). Figures 9A and 9B are schematic diagrams of the steps of a method for positive enrichment / selection of target nucleic acid fragments using catalytically inactive variants of the Cas9 ribonucleoprotein complex with a capture label described above. Fragmented double-stranded DNA fragments in a sample (e.g., mechanically sheared, acoustically fragmented, cell-free DNA, etc.) can be positively enriched / selected via target-directed binding by one or more catalytically inactive Cas9 ribonucleoprotein complexes in solution (Figure 9A).

[0110] In some embodiments, methods may include the use of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) capture labels that can be used to differentially tag multiple Cas9 ribonucleic acid protein complexes. For example, a sample can simultaneously enrich for multiple target nucleic acid samples. In some embodiments, it is contemplated that all Cas9 complexes have the same capture label (e.g., biotin), so that all target sequences may be pulled down (affinity purified) together in a single sample. In other embodiments, separation of different target sequences can be facilitated by incorporating substantially unique capture labels with Cas9 complexes intended to target different regions. In some embodiments, at least two capture labels used in a method are different from one another (e.g., small molecules and peptides). In some embodiments, the inclusion of two or more different capture labels allows for the use of both positive enrichment / selection and negative enrichment / selection. The inclusion of two or more capture labels may be particularly useful when it is desired to physically separate nucleic acid fragments containing different target sequences for subsequent nucleic acid interrogation (e.g., sequencing).

[0111] The reaction mixture is contacted with a functionalized surface(s) to which one or more extraction moieties are attached, the extraction moieties being capable of binding to a capture label (e.g., streptavidin beads when the capture label is biotin) for immobilization and separation of molecules bearing the capture label (Figure 9B).

[0112] In some embodiments, it is desirable to enrich or isolate target nucleic acid material from a sample containing fragments of various sizes, including small fragment sizes that may otherwise be lost during processing steps (e.g., steps of a DS process). Figure 10 is a schematic diagram illustrating steps of a method for positive enrichment / selection of target nucleic acid fragments using a catalytically inactive variant of a Cas9 ribonucleoprotein complex bearing a capture label. Panel A shows multiple fragmented double-stranded DNA fragments of various sizes in a sample, including molecule 2, which is too small to be reliably enriched by size selection or affinity-based methods. In this embodiment, adapters (e.g., sequencing adapters) may be ligated / attached to the fragment ends using known sequencing library preparation processes. In this manner, specific small nucleic acid fragments are extended by adjacent adapter molecules. Positive enrichment of target fragments from solution can proceed as described above with respect to Figures 9A and 9B. For example, panel B of Figure 10 shows further lengthening of such DNA fragments by ligating adapters to the 5' and 3' ends of molecules in the sample. Panel C shows the affinity purification after a positive enrichment / selection step of molecule 2 via targeted binding by a catalytically inactive Cas9 ribonucleoprotein complex bearing a capture label in solution.

[0113] 11 is a schematic diagram illustrating method steps for enriching target nucleic acid material using a negative enrichment scheme (Panel A) and a positive enrichment scheme (Panel B) according to one embodiment of the present technology. Panel A shows ligation of hairpin adapters to the 5' and 3' ends of double-stranded target DNA molecules to generate adapter-nucleic acid complexes with no exposed ends. The adapter-nucleic acid complexes are treated with an exonuclease in the negative enrichment / selection scheme to remove nucleic acid material fragments and adapters with unprotected 5' and 3' ends (e.g., adapter-nucleic acid complexes without four ligated phosphodiester bonds, unligated DNA, single-stranded nucleic acid material, free adapters, etc.), as shown on the right side of Panel B.

[0114] As shown in Figure 11, hairpin adapters may contain a cleavable moiety, such as a uracil group, or any other enzymatically, chemically, or photoelectrically cleavable group in the linker portion. When treated with a combination of uracil DNA glycosylase (UDG) and an enzyme with abasic site DNA lyase activity, such as endonuclease VIII or formamidopyrimidine [fapy]-DNA glycosylase (FPG), or a commercially available premixed combination (e.g., USER™ enzyme), cleavage at the uracil can convert the hairpin adapter into a Y-shaped adapter suitable for polony formation (bridge amplification) and certain sequencing formats.

[0115] Exonuclease-resistant adaptor-nucleic acid complexes can be further enriched by size selection or target sequence (e.g., CRISPR / Cas9 pulldown) (Figure 11, panel B, left). In another embodiment, hairpin adaptors with capture labels may be used (as shown in Figure 12), which are directly suitable for affinity-based enrichment using functionalized surfaces with exposed extraction moieties.

[0116] In embodiments following negative enrichment of target nucleic acid fragments ligated to hairpin adapters as described in Figure 11, a further positive enrichment step may be performed. For example, Figure 13 is a schematic diagram showing the steps of a method for rolling circle amplification (Panels B and C) following positive enrichment of adapter-target nucleic acid complexes using hairpin adapters (Panel A). The rolling circle amplification step can be used to (1) provide a substantially 1:1 ratio of first-strand amplicons to second-strand amplicons and (2) prevent strand dissociation prior to tagging and / or during the library removal step. Long molecule sequencing platforms may be suitable for directly sequencing the rolling circle amplicons (Panel C). However, for short-read sequencing platforms, either (1) a hairpin linker segment containing a cleavage site (e.g., a restriction endonuclease recognition site) can be enzymatically cleaved to create first-strand and second-strand amplicons in approximately equal proportions (Panel D, left), or (2) PCR amplification can be used to create multiple short amplicons containing the first and second sequences in substantially equal proportions (Panel D, right).

[0117] Figure 14 is a schematic diagram showing steps of a method for generating target nucleic acid fragments of known / selected lengths with distinct 5' and 3' ligatable ends using CRISPR / Cpf1 site-directed binding and cleavage. In various embodiments, the 5' and 3' ligatable ends comprise single-stranded overhang regions with known nucleotide lengths and sequences. Cpf1 in the targeting endonuclease recognizes a T-rich PAM 5' of the guide and performs a sticky cleavage in the double-stranded DNA target sequence. For example, as shown in Figure 14, a Cpf1 variant cleaved 19 bp after the PAM on the sense strand and 23 bp on the antisense strand. Panel A shows gRNA-promoted binding of Cpf1 at the target DNA site. Cpf1-directed cleavage generates a sticky cleavage and provides a 4 (as shown) or 5-nucleotide overhang (e.g., "sticky end"). Site-directed Cpf1 cleavage adjacent to the target DNA sequence generates double-stranded target DNA fragments of known length (e.g., which may be further optionally enriched by size selection) with sticky end 1 at the 5' end of the fragment and sticky end 2 at the 3' end (Panel B). Panel B further shows that adaptor 1 is attached to the 5' end of the fragment and adaptor 2 is attached to the 3' end, with adaptors 1 and 2 comprising overhanging sequences at least partially complementary to sticky ends 1 and 2 of the fragment, respectively.

[0118] By design, the sequence of sticky end 1 (the overhang at the 5' end of the target fragment) is known. Similarly, the sequence of sticky end 2 (the overhang at the 3' end of the target fragment) is known. Specific adapters containing substantially complementary sequences can be synthesized so that fragments can be ligated to the adapters at both ends. In one embodiment, the adapters can be the same type of adapter (e.g., Y-shaped, U-shaped, barcoded, etc.). In another embodiment, the adapters can be different (e.g., adapter 1 can contain a Y-shaped adapter and adapter 2 can contain a U-shaped adapter). Other unique features can include different primer sites for amplification, different types or positions of barcodes or other unique molecular identifiers, adapters with and without capture labels, specific adapters can contain fluorescent tags, etc. There are identified advantages in some applications for designing specific adapters to be located at either the 5' or 3' end of a fragment. The specificity of the substantially unique sticky ends on the target fragments facilitates these types of applications. Furthermore, positive selection of successfully cleaved and adaptor-ligated target fragments can ensure amplification and sequencing of only enriched target nucleic acid regions.

[0119] In some embodiments, the substantially unique sticky ends generated by Cpf1 cleavage can be used in further positive enrichment schemes. For example, Figure 15 is a schematic diagram illustrating the steps of a method for affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments generated by the method of Figure 14) according to one embodiment of the present technology. Panel A shows the stepwise addition of a functionalized surface capable of binding sticky ends associated with cleaved target DNA fragments in solution. For example, the functionalized surface may have one or more attached extraction moieties suitable as binding pairs for one or more target DNA overhang sequences. The provided extraction moieties may be, for example, synthetic oligonucleotides having predetermined or known oligonucleotide sequences at least partially complementary to the generated sticky end(s) of the Cpf1-cleaved target sequence. The oligonucleotides may contain DNA, RNA, or LNA sequences (e.g., sticky ends) capable of binding to a capture label for immobilization and isolation of targets containing sticky end(s). Upon binding to a functionalized surface, the affinity interaction facilitates the pull-down (e.g., affinity purification) of desired double-stranded DNA fragments while discarding non-target fragments, as shown in panel B.

[0120] 16 is a schematic diagram illustrating steps in a method for affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments produced by the method of FIG. 14 ) according to another embodiment of the present technology. Panel A shows the stepwise addition of oligonucleotides bearing capture labels with predetermined or known oligonucleotide sequences at least partially complementary to the portion of the sticky end that associates with cleaved target DNA fragments in solution. In certain examples, the oligonucleotide chain may be synthesized in the 3′ to 5′ direction (e.g., on controlled pore glass (CPG) fragments, etc.), e.g., by phosphoramidite methods, and the chemical moiety may be attached (e.g., by covalent, non-covalent, ionic, or other linking chemistry) to the 5′ end after synthesis of the oligonucleotide, or as part of the synthesis of the oligonucleotide, e.g., by incorporation of a non-canonical phosphoramidite molecule at the 5′ end, near the 5′ end, or at an internal position within the oligonucleotide.

[0121] As shown in panel B, the further addition of a functionalized surface capable of binding a capture label facilitates pull-down (e.g., affinity purification) of desired double-stranded DNA fragments while discarding non-target fragments.

[0122] Referring also to Figures 15 and 16, in a next step (not shown), elution of the target fragments may occur via release from the extraction moiety. In some non-limiting examples, a cleavable moiety may be incorporated adjacent to the bound end of the oligonucleotide extraction moiety. In another embodiment, temperature or other conditions may be altered to denature the short capture label / extraction bond while maintaining the double-stranded nature of the target nucleic acid fragments. In yet another embodiment, a hairpin adapter may be used at the second sticky end of the target fragment to tether the double-stranded fragment together during elution and further processing. In various embodiments, after the enrichment step, the sticky ends may be polished, trimmed, or biocomputationally filtered as described herein to avoid spurious reaction errors.

[0123] 17 is a schematic diagram showing steps of a method for target fragment enrichment of a nucleic acid entity of known length with distinct 5' and 3' ligatable ends containing long single-stranded overhang regions of known nucleotide length and sequence using Cas9 nickase, according to one embodiment of the present technology. Panel A shows gRNA-targeted binding of paired Cas9 nickase at a target DNA region. A double-stranded break can be introduced by the use of paired nickases to cleave the target DNA region, and when paired Cas9 nickases are used, long overhangs (sticky ends 1 and 2) are generated for each cleaved end, as shown in panel B. Thus, in contrast to cleavage using catalytically active Cas9, which generates blunt ends, strategic pairing of Cas9 nickases provides single-stranded cohesive cuts in opposing DNA strands, which can generate long overhangs, as shown in panel B. As described above with respect to Figure 15, the stepwise addition of functionalized surfaces capable of binding long sticky ends (e.g., sticky end 1) associated with cleaved target DNA fragments in solution provides a positive enrichment step for the target DNA fragments in solution. For example, the extracting moiety may be an oligonucleotide having a predetermined or known oligonucleotide sequence substantially complementary to the predetermined or known sequence of the long sticky end of the fragment. Upon binding to the functionalized surface, the affinity interaction facilitates the pull-down (e.g., affinity purification) of the desired double-stranded DNA fragments while discarding non-target fragments, as shown in panel D.

[0124] Panel E of Figure 17 shows a variation of the positive enrichment step, involving the addition and annealing of a capture-labeled oligonucleotide with a predetermined or known oligonucleotide sequence at least partially complementary to a portion of the long sticky end (e.g., sticky end 1) associated with the cleaved target DNA fragment in solution. Panel F shows the annealing of a second oligonucleotide strand at least partially complementary to a portion of the capture-labeled oligonucleotide. Enzymatic extension of the second oligonucleotide strand and ligation to the template DNA fragment produces an adapter-target DNA complex. As shown, the first and second oligonucleotide strands contain single-stranded portions such that the resulting adapter complex contains asymmetry for DS processing. Additionally, the first oligonucleotide strand may contain a denatured or semi-denatured SMI sequence such that when the second oligonucleotide strand is extended, the first oligonucleotide strand serves as the template strand and the SMI sequence becomes double-stranded. A further step may involve the introduction of a functionalized surface (not shown) capable of binding a capture label to facilitate pull-down (e.g., affinity purification) of the desired adaptor-double-stranded DNA complex while discarding non-target fragments.

[0125] Various aspects of the present technology include methods for negatively enriching nucleic acid regions by providing exonuclease and endonuclease resistance through protein binding. In one embodiment, as shown in Figure 18, site-selective protein binding to target DNA can be used to provide exonuclease and endonuclease resistance. As shown, the target nucleic acid enrichment scheme uses a catalytically inactive Cas9 ribonucleoprotein complex to protect the target genomic region. Cas9 can be targeted to the desired sequence in the sample by a gRNA. One or more catalytically inactive ribonucleoprotein complexes carrying one or more capture labels can be positioned adjacent to and / or adjacent to protect the region of genomic DNA from enzymatic digestion. In some embodiments, as shown, the ribonuclease complex can be designed so that another protein complex structure is directed to the target DNA region. When this protein complex structure covers the target DNA region, exonuclease resistance is provided. After treatment with an exonuclease or a combination of an endonuclease and an exonuclease, affinity purification of the protein complex (e.g., via capture labels bound to functionalized surfaces, antibody pull-down, etc.), the target DNA fragments are separated from other undesired nucleic acid material or unbound proteins in solution. The target nucleic acid fragments may then be released from the ribonucleotide complex binding.

[0126] Nucleic acid libraries and methods for making and using nucleic acid libraries In some embodiments, the provided methods may include providing a nucleic acid material and directing a plurality of catalytically inactive target endonucleases (e.g., ribonucleoprotein complexes) to a plurality of regions distributed along the nucleic acid material to create a nucleic acid library that can be interrogated at any time via selection probes.

[0127] Figures 19A and 19B are conceptual diagrams of prepared DNA libraries and reagents that can be used as tools to selectively interrogate DNA regions of interest, according to one embodiment of the present technology. Uniquely tagged catalytically inactive Cas9s are directed to target multiple (e.g., spaced) regions of isolated / unfragmented genomic DNA (or other large fragments of DNA) (Figure 19A). Each catalytically inactive Cas9 ribonucleoprotein contains a known oligonucleotide tag with a known sequence (e.g., a coding sequence) and binds to a pre-designed region of the genome. As shown schematically in Figure 19A, multiple inactive Cas9 ribonucleoprotein complexes (e.g., iCas9) are synthesized. A , iCas9 B , iCas9 C , iCas9 N ) are genomic sites distributed across a genomic region (e.g., a large selected region, the entire genome, etc.). A , Site B , Site C , Site N Each iCas9 complex contains an oligonucleotide tag that includes an oligonucleotide code sequence (AAAAAAA), where "A" is any nucleotide (unmodified or modified) that contains a substantially unique code that can be recorded and later looked up in a lookup table.

[0128] If it is desired to interrogate (e.g., sequence) a specific target sequence or smaller region, the library may be probed with a specifically designed capture probe engineered to pull down the desired region. Fragmentation methods can be used to fragment genomic DNA into various sizes (e.g., restriction enzyme digestion, mechanical shearing, etc.). Because each iCas9 complex contains a substantially unique oligonucleotide tag computationally associated with a DNA site, users can incrementally add one or more probes containing the complement of a coding sequence (e.g., an anti-coding sequence) corresponding to a genomic region of interest. For example, as shown in Figure 19B, an anti-coding sequence is a nucleotide sequence substantially complementary to the coding sequence of interest. For example, site A To extract the region containing site A The coding sequence (AAAAAAA) associated with the iCas9A complex bound to the target region is then probed. Using an oligonucleotide probe containing the anticoding sequence (A'A'A'A'A'A'A'), with a capture label immobilized or incorporated therein, the region of interest may then be functionally selected and enriched by the introduction of a functionalized surface bearing an appropriate extraction moiety (e.g., streptavidin if biotin is the capture label).

[0129] In various embodiments, a nucleic acid library can be used as a resource for interrogation using several probes. In addition, several libraries containing multiple pre-bound CRISPR / Cas site-directed complexes can be prepared. Furthermore, some libraries can be pre-fragmented or cleaved using either mechanical shearing or endonuclease cleavage (using one or more restriction endonucleases). When the desired target region is cleaved (e.g., via targeted endonuclease digestion (e.g., CRISPR / Cas, restriction enzymes, etc.)), the length of the target fragment is known, and after pull-down using the probe, the target fragment can be further enriched by size selection.

[0130] More ways Some aspects of the present technology are suitable for use with long sequence sequencing technology, such as direct digital sequencing (DDS) platform.In some embodiments, it is desirable to enrich the target sequence of interest for use with DDS.In such embodiments, it is desirable to enrich the target sequence without amplification.In addition, it is further desirable to generate double-stranded sequencing data on such platform.

[0131] Figure 20 shows steps of a method for affinity-based enrichment and sequencing of target DNA fragments for use with direct digital sequencing methods, according to one embodiment of the present technology. Panel A shows the attachment of selected adapters to a target DNA fragment containing sticky end(s) (e.g., a target DNA fragment created by the method of Figure 14 or Figure 17). Panel A further shows the attachment of adapter 1 to the 5' end of the fragment and adapter 2 to the 3' end, where adapters 1 and 2 contain overhanging sequences at least partially complementary to sticky ends 1 and 2 of the fragment, respectively. Adapter 1 has a Y-shape and includes 5' and 3' single-stranded arms bearing different labels (A and B) with distinct properties. Adapter 2 is a hairpin-shaped adapter.

[0132] Panel B shows a step in a direct digital sequencing method in which label A is configured to bind to a functionalized surface. Label B provides a physical property (e.g., charge, magnetic property, etc.) that triggers electroextension of the DNA fragments after denaturation of the first and second strands of the double-stranded adapter-DNA complex by application of an electric or magnetic field. The first and second strands remain tethered by the hairpin adapter, so that sequence information from the enriched / targeted strand provides double-stranded sequence information for error correction and other nucleic acid interrogation (e.g., assessment of DNA damage). For example, the sequence generated from the first strand may be compared with the sequence generated from the second strand for error correction, or, in another example, to determine the site and characteristics of DNA damage. In some embodiments, the target genomic region to be enriched may have a length of approximately 1 to 1,000,000 bases. For example, in some embodiments, the length of the enriched nucleic acid fragments, when denatured and sequenced, may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, or 50,000 bases in length. In some embodiments, the length of the fragments may be up to 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000 or 1,000,000 bases.

[0133] Figure 21 shows steps of a method for affinity-based enrichment for sequencing of target DNA fragments using a DDS method according to another embodiment of the present technology. Panel A shows affinity-based enrichment of target DNA fragments containing sticky end(s) (e.g., target DNA fragments produced by the methods of Figure 14 or Figure 17). As shown, hairpin adapters are attached to the 3' ends of the double-stranded DNA fragments in a sequence-dependent manner. The target DNA molecule(s) may be flowed over a functionalized surface capable of binding sticky ends associated with cleaved target DNA fragments (e.g., with attached oligonucleotides). In addition, a second oligonucleotide strand containing label B and at least partially complementary to a portion of the attached oligonucleotide is added to the solution. Annealing and ligation of the adapter / DNA fragment components provides a surface-bound adapter-target double-stranded DNA complex suitable for direct digital sequencing (Panel B). Application of an electric or magnetic field and electroextension of the adapter-DNA complex for the sequencing process can be performed, for example, as described in Figure 20.

[0134] Reagents and Methods Adapter Type Although most of the examples in the present disclosure show Y-shaped or loop adapters, any known adapter structure may be used according to various embodiments, such as, for example, those described in WO2017 / 100441, which is incorporated herein by reference in its entirety. Various adapter shapes, including, for example, bubbles (e.g., internal regions of non-complementarity), are further envisioned.

[0135] separation As described herein, various methods include at least one separation step. It is specifically contemplated that any of a variety of separation steps may be included in various embodiments. For example, in some embodiments, the separation may be or include physical separation, size separation, magnetic separation, solubility separation, charge separation, hydrophobic separation, polarity separation, electrophoretic mobility separation, density separation, chemical elution separation, SBIR bead separation, etc. For example, the physical group may have magnetic properties, charge properties, or insolubility properties. In an embodiment, when a physical group has magnetic properties and a magnetic field is applied, related adapter nucleic acid sequences that include the physical group are separated from adapter nucleic acid sequences that do not include the physical group. In another embodiment, when a physical group has charge properties and an electric field is applied, related adapter nucleic acid sequences that include the physical group are separated from adapter nucleic acid sequences that do not include the physical group. In an embodiment, when a physical group has insolubility properties and adapter nucleic acid sequences are contained in a solution in which the physical group is insoluble, related adapter nucleic acid sequences that include the physical group precipitate away from adapter nucleic acid sequences that do not include the physical group and remain in solution.

[0136] Any of a variety of physical separation methods may be included in various embodiments, with a specific, non-limiting set of methods including size-selective filtration, density centrifugation, HPLC separation, gel filtration separation, FPLC separation, density gradient centrifugation, and gel chromatography, among others.

[0137] Any of a variety of magnetic separation methods may be included in various embodiments. Typically, magnetic separation methods involve the inclusion or attachment of one or more physical groups with magnetic properties, such that, upon application of a magnetic field, molecules containing such physical group(s) are separated from molecules that do not contain such physical groups. Specific examples of physical groups exhibiting magnetic properties include, but are not limited to, paramagnetic materials, such as iron, nickel, cobalt, dysprosium, gadolinium, and alloys thereof. Paramagnetic beads, commonly used for chemical and biochemical separations, may be functionalized with such materials embedded within their surface to reduce chemical interactions between the material and the chemicals being manipulated (e.g., polystyrene) and for the affinity properties described above.

[0138] capture beacon As described herein, in some embodiments, the capture label may be present on the protein in any of a variety of configurations, such as along an oligonucleotide probe, adapter, ribonucleotide sequence, ribonucleic acid protein complex, etc. In some embodiments, the capture label may be incorporated into or immobilized on the oligonucleotide strand in the 5' region of the sequence. In some embodiments, the capture label may be located somewhere in the middle of the oligonucleotide strand (i.e., not at the 5' or 3' end of the oligonucleotide). In embodiments including two or more capture labels, each capture label may be located at a different position along the oligonucleotide.

[0139] In some embodiments, the capture label is, inter alia, biotin, biotin deoxythymidine dT, biotin NHS, biotin TEG, biotin-6-aminoallyl-2'-deoxyuridine-S'-triphosphate, biotin-16-aminoallyl-2-deoxycytidine-5'-triphosphate, biotin 16-aminoallyl cytidine-5'-triphosphate, N4-biotin-OBEA-2'-deoxycytidine-5'-triphosphate, biotin-16-amino The nucleic acid sequence is selected from the group consisting of allyluridine-5'-triphosphate, biotin-16-7-deaza-7-aminoallyl-2'-deoxyguanosine-5'-triphosphate, 5'-biotin-G-monophosphate, 5'-biotin-A-monophosphate, 5'-biotin-dG-monophosphate, 5'-biotin-dA-monophosphate, desthiobiotin NHS, desthiobiotin-6-aminoallyl-2'-deoxycytidine-5'-triphosphate, digoxigenin NHS, DNP TEG, thiol, colicin E2, Im2, glutathione, glutathione-s-transferase (GST), nickel, polyhistidine, FLAG tag, and myc tag. In some embodiments, capture labels include, but are not limited to, biotin, avidin, streptavidin, haptens recognized by antibodies, specific nucleic acid sequences, and / or magnetically attractable particles. In some embodiments, one or more chemical modifications of nucleic acid molecules (e.g., Acridite™ modifications, among many other modifications, some of which are described elsewhere in this application) can function as capture labels.

[0140] extraction part An extracting moiety may be a physical binding partner or pair for a target capture label and refers to an isolatable moiety or any type of molecule that allows affinity separation of a nucleic acid bearing a capture label or bound by a molecule bearing a capture label (e.g., an oligonucleotide, a protein, a ribonucleic acid-protein complex, etc.) from a nucleic acid lacking the capture label. The extracting moiety may be directly or indirectly linked to a substrate, e.g., a solid surface (e.g., via a nucleic acid, an antibody, an aptamer, etc.). In some embodiments, the extracting moiety is selected from a group including a small molecule, a nucleic acid, a peptide, an antibody, or any uniquely binding moiety. The extracting moiety may be linked or linkable to a solid phase or other surface to form a functionalized surface. In some embodiments, the extracting moiety is a sequence of nucleotides linked to a surface (e.g., a solid surface, a bead, a magnetic particle, etc.). In some embodiments, when the capture label is biotin, the extracting moiety is selected from the group of avidin or streptavidin. It will be understood by those skilled in the art that any of a variety of affinity binding pairs may be used according to various embodiments.

[0141] In certain embodiments, the extraction moiety may be a physical or chemical property that interacts with the target capture label. For example, the extraction moiety may be a magnetic field, an electric field, or a liquid solution in which the target capture label is insoluble. Such physical or chemical properties may be applied, and the adapter nucleic acid bearing the capture label may be immobilized in / to a container (surface) or column. Depending on the desired positive enrichment / selection or negative enrichment / selection results, the immobilized molecules may be retained (positive enrichment), or the non-immobilized molecules may be retained (negative enrichment) for further purification / processing or use.

[0142] solid surface If the affinity partner / extraction moiety is attached to a solid surface or substrate and binds to a capture label, the adapter nucleic acid sequence containing the capture label can be separated from the adapter nucleic acid sequence that does not contain the affinity label. The solid surface or substrate may be a bead, isolatable particle, magnetic particle, or another immobilized structure.

[0143] As described herein and understood by those skilled in the art, any of a variety of functionalized surfaces may be used in accordance with various embodiments. For example, in some embodiments, the functionalized surface may be or include beads (e.g., controlled pore size glass beads, macroporous polystyrene beads, etc.). However, it will be understood by those skilled in the art that many other chemical moiety / surface pairs can be similarly used to achieve the same purpose. It will be understood that the specific functionalized surfaces described herein are meant to be merely exemplary, and that any other suitable immobilized structure or substrate capable of associating (e.g., linking, binding, etc.) with one or more extracting moieties may be used.

[0144] Nucleic acid cleavage Various aspects of the present technology include enzymatic cleavage, which may incorporate enzymatic cleavage of single strands, enzymatic cleavage of double strands, enrichment of nucleic acid material using adaptors, oligonucleotides and capture labels, incorporation of modified nucleic acids followed by enzymatic treatment to cleave one or both strands, incorporation of photocleavable linkers, incorporation of uracil, incorporation of ribose bases, incorporation of 8-oxo-guanine adducts, use of restriction endonucleases, use of site-directed cleavage enzymes, etc. In other embodiments, endonucleases may be used, such as ribonucleoprotein endonucleases (e.g., Cas enzymes, e.g., Cas9 or CPF1), or other programmable endonucleases (e.g., homing endonucleases, zinc finger nucleases, TALENs, meganucleases (e.g., megaTAL nucleases), Argonaute nucleases, etc.), and any combination thereof.

[0145] As described herein, various embodiments include the use of one or more endonucleases that recognize unique nucleotide sequences or modifications or other entities that recognize bases or other backbone chemical modifications to cleave and / or cleave double-stranded nucleic acids (e.g., DNA or RNA) at specific positions in one or more strands. Examples include uracil (which can be recognized and cleaved using a combination of uracil DNA glycosylase and an abasic site lyase (e.g., endonuclease VIII or FPG)) and ribose nucleotides (which, when paired with DNA bases, can be recognized and cleaved by RNAse H2). The nucleic acid may be DNA, RNA, or a combination thereof, and optionally includes peptide nucleic acid (PNA) or locked nucleic acid (LNA), or other modified nucleic acids. In some embodiments, cleavage may be achieved by the use of one or more restriction endonucleases. In some embodiments, cleavage may be achieved using a cleavable linker, e.g., uracil-desthiobotin-TEG, ribose cleavage, or other methods. In some embodiments, the cleavable linker may be, or may partially be, a photocleavable linker or a chemically cleavable linker that does not require an enzyme.

[0146] It will be understood by those skilled in the art that a variety of restriction endonucleases (i.e., restriction enzymes) that cleave DNA at or near their recognition sites (e.g., EcoRI, BamHI, XbaI, HindIII, AluI, AvaII, BsaJI, BstNI, DsaV, Fnu4HI, HaeIII, MaeIII, N1aIV, NSiI, MspJI, FspEI, NaeI, Bsu36I, NotI, HinF1, Sau3AI, PvuII, SmaI, HgaI, AluI, EcoRV, etc.) may be used in accordance with various embodiments of the present technology. Lists of several restriction endonucleases are available in both printed and computer-readable form and are provided by many commercial suppliers (e.g., New England Biolabs, Ipswich, MA). A non-limiting list of restriction endonucleases and associated recognition sites may be found at www.neb.com / tools-and-resources / selection-charts / alphabetized-list-of-recognition-specificities.

[0147] In some embodiments, the modified nucleotide or non-nucleotide can provide a cleavable moiety, such as a uracil base (which can be cleaved, for example, using a combination of UGD and endonuclease VIII or FPG), an abasic site (which can be cleaved, for example, by endonuclease VIII), 8-oxo-guanine (which can be cleaved, for example, by FPG or OGG1), and a ribose nucleotide (which can be cleaved, for example, by RNAse H2 when paired with DNA).

[0148] Ligable ends In some embodiments, the adapter products are generated with a ligatable 3' end suitable for ligation to a target double-stranded nucleic acid sequence (e.g., for sequencing library preparation). The ligation domain present in each double-stranded adapter product may be ligatable to one corresponding strand of a double-stranded target nucleic acid sequence. In some embodiments, one of the ligation domains comprises a T-overhang, an A-overhang, a CG-overhang, a multi-nucleotide overhang, a blunt end, or another ligatable nucleic acid sequence. In some embodiments, the double-stranded 3' ligation domain comprises a blunt end. In certain embodiments, at least one of the ligation domain sequences comprises a modified or non-standard nucleic acid. In some embodiments, the modified nucleotide may be an abasic site, uracil, tetrahydrofuran, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-A), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-G), deoxyinosine, 5'-nitroindole, 5-hydroxymethyl-2'-deoxycytidine, iso-cytosine, 5'-methyl-isocytosine, or iso-guanosine. In some embodiments, at least one strand of the ligation domain comprises a dephosphorylated base. In some embodiments, at least one of the ligation domains comprises a dehydroxylated base. In some embodiments, at least one strand of the ligation domain is chemically modified to render it unable to ligate (e.g., until further work is done to render the ligation domain ligatable). In some embodiments, the 3' overhang is obtained by use of a polymerase with terminal transferase activity. In one example, Taq polymerase can add a single base pair overhang, which in some embodiments is an "A."

[0149] Non-canonical nucleotides In some embodiments, the provided template and / or extension strand may include one or more non-standard / non-canonical nucleotides. In some embodiments, the non-standard nucleotide may be or include uracil, methylated nucleotides, RNA nucleotides, ribose nucleotides, 8-oxo-guanine, biotinylated nucleotides, desthiobiotin nucleotides, thiol-modified nucleotides, acrydite-modified nucleotides, iso-dC, iso-dG, 2'-O-methyl nucleotides, inosine nucleotide-locked nucleic acids, peptide nucleic acids, 5-methyl-dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotides, abasic nucleotides, 5-nitroindole nucleotides, adenylated nucleotides, azido nucleotides, digoxigenin nucleotides, I-linkers, 5' hexynyl-modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non-photocleavable spacers, click chemistry compatible modified nucleotides, fluorescent dyes, biotin, furan, BrdU, fluoro-dU, roto-dU, and any combination thereof.

[0150] Further Aspects According to aspects of the present disclosure, some embodiments provide high-quality sequencing information from very small amounts of nucleic acid material. In some embodiments, the provided methods and compositions may be used with starting amounts of nucleic acid material of up to about 1 picogram (pg), 10 pg, 100 pg, 1 nanogram (ng), 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng. In some embodiments, the provided methods and compositions may be used with input amounts of nucleic acid material of up to 1 molar copy or genome equivalent, 10 molar copies or genome equivalent, 100 molar copies or genome equivalent, 1,000 molar copies or genome equivalent, 10,000 molar copies or genome equivalent, 100,000 molar copies or genome equivalent, or 1,000,000 molar copies or genome equivalent. For example, in some embodiments, up to 1,000 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 10 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 ng of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 100 pg of nucleic acid material is initially provided for a particular sequencing process. For example, in some embodiments, up to 1 pg of nucleic acid material is initially provided for a particular sequencing process.

[0151] According to other aspects of the present technology, some provided methods may be useful for sequencing any variety of suboptimal (e.g., damaged or degraded) samples of nucleic acid material, e.g., in some embodiments, at least a portion of the nucleic acid material is damaged. In some embodiments, the damage is oxidation, alkylation, deamination, methylation, hydrolysis, nicking, intrastrand crosslinks, interstrand crosslinks, blunt-end strand breaks, sticky-end double-strand breaks, phosphorylation, dephosphorylation, sumoylation, glycosylation, single-strand gaps, damage from heat, damage from desiccation, damage from UV exposure, damage from gamma radiation, damage from X-rays, damage from ionizing radiation, damage from non-ionizing radiation, damage from heavy particle radiation, damage from nuclear decay, damage from beta radiation, damage from alpha radiation, damage from neutron radiation, damage from proton radiation, damage from cosmic radiation, damage from high pH, ​​damage from low pH, damage from reactive oxidative species, damage from free radicals, damage from peroxides, damage from hypochlorite, damage from tissue fixation such as formalin or formaldehyde, damage from reactive iron, damage from low ionic conditions, damage from high ionic conditions, damage from unbuffered conditions, damage from nucleases, damage from environmental exposure. The damage may be or include at least one of the following: scratches, damage from fire, damage from mechanical stress, damage from enzymatic degradation, damage from microorganisms, damage from mechanical shear in preparation, damage from enzymatic fragmentation in preparation, damage occurring naturally in vivo, damage occurring during nucleic acid extraction, damage occurring during sequencing library preparation, damage introduced by polymerases, damage introduced during nucleic acid repair, damage occurring during nucleic acid end tailing, damage occurring during nucleic acid ligation, damage occurring during sequencing, damage occurring from mechanical handling of DNA, damage occurring during passage through a nanopore, damage occurring as part of aging in an organism, damage occurring as a result of chemical exposure of an individual, damage caused by mutagens, damage caused by carcinogens, damage caused by clastogens, damage resulting from inflammatory damage in vivo due to oxygen exposure, damage resulting from one or more strand breaks, and any combination thereof.

[0152] II. SELECTED EMBODIMENTS OF DOUBLE-STRANDED SEQUENCING METHODS AND ASSOCIATED ADAPTER AND REAGENTS Double-stranded sequencing is a method for generating error-corrected DNA sequences from double-stranded nucleic acid molecules, and was originally described in International Patent Publication No. WO2013 / 142389 and U.S. Patent No. 9,752,188 and WO2017 / 100441, Schmitt et al., PNAS, 2012 [1], Kennedy et al., PLOS Genetics, 2013 [2], Kennedy et al., Nature Protocols, 2014 [3], and Schmitt et al., Nature Methods, 2015 [4]. Each of the above-mentioned patents, patent applications, and publications is incorporated herein by reference in its entirety. As shown in Figures 1A-1C, in certain embodiments of the present technology, double-stranded sequencing can be used to independently sequence both strands of individual DNA molecules in a manner that allows derivative sequence reads to be recognized as originating from the same double-stranded nucleic acid parent molecule during massively parallel sequencing (MPS) (also commonly known as next-generation sequencing (NGS)), but also allows both strands to be independently sequenced in such a way that they are distinguished from one another as distinct entities after sequencing. The sequence reads obtained from each strand are then compared for the purpose of obtaining an error-corrected sequence of the original double-stranded nucleic acid molecule, known as a double-stranded consensus sequence (DCS). The process of double-stranded sequencing allows for explicit confirmation that both strands of the original double-stranded nucleic acid molecule appear in the generated sequencing data used to form the DCS.

[0153] In certain embodiments, a method of incorporating a DS may include ligating one or more sequencing adaptors to a target double-stranded nucleic acid molecule comprising a first strand target nucleic acid sequence and a second strand target nucleic acid sequence to generate a double-stranded target nucleic acid complex (e.g., Figure 22A).

[0154] In various embodiments, the resulting target nucleic acid complex may contain at least one SMI sequence, which may be accompanied by an externally applied denatured or semi-denatured sequence (e.g., the randomized double-stranded tags shown in Figure 22A, sequences identified as α and β in Figure 22A), endogenous information related to the specific shear point of the target double-stranded nucleic acid molecule, or a combination thereof. SMIs can render a target nucleic acid molecule substantially distinguishable from multiple other molecules in a sequenced population, either alone or in combination with distinguishing elements of the nucleic acid fragment to which they are ligated. The substantially distinguishing characteristics of SMI elements may be independently possessed by each single strand forming the double-stranded nucleic acid molecule, such that the derivative amplification products of each strand can be recognized as originating from the same original, substantially unique double-stranded nucleic acid molecule after sequencing. In other embodiments, SMIs may contain additional information and / or may be used in other ways in which such molecular distinguishing capabilities are useful, such as those described in the publications cited above. In another embodiment, SMI elements may be incorporated after adapter ligation. In some embodiments, SMIs are essentially double-stranded. In other embodiments, the SMI is essentially single-stranded (e.g., the SMI may be on the single-stranded portion(s) of the adapter). In other embodiments, the SMI is a combination of single-stranded and double-stranded in nature.

[0155] In some embodiments, each double-stranded target nucleic acid sequence complex may further comprise an element (e.g., an SDE) that renders the amplification products of the two single-stranded nucleic acids that form the target double-stranded nucleic acid molecule substantially distinguishable from one another after sequencing. In one embodiment, the SDE may comprise an asymmetric primer site contained within the sequencing adaptor, or in other arrangements, the sequence asymmetry may be introduced within the adaptor molecule rather than within the primer sequence, such that at least a portion of the nucleotide sequences of the first strand target nucleic acid sequence complex and the second strand of the target nucleic acid sequence complex differ from one another after amplification and sequencing. In other embodiments, the SDE may comprise another biochemical asymmetry between the two strands that differs from the canonical nucleotide sequence A, T, C, G, or U, but that translates into at least one canonical nucleotide sequence difference in the two amplified and sequenced molecules. In yet another embodiment, the SDE may be a means for physically separating the two strands prior to amplification, such that the derivative amplification products from the first strand target nucleic acid sequence and the second strand target nucleic acid sequence remain substantially physically isolated from one another for the purpose of maintaining distinction between these two sequences. Other such configurations and methodologies for providing an SDE function that allows for distinction between the first and second strands, such as those described in the publications cited above, or other methods that accomplish the described functional purpose, may also be utilized.

[0156] After creating a double-stranded target nucleic acid complex containing at least one SMI and at least one SDE, or if one or both of these elements are subsequently introduced, the complex may be subjected to DNA amplification (e.g., using PCR) or any other biochemical method of DNA amplification (e.g., rolling circle amplification, multiple displacement amplification, isothermal amplification, bridge amplification, or surface-bound amplification), resulting in the production of one or more copies of the first-strand target nucleic acid sequence and one or more copies of the second-strand target nucleic acid sequence (e.g., Figure 22B).The one or more amplified copies of the first-strand target nucleic acid molecule and one or more amplified copies of the second target nucleic acid molecule can then be subjected to DNA sequencing, preferably using a "next-generation" massively parallel DNA sequencing platform (e.g., Figure 22B).

[0157] Sequence reads generated from either the first strand target nucleic acid molecule or the second strand target nucleic acid molecule derived from an original double-stranded target nucleic acid molecule can be identified based on a common, related, substantially unique SMI and distinguished from the opposite strand target nucleic acid molecule by SDE. In some embodiments, the SMI can be a sequence based on a mathematically based error-correcting code (e.g., a Hamming code), whereby certain amplification errors, sequencing errors, or SMI synthesis errors can be tolerated for the purpose of relating the sequence of the SMI sequence to the complementary strand of the original double strand (e.g., a double-stranded nucleic acid molecule). For example, if the SMI includes a double-stranded exogenous SMI containing 15 base pairs of a fully denatured sequence of canonical DNA bases, then approximately 4 to the power of 15 = 1,073,741,824 SMI variants exist in the set of fully denatured SMIs. If two SMIs are recovered from a sequencing data read that differs by only one nucleotide in the SMI sequence from a set of 10,000 sampled SMIs, the probability of this occurring by chance can be mathematically calculated, and a determination can be made as to whether this single base pair difference is likely to reflect one of the types of errors described above, and whether the SMI sequences are derived from the same original double-stranded molecule. In some embodiments where the SMI is at least partially an externally applied sequence, and its sequence variants are not completely degenerate from each other, but are at least partially a known sequence, the identity of the known sequence can be designed in such a way that one or more of the above-mentioned types of errors do not convert the identity of one known SMI sequence to the identity of another SMI sequence, thereby reducing the probability that one SMI will be mistaken for another SMI. In some embodiments, this SMI design strategy involves a Hamming code approach or its derivatives. Once identified, one or more sequence reads made from the first strand target nucleic acid molecule are compared with one or more sequence reads made from the second strand target nucleic acid molecule to generate an error-corrected target nucleic acid molecule sequence (e.g., Figure 22C).For example, nucleotide positions where bases from both the first strand target nucleic acid sequence and the second strand target nucleic acid sequence match are considered to be true sequences, while nucleotide positions where there is no match between the two strands are considered to be potential technical error sites, which may be ignored, excluded, corrected, or otherwise identified. In this manner, an error-corrected sequence of the original double-stranded target nucleic acid molecule can be generated (as shown in Figure 22C). In some embodiments, after separately grouping each sequencing read generated from the first strand target nucleic acid molecule and the second strand target nucleic acid molecule, a single-stranded consensus sequence can be created for each of the first and second strands. The single-stranded consensus sequences derived from the first strand target nucleic acid molecule and the second strand target nucleic acid molecule can then be compared to generate an error-corrected target nucleic acid molecule sequence (e.g., Figure 22C).

[0158] Alternatively, in some embodiments, the sequence mismatch between the two strands can be recognized as a potential biologically derived mismatch in the original double-stranded target nucleic acid molecule. Alternatively, in some embodiments, the sequence mismatch between the two strands can be recognized as a potential DNA synthesis-derived mismatch in the original double-stranded target nucleic acid molecule. Alternatively, in some embodiments, the sequence mismatch between the two strands can be recognized as a potential site where damaged or modified nucleotide bases are present in one or both strands and have been converted into mismatches by an enzymatic process (e.g., DNA polymerase, DNA glycosylase, or another nucleic acid-modifying enzyme or chemical process). In some embodiments, this latter finding can be used to infer the presence of nucleic acid damage or nucleotide modification prior to the enzymatic process or chemical treatment.

[0159] In some embodiments, according to aspects of the present technology, sequencing reads generated from the double-stranded sequencing process described herein can be further filtered to remove sequencing reads from DNA-damaging molecules (e.g., damaged during storage, transportation, during or after tissue or blood extraction, during or after library preparation, etc.). For example, DNA repair enzymes, such as uracil-DNA glycosylase (UDG), formamidopyrimidine DNA glycosylase (FPG), and 8-oxoguanine DNA glycosylase (OGG1), can be used to remove or correct DNA damage (e.g., in vitro DNA damage or in vivo damage). These DNA repair enzymes are glycosylases that remove damaged bases from DNA. For example, UDG removes uracil resulting from cytosine deamination (resulting from spontaneous hydrolysis of cytosine), and FPG removes 8-oxo-guanine (e.g., a common DNA lesion resulting from reactive oxygen species). FPG also possesses lyase activity, which can generate one-base gaps at abasic sites. Such abasic sites generally cannot be subsequently amplified by PCR, for example, because the polymerase cannot copy the template. Therefore, the use of such DNA damage repair / removal enzymes can effectively remove damaged DNA that does not contain true mutations but may otherwise go undetected as errors after sequencing and double-stranded sequence analysis. While errors resulting from damaged bases can often be corrected by double-stranded sequencing, it is theoretically possible that complementary errors may occur at the same position on both strands, thereby reducing the probability of artifacts by reducing the damage that increases errors. Furthermore, during library preparation, certain fragments of DNA to be sequenced may be single-stranded from their source or from processing steps (e.g., mechanical DNA shearing). These regions are typically converted to double-stranded DNA during the "end repair" process known in the art, in which DNA polymerase and nucleoside substrates are added to the DNA sample to extend the 5' recessed end.Mutagenic sites of DNA damage in the single-stranded portions of the DNA being copied (i.e., single-stranded 5' overhangs at one or both ends of the DNA duplex or internal single-stranded nicks or gaps) can generate errors during the end-blunting reaction, resulting in single-strand mutations, synthesis errors, or nucleic acid damage sites in double-stranded form that can be mistakenly interpreted as true mutations in the final double-stranded consensus sequence, even though true mutations were not actually present in the original double-stranded nucleic acid molecule. This situation is called a "false duplex" and can be reduced or prevented by the use of enzymes that destroy / repair such damage. In other embodiments, this occurrence can be reduced or eliminated by the use of strategies to destroy or prevent single-stranded sites from forming in the original double-stranded molecule (e.g., the use of specific enzymes used to fragment the original double-stranded nucleic acid material rather than mechanical shearing or certain other enzymes that may leave nicks or gaps). In other embodiments, processes to eliminate the single-stranded portions of the original double-stranded nucleic acid (e.g., the use of single-strand specific nucleases such as S1 nuclease or mung bean nuclease) can be utilized for similar purposes.

[0160] In a further embodiment, the sequencing reads generated from the double-strand sequencing process described herein can be further filtered to eliminate false mutations by trimming the ends of the reads that are most prone to false double-strand artifacts. For example, DNA fragmentation can generate single-stranded portions at the ends of double-stranded molecules. These single-stranded portions can be blunted during end repair (e.g., by Klenow or T4 polymerase). In some cases, the polymerase may cause copying errors in these end-repaired regions, resulting in the creation of "false double-stranded molecules." These library preparation artifacts may erroneously appear to be true mutations when sequenced. Errors resulting from these end-repair mechanisms can be eliminated or reduced from post-sequencing analysis by trimming the ends of sequencing reads to exclude mutations that may occur in regions with higher risk, thereby reducing the number of false mutations. In one embodiment, such trimming of sequencing reads can be achieved automatically (e.g., as a routine processing step). In another embodiment, variant frequencies can be assessed for fragment end regions, and if a threshold level of mutations is observed in this fragment end region, trimming of the sequencing reads can be performed before generating double-stranded consensus sequence reads for the DNA fragment.

[0161] As a specific example, in some embodiments, provided herein are methods for generating error-corrected sequence reads for a double-stranded target nucleic acid material, the methods comprising: ligating the double-stranded target nucleic acid material to at least one adapter sequence to form an adapter-target nucleic acid material complex, wherein the at least one adapter sequence comprises: (a) a denatured or semi-denatured single molecule identifier (SMI) sequence that uniquely labels each molecule of the double-stranded target nucleic acid material; and (b) a first nucleotide adapter sequence that tags a first strand of the adapter-target nucleic acid material complex and a second nucleotide adapter sequence that is at least partially non-complementary to the first nucleotide sequence that tags the second strand of the adapter-target nucleic acid material complex, such that each strand of the adapter-target nucleic acid material complex has a nucleotide sequence that is separately distinguishable from its complementary strand. The methods may then comprise amplifying each strand of the adapter-target nucleic acid material complex to generate a plurality of first strand adapter-target nucleic acid complex amplicons and a plurality of second strand adapter-target nucleic acid complex amplicons. The method may further include amplifying both the first strand and the second strand to provide a first nucleic acid product and a second nucleic acid product. The method may also include sequencing each of the first nucleic acid product and the second nucleic acid product to generate a plurality of first strand sequence reads and a plurality of second strand sequence reads, and confirming the presence of at least one first strand sequence read and at least one second strand sequence read. The method may further include generating an error-corrected sequence read of the double-stranded target nucleic acid material by comparing the at least one first strand sequence read with the at least one second strand sequence read and disregarding mismatched nucleotide positions, or alternatively, removing the compared first strand sequence read and the second strand sequence read where the compared first strand sequence read and the second strand sequence read have one or more non-complementary nucleotide positions.

[0162] As a further specific example, in some embodiments, provided herein are methods for identifying DNA variants from a sample, the methods including: ligating both strands of a nucleic acid material (e.g., a double-stranded target DNA molecule) to at least one asymmetric adapter molecule to form an adapter-target nucleic acid material complex having a first nucleotide sequence associated with a first strand (e.g., the top strand) of the double-stranded target DNA molecule and a second nucleotide sequence that is at least partially non-complementary to the first nucleotide sequence associated with a second strand (e.g., the bottom strand) of the double-stranded target DNA molecule; and amplifying each strand of the adapter-target nucleic acid material to generate a separate yet related set of amplified adapter-target nucleic acid products for each strand. The method may further include sequencing each of the plurality of first strand adaptor-target nucleic acid products and the plurality of second strand adaptor-target nucleic acid products; confirming the presence of at least one amplified sequence read from each strand of the adaptor-target nucleic acid material complex; and comparing the at least one amplified sequence read obtained from the first strand with the at least one amplified sequence read obtained from the second strand to form a consensus sequence read for the nucleic acid material (e.g., the double-stranded target DNA molecule) having only nucleotide bases that match in sequence on both strands of the nucleic acid material (e.g., the double-stranded target DNA molecule), such that a variant occurring at a particular position in the consensus sequence read (e.g., compared to a reference sequence) is identified as a true DNA variant.

[0163] In some embodiments, provided herein are methods for generating high-precision consensus sequences from double-stranded nucleic acid material, the methods comprising: tagging individual double-stranded DNA molecules with adapter molecules to form tagged DNA material, wherein each adapter molecule comprises: (a) a denatured or semi-denatured single molecule identifier (SMI) that uniquely labels the double-stranded DNA molecule; and (b) for each tagged DNA molecule, first and second non-complementary nucleotide adapter sequences that distinguish the original top strand from the original bottom strand of each individual DNA molecule within the tagged DNA material; and generating a set of overlaps of the original top strands of the tagged DNA molecules and a set of overlaps of the original bottom strands of the tagged DNA molecules to form amplified DNA material. The method may further include the steps of creating a first single-stranded consensus sequence (SSCS) from the overlap of the original top strand and a second single-stranded consensus sequence (SSCS) from the overlap of the original bottom strand, comparing the first SSCS of the original top strand with the second SSCS of the original bottom strand, and creating a highly accurate consensus sequence having only nucleotide bases that are complementary in sequence to both the first SSCS of the original top strand and the second SSCS of the original bottom strand.

[0164] In a further embodiment, provided herein is a method for detecting and / or quantifying DNA damage from a sample containing double-stranded target DNA molecules, the method comprising: ligating both strands of each double-stranded target DNA molecule to at least one asymmetric adapter molecule to form a plurality of adapter-target DNA complexes, wherein each adapter-target DNA complex has a first nucleotide sequence associated with a first strand of the double-stranded target DNA molecule and a second nucleotide sequence that is at least partially non-complementary to the first nucleotide sequence associated with a second strand of the double-stranded target DNA molecule; and, for each adapter-target DNA complex, amplifying each strand of the adapter-target DNA complex to produce a separate yet related set of amplified adapter-target DNA amplicons for each strand. The method may further include sequencing each of the plurality of first strand adaptor-target DNA amplicons and the plurality of second strand adaptor-target DNA amplicons; confirming the presence of at least one sequence read from each strand of the adaptor-target DNA complex; and comparing the at least one sequence read obtained from the first strand with the at least one sequence read obtained from the second strand to detect and / or quantify mismatched (e.g., non-complementary) nucleotide bases in the sequence read of one strand of the double-stranded DNA molecule, such that site(s) of DNA damage can be detected and / or quantified. In some embodiments, the method may further include generating a first single-stranded consensus sequence (SSCS) from the first strand adaptor-target DNA amplicon and a second single-stranded consensus sequence (SSCS) from the second strand adaptor-target DNA amplicon; comparing the first SSCS of the original first strand with the second SSCS of the original second strand; and identifying nucleotide bases where the sequences of the first SSCS and the second SSCS are non-complementary to detect and / or quantify DNA damage associated with the double-stranded target DNA molecule in the sample.

[0165] Single Molecule Identifier (SMI) According to various embodiments, the provided methods and compositions include one or more SMI sequences on each strand of a nucleic acid material. SMIs may be independently present on each single strand obtained from a double-stranded nucleic acid molecule, such that the derivative amplification products of each strand can be recognized as originating from the same original, substantially unique double-stranded nucleic acid molecule after sequencing. In some embodiments, SMIs may contain additional information and / or may be used in other ways in which the ability to distinguish such molecules is useful, as will be recognized by those skilled in the art. In some embodiments, SMI elements may be incorporated before, substantially simultaneously with, or after ligation of an adapter sequence to the nucleic acid material.

[0166] In some embodiments, the SMI sequence may comprise at least one denatured or semi-denatured nucleic acid. In other embodiments, the SMI sequence may not be denatured. In some embodiments, the SMI may be a sequence associated with or near the fragment end of a nucleic acid molecule (e.g., a randomly or semi-randomly sheared end of a ligated nucleic acid material). In some embodiments, an exogenous sequence may be considered in combination with a sequence corresponding to a randomly or semi-randomly sheared end of a ligated nucleic acid material (e.g., DNA) to obtain an SMI sequence that can distinguish single DNA molecules from each other. In some embodiments, the SMI sequence is part of an adapter sequence ligated to a double-stranded nucleic acid molecule. In certain embodiments, the adapter sequence containing the SMI sequence is double-stranded, such that each strand of the double-stranded nucleic acid molecule contains an SMI after ligation to the adapter sequence. In another embodiment, the SMI sequence may be single-stranded before or after ligation to a double-stranded nucleic acid molecule, and a complementary SMI sequence may be created by extending the opposite strand with a DNA polymerase to obtain a complementary double-stranded SMI sequence. In other embodiments, the SMI sequence is in the single-stranded portion of the adaptor (e.g., the arm of a Y-shaped adaptor). In such embodiments, the SMI may facilitate grouping of families of sequence reads derived from the original strand of the double-stranded nucleic acid molecule, and in some cases, may provide a relationship between the original first and second strands of the double-stranded nucleic acid molecule (e.g., all or part of the SMI may be related by a lookup table). In embodiments, when the first and second strands are labeled with different SMIs, the sequence reads from the two original strands may be related using one or more endogenous SMIs (e.g., fragment-specific features such as sequences associated with or near the fragment ends of a nucleic acid molecule), or may be related through the use of additional molecular tags shared by the two original strands (e.g., barcodes in the double-stranded portions of the adapters), or a combination thereof.In some embodiments, each SMI sequence may comprise from about 1 to about 30 nucleic acids (eg, 1, 2, 3, 4, 5, 8, 10, 12, 14, 16, 18, 20, or more denatured or semi-denatured nucleic acids).

[0167] In some embodiments, the SMI can be ligated to one or both of the nucleic acid material and the adapter sequence. In some embodiments, the SMI can be ligated to at least one of a T-overhang, an A-overhang, a CG-overhang, an overhang including a "sticky end" or single-stranded overhang region having a known nucleotide length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides), a dehydroxylated base, and a blunt end of the nucleic acid material.

[0168] In some embodiments, the sequence of the SMI may be conceived (or designed) in combination with sequences corresponding to randomly or semi-randomly sheared ends of, for example, nucleic acid materials (e.g., ligated nucleic acid materials) to obtain SMI sequences that are capable of distinguishing single nucleic acid molecules from one another.

[0169] In some embodiments, at least one SMI may be an endogenous SMI (e.g., an SMI associated with a shear point (e.g., a fragment end)), e.g., with the shear point itself or with a predetermined number of nucleotides in the nucleic acid material immediately adjacent to the shear point (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides from the shear point). In some embodiments, at least one SMI may be an exogenous SMI (e.g., an SMI comprising a sequence not found in the target nucleic acid material).

[0170] In some embodiments, the SMI may be or include an imaging moiety (e.g., a fluorescent or otherwise optically detectable moiety), hi some embodiments, such an SMI allows for detection and / or quantification without the need for an amplification step.

[0171] In some embodiments, the SMI element may comprise two or more separate SMI elements located at different locations on the adaptor-target nucleic acid complex.

[0172] Various embodiments of SMIs are further disclosed in International Patent Publication No. WO2017 / 100441, which is incorporated herein by reference in its entirety.

[0173] Chain Definition Element (SDE) In some embodiments, each strand of a double-stranded nucleic acid material may further comprise an element that renders the amplification products of the two single-stranded nucleic acids that form the target double-stranded nucleic acid material substantially distinguishable from one another after sequencing. In some embodiments, the SDE may be or comprise an asymmetric primer site contained within a sequencing adaptor, or in other arrangements, the sequence asymmetry may be introduced within the adaptor sequence rather than within the primer sequence, such that at least a portion of the nucleotide sequences of the first strand target nucleic acid sequence complex and the second strand of the target nucleic acid sequence complex differ from one another after amplification and sequencing. In other embodiments, the SDE may comprise another biochemical asymmetry between the two strands that differs from the canonical nucleotide sequence A, T, C, G, or U, but that translates into at least one canonical nucleotide sequence difference in the two amplified and sequenced molecules. In yet another embodiment, the SDE may be or include a means for physically separating the two strands prior to amplification, such that the derivative amplification products from the first strand target nucleic acid sequence and the second strand target nucleic acid sequence remain substantially physically isolated from one another for the purposes of maintaining distinction between the two derivative amplification products. Other such arrangements or methodologies for providing an SDE function that allows for distinction between the first and second strands may also be utilized.

[0174] In some embodiments, the SDE may be capable of forming a loop (e.g., a hairpin loop). In some embodiments, the loop may include at least one endonuclease recognition site. In some embodiments, the target nucleic acid complex may contain an endonuclease recognition site that facilitates a cleavage event within the loop. In some embodiments, the loop may include a non-canonical nucleotide sequence. In some embodiments, the contained non-canonical nucleotides may be recognizable by one or more enzymes that facilitate strand cleavage. In some embodiments, the contained non-canonical nucleotides may be targeted by one or more chemical processes that facilitate strand cleavage within the loop. In some embodiments, the loop may contain a modified nucleic acid linker that can be targeted by one or more enzymatic, chemical, or physical processes that facilitate strand cleavage within the loop. In some embodiments, the modified linker is a photocleavable linker.

[0175] Various other molecular tools can function as SMIs and SDEs. In addition to shear points and DNA-based tags, single-molecule compartmentalization methods that maintain paired strands in physical proximity, or other non-nucleic acid tagging methods, can perform strand-related functions. Similarly, asymmetric chemical labeling of adapter strands in a manner that can physically separate them, can serve as an SDE. A recently described variant of double-stranded sequencing uses bisulfite conversion to convert naturally occurring strand asymmetry in the form of cytosine methylation into sequence differences that distinguish the two strands. While this embodiment is limited in the types of mutations that can be detected, this concept of utilizing natural asymmetry is noteworthy in terms of the emergence of sequencing technologies that can directly detect modified nucleotides. Various embodiments of SDEs are further disclosed in International Patent Publication No. WO2017 / 100441, the entire contents of which are incorporated herein by reference.

[0176] Adapters and adapter sequences Adapter molecules containing SMIs (e.g., molecular barcodes), SDEs, primer sites, flow cell sequences, and / or other features in various configurations are contemplated for use with many of the embodiments disclosed herein. In some embodiments, the provided adapters may be or include one or more sequences complementary or at least partially complementary to PCR primers (e.g., primer sites) that have at least one of the following properties: (1) high target specificity, (2) the ability to be multiplexed, and (3) the ability to exhibit stable, minimally biased amplification.

[0177] In some embodiments, adapter molecules may be "Y" shaped, "U" shaped, "hairpin" shaped, have a bubble (e.g., a portion of the sequence that is not complementary), or have other features. In other embodiments, adapter molecules may be "Y" shaped, "U" shaped, "hairpin" shaped, or contain a bubble. Certain adapters may contain modified or non-standard nucleotides, restriction sites, or other features for in vitro structural or functional engineering. Adapter molecules may be ligated to various nucleic acid entities having ends. For example, adapter molecules may be suitable for ligating to T-overhangs, A-overhangs, CG-overhangs, multi-nucleotide overhangs (also referred to herein as "sticky ends" or "sticky overhangs"), dehydroxylated bases, or blunt ends of nucleic acid entities, or the ends of the molecules may be 5' dephosphorylated or otherwise blocked from conventional ligation. In other embodiments, the adapter molecule may contain dephosphorylation or another modification on the 5' strand of the ligation site that prevents ligation. In the latter two embodiments, such a strategy may be useful to prevent dimerization of the library fragments or the adapter molecule.

[0178] In some embodiments, the adapter molecule may comprise a capture moiety suitable for isolating a desired target nucleic acid molecule ligated to the capture molecule.

[0179] An adapter sequence may refer to a single-stranded sequence, a double-stranded sequence, a complementary sequence, a non-complementary sequence, a partially complementary sequence, an asymmetric sequence, a sequence that binds to a primer, a flow cell sequence, a ligation sequence, or other sequence provided by an adapter molecule. In certain embodiments, an adapter sequence may refer to a sequence used for amplification by complementarity to an oligonucleotide.

[0180] In some embodiments, the methods and compositions provided include at least one adapter sequence (e.g., two adapter sequences, one for each of the 5' and 3' ends of the nucleic acid material). In some embodiments, the methods and compositions provided may include two or more adapter sequences (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, at least two of the adapter sequences differ from each other (e.g., by sequence). In some embodiments, each adapter sequence differs from each other (e.g., by sequence). In some embodiments, at least one adapter sequence is at least partially non-complementary to at least a portion of at least one other adapter sequence (e.g., not complementary by at least one nucleotide).

[0181] In some embodiments, the adapter sequence comprises at least one non-standard nucleotide, in some embodiments, the non-standard nucleotide is an abasic site, uracil, tetrahydrofuran, 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-A), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-G), deoxyinosine, 5'nitroindole, 5-hydroxymethyl-2'-deoxycytidine, iso-cytosine, 5'-methyl-isocytosine, or isoguanosine, a methylated nucleotide, an RNA nucleotide, a ribose nucleotide, an 8-oxo-guanine, a photocleavable linker, a biotinylated nucleotide, a desthiobiotin nucleotide, a thiol-modified nucleotide, or a nucleotide sequence. The nucleotides are selected from nucleotides such as oxidase, acrydite modified nucleotides, iso-dC, isodG, 2'-O-methyl nucleotides, inosine nucleotide locked nucleic acids, peptide nucleic acids, 5 methyl dC, 5-bromodeoxyuridine, 2,6-diaminopurine, 2-aminopurine nucleotides, abasic nucleotides, 5-nitroindole nucleotides, adenylated nucleotides, azido nucleotides, digoxigenin nucleotides, I-linkers, 5' hexynyl modified nucleotides, 5-octadiynyl dU, photocleavable spacers, non-photocleavable spacers, click chemistry compatible modified nucleotides, and any combination thereof.

[0182] In some embodiments, the adapter sequence includes a portion having magnetic properties (i.e., a magnetic portion). In some embodiments, the magnetic property is paramagnetic. In some embodiments, where the adapter sequence includes a magnetic portion (e.g., a nucleic acid substance that ligates to an adapter sequence that includes a magnetic portion), when a magnetic field is applied, the adapter sequence that includes the magnetic portion is substantially separated from the adapter sequence that does not include the magnetic portion (e.g., a nucleic acid substance that ligates to an adapter sequence that does not include the magnetic portion).

[0183] In some embodiments, at least one adapter sequence is located 5' to the SMI. In some embodiments, at least one adapter sequence is located 3' to the SMI.

[0184] In some embodiments, the adaptor sequence may be connected to at least one of the SMI and the nucleic acid material via one or more linker domains. In some embodiments, the linker domain may be composed of nucleotides. In some embodiments, the linker domain may include at least one modified nucleotide or non-nucleotide molecule (e.g., those described elsewhere in this disclosure). In some embodiments, the linker domain may be or include a loop.

[0185] In some embodiments, the adapter sequences on one or both ends of each strand of a double-stranded nucleic acid material may further comprise one or more elements that provide an SDE, which in some embodiments may be or include an asymmetric primer site contained within the adapter sequence.

[0186] In some embodiments, the adapter sequence may be or may include at least one SDE and at least one ligation domain (i.e., at least one domain amendable to ligase activity, e.g., a domain suitable for ligation to a nucleic acid material by ligase activity). In some embodiments, from 5' to 3', the adapter sequence may be or may include a primer binding site, an SDE, and a ligation domain.

[0187] Various methods for synthesizing double-stranded sequencing adapters have been previously described, for example, in U.S. Pat. No. 9,752,188, International Patent Publication No. WO2017 / 100441, and International Patent Application No. PCT / US18 / 59908 (filed November 8, 2018), all of which are incorporated herein by reference in their entireties.

[0188] Primer In some embodiments, one or more PCR primers having at least one of the following characteristics are contemplated for use in various embodiments according to the present technology: (1) high target specificity, (2) multiplexing capability, and (3) stable amplification with minimal bias. Many conventional tests and commercial products have designed primer mixtures that meet these specific criteria for conventional PCR-CE. However, it should be noted that these primer mixtures are not always suitable for use with MPS. In fact, developing a highly multiplexed primer mixture can be a challenging and time-consuming process. Conveniently, both Illumina and Promega have recently developed multiplex-compatible primer mixtures for the Illumina platform that demonstrate stable and efficient amplification of various standard and non-standard STR and SNP loci. Because these kits use PCR to amplify their target regions before sequencing, the 5' end of each read in paired-end sequencing data corresponds to the 5' end of the PCR primer used to amplify the DNA. In some embodiments, the provided methods and compositions include primers designed to ensure uniform amplification, which may involve varying reaction concentrations, melting temperatures, and minimizing secondary structure and intra- or inter-primer interactions. Many techniques have been described for highly multiplexed primer optimization for MPS applications. In particular, these techniques are often known as ampliseq methods, as they are well documented in the art.

[0189] amplification The provided methods and compositions, in various embodiments, utilize or are used in at least one amplification step that amplifies nucleic acid material (or a portion thereof, e.g., a specific target region or locus) to form amplified nucleic acid material (e.g., some members of an amplicon product).

[0190] In some embodiments, amplifying the nucleic acid material includes amplifying the nucleic acid material derived from each of the first and second nucleic acid strands from the original double-stranded nucleic acid material using at least one single-stranded oligonucleotide that is at least partially complementary to a sequence present in the first adapter sequence, such that the SMI sequence is at least partially maintained. The amplifying step further includes amplifying each strand of interest using a second single-stranded oligonucleotide, where such second single-stranded oligonucleotide may (a) be at least partially complementary to the target sequence of interest, or (b) be at least partially complementary to a sequence present in the second adapter sequence, such that the at least one single-stranded oligonucleotide and the second single-stranded oligonucleotide are oriented in a manner to effectively amplify the nucleic acid material.

[0191] In some embodiments, amplifying nucleic acid material in a sample may include amplifying the nucleic acid material in a "tube" (e.g., a PCR tube), in an emulsion droplet, in a microchamber, and other examples described above or in other known containers. In some embodiments, amplifying nucleic acid material may include amplifying nucleic acid material in two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more samples) physically separated samples (e.g., tubes, droplets, chambers, containers, etc.). For example, prior to the amplification step, the initial sample may be separated into multiple containers. In some embodiments, each sample contains substantially the same amount of amplified nucleic acid material as each other sample, and in some embodiments, at least two samples contain substantially different amounts of amplified nucleic acid material.

[0192] In some embodiments, at least one amplifying step includes at least one primer that is or includes at least one non-standard nucleotide, in some embodiments, the non-standard nucleotide is selected from uracil, a methylated nucleotide, an RNA nucleotide, a ribose nucleotide, an 8-oxo-guanine, a biotinylated nucleotide, a locked nucleic acid, a peptide nucleic acid, a high Tm nucleic acid variant, an allele-distinguishing nucleic acid variant, any other nucleotide or linker variant described elsewhere herein, or any combination thereof.

[0193] While it is contemplated that any amplification reaction suitable for any application is compatible with some embodiments, by way of example, in some embodiments the amplification step may be or include polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), isothermal amplification, polony amplification in emulsion, bridge amplification on a surface, on a bead or on a surface in a hydrogel, and any combination thereof.

[0194] In some embodiments, amplifying the nucleic acid material involves the use of single-stranded oligonucleotides that are at least partially complementary to regions of an adapter sequence on the 5' and 3' ends of each strand of the nucleic acid material. In some embodiments, amplifying the nucleic acid material involves the use of at least one single-stranded oligonucleotide that is at least partially complementary to a target region or sequence of interest (e.g., a genomic sequence, a mitochondrial sequence, a plasmid sequence, a synthetically produced target nucleic acid, etc.) and a single-stranded oligonucleotide that is at least partially complementary to a region of the adapter sequence (e.g., a primer site).

[0195] In general, stable amplification (e.g., PCR amplification) can be highly dependent on reaction conditions. Multiplex PCR can be sensitive to, for example, buffer composition, monovalent or divalent cation concentration, detergent concentration, crowding agent (i.e., PEG, glycerol, etc.) concentration, primer concentration, primer Tm, primer design, primer GC content, primer modified nucleotide characteristics, and cycling conditions (i.e., temperature and extension time, and temperature change rate). Optimizing buffer conditions can be a difficult and time-consuming process. In some embodiments, amplification reactions may use at least one of buffer, primer pool concentration, and PCR conditions according to an already known amplification protocol. In some embodiments, new amplification protocols may be created and / or amplification reaction optimization may be used. As a specific example, in some embodiments, PCR optimization kits, such as those from Promega®, may be used, which include many pre-formulated buffers partially optimized for various PCR applications (e.g., multiplex, real-time, GC-rich, and inhibitor-resistant amplification). These pre-formulated buffers contain various Mg 2+ The concentration and primer concentration, as well as the primer pool ratio, can be rapidly replenished. In addition, in some embodiments, various cycling conditions (e.g., thermal cycling) may be evaluated and / or used. When evaluating whether a particular embodiment is suitable for a particular desired application, one or more of the following may be evaluated: specificity, allele coverage ratio to heterozygous loci, interlocus balance, and depth, among other aspects. Measurement of amplification success may include DNA sequencing of the product, evaluation of the product by visualization of fragments after gel or capillary electrophoresis or HPLC or other size separation methods, melting curve analysis using dyes or fluorescent probes that bind to double-stranded nucleic acids, mass spectrometry, or other methods known in the art.

[0196] According to various embodiments, any of a variety of factors can affect the length of a particular amplification step (e.g., the number of cycles in a PCR reaction). For example, in some embodiments, the provided nucleic acid material may be damaged or otherwise suboptimal (e.g., degraded and / or contaminated). In such cases, a longer amplification step may help ensure that the desired product is amplified to an acceptable degree. In some embodiments, the amplification step may provide an average of 3 to 10 sequenced PCR copies from each starting DNA molecule, while in other embodiments, only one copy of each of the first and second strands is necessary. While not wishing to be bound by theory, too many or too few PCR copies may reduce assay efficiency and ultimately reduce depth. In general, the number of nucleic acid (e.g., DNA) fragments used in an amplification (e.g., PCR) reaction is the first adjustable variable, which can determine the number of reads sharing the same SMI / barcode sequence.

[0197] Nucleic acid material kinds According to various embodiments, any of a variety of nucleic acid agents may be used. In some embodiments, the nucleic acid agent may include at least one modification to a polynucleotide in the canonical sugar-phosphate backbone. In some embodiments, the nucleic acid agent may include at least one modification in any base within the nucleic acid agent. For example, and not by way of limitation, in some embodiments, the nucleic acid agent is or includes at least one of double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA).

[0198] source It is contemplated that the nucleic acid material may be derived from any of a variety of sources. For example, in some embodiments, the nucleic acid material is provided from a sample from at least one subject (e.g., a human or animal subject) or other biological source. In some embodiments, the nucleic acid material is provided from a preserved / stored sample. In some embodiments, the sample is blood, serum, sweat, saliva, cerebrospinal fluid, mucus, uterine washing, vaginal swab, nasal swab, oral swab, tissue scraping, hair, fingerprint, urine, stool, vitreous fluid, peritoneal washing, sputum, bronchial lavage, oral wash, pleural lavage, gastric lavage, gastric juice, bile, pancreatic duct lavage, bile duct lavage, common bile duct lavage, gallbladder fluid, synovial fluid, infected wound, non-infected wound, archaeological sample, forensic sample, water sample, tissue sample, food sample, bioreactor sample, etc. In other embodiments, the sample is or comprises at least one of a human sample, a plant sample, nail polish, semen, prostatic fluid, fallopian tube washing, cell-free nucleic acid, intracellular nucleic acid, metagenomics sample, implanted foreign body washing, nasal wash, intestinal fluid, epithelial brushing, epithelial wash, tissue biopsy, autopsy sample, necrosis sample, organ sample, human identification ampoule, artificially created nucleic acid sample, synthetic gene sample, nucleic acid data repository sample, tumor tissue, and any combination thereof. In other embodiments, the sample is or comprises at least one of a microorganism, a plant-based organism, or any collected environmental sample (e.g., water, soil, archaeological sample, etc.).

[0199] qualification According to various embodiments, the nucleic acid material may undergo one or more modifications before, substantially simultaneously with, or after any particular step, depending on the application for which the particular provided method or composition is to be used.

[0200] In some embodiments, the modification may be or may include repair of at least a portion of the nucleic acid material. While it is contemplated that methods suitable for any application of nucleic acid repair are compatible with some embodiments, certain exemplary methods and compositions are described below and in the Examples.

[0201] As a non-limiting example, in some embodiments, DNA repair enzymes, such as uracil-DNA glycosylase (UDG), formamidopyrimidine DNA glycosylase (FPG), and 8-oxoguanine DNA glycosylase (OGG1), can be used to repair DNA damage (e.g., in vitro DNA damage). As described above, these DNA repair enzymes are glycosylases that remove damaged bases from DNA. For example, UDG removes uracil resulting from cytosine deamination (caused by spontaneous hydrolysis of cytosine), and FPG removes 8-oxoguanine (e.g., the most common DNA lesion resulting from reactive oxygen species). FPG also has lyase activity and can generate one-base gaps at abasic sites. Such abasic sites cannot be subsequently amplified by PCR, for example, because the polymerase cannot copy the template. Therefore, the use of such DNA damage repair enzymes can effectively remove damaged DNA that does not harbor true mutations but may not otherwise be detected as errors after sequencing and double-strand sequence analysis.

[0202] As mentioned above, in further embodiments, the sequencing reads produced from the processing steps described herein can be further filtered to eliminate false mutations by trimming the ends of the reads that are most prone to artifacts. For example, DNA fragmentation can generate single-stranded portions at the ends of double-stranded molecules. These single-stranded portions can be blunted during end repair (e.g., by Klenow). In some cases, polymerases can cause copying errors in these end-repaired regions, resulting in the creation of "false double-stranded molecules." These artifacts may appear to be true mutations when sequenced. Errors resulting from these end-repair mechanisms can be excluded from post-sequencing analysis by trimming the ends of sequencing reads to eliminate potential mutations, thereby reducing the number of false mutations. In some embodiments, such trimming of sequencing reads can be achieved automatically (e.g., as part of a routine processing step). In some embodiments, variant frequencies can be assessed for fragment end regions, and if a threshold level of mutations is observed in this fragment end region, trimming of the sequencing reads can be performed before generating double-stranded consensus sequence reads for the DNA fragment.

[0203] Some embodiments of the DS method provide a PCR-based targeted enrichment strategy that is compatible with the use of molecular barcodes for error correction. For example, sequencing enrichment strategies utilizing steps from the Separated PCRs of Linked Templates for Sequencing ("SPLiT-DS") method can also benefit from pre-enriched nucleic acid material using one or more of the embodiments described herein. SPLiT-DS was originally described in International Patent Publication No. WO / 2018 / 175997, the entire contents of which are incorporated herein by reference. The SPLiT-DS approach may begin with labeling (e.g., tagging) fragmented double-stranded nucleic acid material (e.g., from a DNA sample) with molecular barcodes in a manner similar to that described above, as per standard DS library construction protocols. In some embodiments, the double-stranded nucleic acid material may be fragmented (e.g., cell-free DNA, damaged DNA, etc.). However, in other embodiments, various steps may include fragmenting the nucleic acid material using mechanical shearing, such as sonication, or other DNA cleavage methods (e.g., those described further herein). Labeling of the fragmented double-stranded nucleic acid material may include end-repair and 3'-dA tailing, followed by ligation of the double-stranded nucleic acid fragments to a DS adapter containing an SMI, if required for a particular application. In other embodiments, the SMI may be endogenous or a combination of exogenous and endogenous sequences, due to uniquely associated information from both strands of the original nucleic acid molecule. After ligation of the adapter molecule to the double-stranded nucleic acid material, the method may be followed by amplification (e.g., PCR amplification, rolling circle amplification, multiple displacement amplification, isothermal amplification, bridge amplification, surface-bound amplification, etc.).

[0204] In certain embodiments, for example, primers specific to one or more adapter sequences may be used to amplify each strand of nucleic acid material resulting in multiple copies of nucleic acid amplicons derived from each strand of the original double-stranded nucleic acid molecule, each amplicon retaining its original associated SMI. After amplification and associated steps for removing reaction by-products, the sample can be divided (preferably, but not necessarily, substantially uniformly) into two or more separate samples (e.g., in tubes, droplets of emulsion, microchambers, isolated droplets on a surface, or other known containers, collectively referred to as "tubes"). After separation, according to one embodiment of the SPLiT-DS process, the method may include amplifying the first strand in the first sample using a primer specific to a first adapter sequence to provide a first nucleic acid product, and amplifying the second strand in the second sample using a primer specific to a second adapter sequence to provide a second nucleic acid product. The method may then include sequencing each of the first and second nucleic acid products and comparing the sequence of the first nucleic acid product with the sequence of the second nucleic acid product. In some embodiments, the nucleic acid material includes an adapter sequence at each of the 5' and 3' ends of each strand of the nucleic acid material. In certain applications, amplification of individual strands in the divided sample can be achieved using single-stranded oligonucleotides at least partially complementary to the target sequence of interest, such that the single molecular identifier sequence is at least partially maintained.

[0205] Selected Applications As described herein, the methods and compositions provided may be used for any of a variety of purposes and / or in any of a variety of contexts. The following describes certain illustrative, non-limiting examples of uses and / or contexts.

[0206] Monitoring response to treatment (e.g., tumor mutations) The advent of next-generation sequencing (NGS) in genomic research has enabled the characterization of tumor mutational landscapes with unprecedented detail, providing a catalog of diagnostic, prognostic, and clinically causative mutations. Collectively, these mutations hold significant promise for improving cancer outcomes through personalized medicine and enabling early cancer detection and screening. Prior to this disclosure, a key limitation in the field was the inability to detect these mutations when they are present at low frequencies. Clinical biopsies often consist largely of normal cells, and detecting cancer cells based on their DNA mutations remains a technical challenge even for modern NGS. Identifying tumor mutations among thousands of normal genomes is like finding a needle in a haystack, requiring a level of sequencing accuracy beyond previously known methods.

[0207] This problem is particularly acute in the case of liquid biopsies, where the challenge is not only to provide the extremely high sensitivity necessary to detect tumor mutations but also to do so using the small amounts of DNA typically present in these biopsies. The term "liquid biopsy" typically refers to blood, which has the ability to provide information about cancer based on the presence of circulating tumor DNA (ctDNA). ctDNA is shed into the bloodstream by cancer cells and shows great promise for monitoring, detecting, and predicting cancer, as well as enabling tumor genotyping and treatment selection. These applications could revolutionize the current management of cancer patients, but progress has been slower than previously expected. A major problem is that ctDNA typically represents a very small fraction of all cell-free DNA (cfDNA) present in plasma. In metastatic cancers, its frequency can exceed 5%, but in localized cancers, it is only 1% to 0.001%. In theory, DNA subpopulations of any size should be detectable by assaying a sufficient number of molecules. However, a fundamental limitation of conventional methods is the high frequency of mis-scored bases. Errors often occur during cluster generation, sequencing cycles, insufficient cluster resolution, and template degradation. As a result, approximately 0.1–1% of sequenced bases are incorrectly called. Further challenges can arise from polymerase errors and amplification bias during PCR, which can result in skewed ensembles or the introduction of erroneous variant allele frequencies (MAFs). Taken together, conventional techniques (including conventional NGS) are unable to perform at the level required to detect low-frequency mutations.

[0208] Due to its high accuracy, DS and the method for improving the conversion efficiency and workflow efficiency of these sequencing platforms have promise in the field of oncology.As described herein, provided methods and compositions enable an innovative approach to DS methodology, incorporating the double-stranded molecular tagging of DS with target nucleic acid enrichment to improve efficiency and scalability while maintaining error correction.

[0209] In addition to the need for highly accurate and efficient assays, the reality of clinical laboratories also requires assays that are rapid, scalable, and reasonably cost-effective. Therefore, various embodiments of the present technology that improve workflow efficiency of DS (e.g., enrichment strategies for DS) are highly desirable. As described herein, digestion / size selection enrichment and affinity-based enrichment of specific target sequences for DS applications provide high target specificity, low DNA input performance, scalability, and minimal cost.

[0210] Some embodiments of the provided methods and compositions are particularly significant for cancer research in general, and for the field of ctDNA in particular, because the techniques developed in the present invention have the ability to identify cancer mutations with unprecedented sensitivity while minimizing DNA input, preparation time and cost.The target nucleic acid enrichment embodiments disclosed herein will be useful for clinical applications that can significantly improve patient management and survival rates through early cancer detection.

[0211] Patient stratification Patient stratification, which generally refers to dividing patients based on one or more treatment-irrelevant factors, is a topic of significant interest in the medical community. Much of this interest stems from the fact that certain therapeutic candidates have failed to receive FDA approval, and in part from previously unrecognized differences between patients undergoing clinical trials. These differences may be, or may include, one or more genetic differences that cause differences in the metabolism of a therapeutic drug or the presence or worsening of side effects in one patient group compared to one or more other patient groups. In some cases, some or all of these differences may be detected as one or more distinct genetic profiles in a patient(s) that cause a different response to a therapeutic drug than other patients who do not display the same genetic profile.

[0212] Thus, in some embodiments, the provided methods and compositions will be useful for determining whether a subject(s) in a particular patient population (e.g., patients suffering from a common disease, disorder, or condition) will respond to a particular therapy. For example, in some embodiments, the provided methods and / or compositions may be used to assess whether a particular subject has a genotype associated with a poor response to treatment. In some embodiments, the provided methods and / or compositions may be used to assess whether a particular subject has a genotype associated with a favorable response to treatment.

[0213] Forensic medicine Traditional approaches to forensic DNA analysis have relied almost entirely on capillary electrophoretic separation of PCR amplicons to identify length polymorphisms in short tandem repeat sequences. This type of analysis has proven extremely valuable since its introduction in 1991. Since then, several publications have introduced standardized protocols, validated their use in laboratories worldwide, detailed their use in many different populations, and introduced more efficient methods, such as miniSTRs.

[0214] Although this approach has proven highly successful, the technology suffers from several drawbacks that limit its usefulness. For example, current approaches to STR genotyping often produce background signal resulting from PCR stutter caused by polymerase sliding on the template DNA. This challenge is particularly significant in samples with two or more contributing factors, as it is difficult to distinguish stutter alleles from true alleles. Another challenge arises when analyzing degraded DNA samples. Fragment length variation often results in significantly fewer or absent longer PCR fragments. As a result, profiles from degraded DNA often have poor discriminatory power.

[0215] The introduction of MPS systems has the potential to address several challenging challenges in forensic analysis. For example, these platforms offer unparalleled capabilities for simultaneous analysis of STRs and SNPs in nuclear and mtDNA, significantly enhancing the ability to discriminate between individuals and potentially determining ethnicity and even physical attributes. Furthermore, unlike PCR-CE, which simply reports the aggregated average genotype of a collection of molecules, MPS technology digitally tabulates the entire nucleotide sequence of many individual DNA molecules, thereby providing the unique ability to detect MAFs in heterogeneous DNA mixtures. Because forensic specimens containing two or more contributing factors remain one of the most problematic challenges in forensic science, the impact of MPS on the field of forensic science could be profound.

[0216] The publication of the human genome highlighted the immense power of MPS platforms. However, until fairly recently, the full power of these platforms was limited in forensic applications due to read lengths significantly shorter than those of STR loci, precluding the ability to call genotypes based on length. Initially, pyrosequencers, such as the Roche 454 platform, were the only platforms with sufficient read lengths to sequence core STR loci. However, as read lengths have increased in the technology race, their utility for forensic applications has begun to emerge. Several studies have demonstrated the potential for MPS genotyping of STR loci. Overall, the general result of all these studies is that, regardless of the platform, STRs can successfully produce genotypes comparable to CE analysis, even from flawed forensic samples.

[0217] While these studies all demonstrate agreement with conventional PCR-CE techniques and additional advantages, such as the detection of SNPs within STRs, they also highlight some current challenges with this technology. For example, current MPS approaches for STR genotyping rely on multiplex PCR to provide sufficient DNA for sequencing and introduction of PCR primers. However, because multiplex PCR kits were designed for PCR-CE, they contain primers for amplicons of various sizes. This variation can lead to coverage imbalances that favor the amplification of smaller fragments and can result in allelic dropout. In fact, recent studies have shown that differences in PCR efficiency can affect the components of a mixture, especially at low MAFs. To address this challenge, several sequencing kits specifically designed for forensic use are now commercially available, and validation studies are beginning to be reported. However, amplification bias remains evident due to the high level of multiplexing.

[0218] Like PCR-CE, MPS is susceptible to the occurrence of PCR stutter. The majority of MPS studies on STRs have reported the occurrence of artificially dropped-in alleles. In recent years, systematic MPS studies have reported that most stutter events manifest as shorter polymorphisms that differ from the true allele by four base pair units, the most common being at n-4, although n-8 and n-12 positions have also been observed. The rate of stutter typically occurred in approximately 1% of reads but can be as high as 3% at some loci, indicating that MPS may exhibit a higher rate of stutter than PCR-CE.

[0219] In contrast, in some embodiments, the provided methods and compositions allow for high-quality and efficient sequencing of low-quality and / or small-volume samples, as described above and in the Examples below. Thus, in some embodiments, the provided methods and / or compositions may be useful for detecting rare variants in DNA from one individual mixed in small amounts with DNA from another individual with a different genotype.

[0220] Forensic DNA samples generally contain non-human DNA. Potential sources of this foreign DNA include the source of the DNA (e.g., microorganisms in saliva or oral samples), the surface environment where the sample was collected, and laboratory contamination (e.g., reagents, work areas, etc.). Another aspect provided by some embodiments is that certain provided methods and compositions enable the identification of contaminating nucleic acid material from other sources (e.g., other species) and / or surface or environmental contamination, such that these materials (and / or their effects) can be removed from the final analysis and do not bias the sequencing results.

[0221] In highly degraded DNA, locus-specific PCR may not function well due to DNA fragments that do not contain the necessary primer annealing sites, which may cause allele dropout. This situation limits the uniqueness of genotype calls, especially in mixed-sample testing, and the reliability of matches is low. However, in some embodiments, the provided methods and compositions allow the use of single nucleotide polymorphisms (SNPs) in addition to or as an alternative to STR markers.

[0222] Indeed, as data on human genetic variation increases, SNPs are becoming increasingly relevant in forensic work. Thus, in some embodiments, the provided methods and compositions use a primer design strategy that allows for the creation of multiple primer panels based on currently available sequencing kits that substantially guarantee reads across one or more SNP positions. Further Examples 1. A method for concentrating target nucleic acid material, comprising: providing a nucleic acid material; cleaving the nucleic acid material with one or more targeting endonucleases such that a predetermined length of the target region is separated from the remainder of the nucleic acid material; enzymatically destroying non-target nucleic acid material; Releasing a predetermined length of the target region from the targeting endonuclease; and analyzing the cleaved target region. 2. The method of Example 1, wherein enzymatically destroying non-target nucleic acid material comprises providing an exonuclease enzyme. 3. The method of Example 1, wherein enzymatically destroying non-target nucleic acid material comprises providing one or more of an exonuclease enzyme and an endonuclease enzyme. 4. The method of Example 1, wherein the disrupting comprises at least one of enzymatic digestion and enzymatic cleavage. 5. The method of any one of Examples 1 to 4, wherein the one or more targeting endonucleases remain bound to the target region during the enzymatic disrupting step. 6. The method of any one of Examples 1-5, wherein the at least one targeting endonuclease is a ribonucleic acid protein complex comprising a capture label, and wherein a predetermined length of the target region is physically separated from the remainder of the nucleic acid via the capture label, while the at least one targeting endonuclease remains bound to the target region. 7. The method of Examples 1-5, wherein at least one targeting endonuclease is a ribonucleoprotein complex comprising a capture label, and the method further comprises capturing the target region with an extraction moiety configured to bind to the capture label. 8. The method of Example 6 or Example 7, wherein the capture label is or comprises at least one of acrydite, azide, azide (NHS ester), digoxigenin (NHS ester), ILinker, Amino modifier C6, Amino modifier C12, Amino modifier C6 dT, Unilink amino modifier, hexynyl, 5-octadiynyl dU, biotin, biotin (azide), biotin dT, biotin TEG, dual biotin, PC biotin, desthiobiotin TEG, thiol modifier C3, dithiol, thiol modifier C6 SS, succinyl group. 9. The method of Example 7, wherein the extracting moiety is or comprises at least one of an aminosilane, an epoxysilane, an isothiocyanate, an aminophenylsilane, an aminopropylsilane, a mercaptosilane, an aldehyde, an epoxide, a phosphonate, streptavidin, avidin, a hapten that recognizes an antibody, a specific nucleic acid sequence, magnetically attractable particles (Dynabeads), and a photosensitive resin. 10. The method of Example 7, wherein the extracting moiety is bound to a surface. 11. The method of Example 7, wherein the target region is physically separated after enzymatic destruction of non-target nucleic acid material. 12. The method of any one of Examples 1-11, wherein the one or more targeting endonucleases are selected from the group consisting of ribonucleoproteins, Cas enzymes, Cas9-like enzymes, Cpf1 enzymes, meganucleases, transcription activator-like effector-based nucleases (TALENs), zinc finger nucleases, Argonaute nucleases, or combinations thereof. 13. The method of any one of Examples 1-12, wherein the one or more targeting endonucleases comprise Cas9, or CPF1, or derivatives thereof. 14. The method of any one of Examples 1-13, wherein cleaving the nucleic acid material comprises cleaving the nucleic acid material with one or more target endonucleases such that two or more target nucleic acid fragments of substantially known length are formed. 15. The method of Example 14, further comprising isolating two or more target nucleic acid fragments based on a predetermined length. 16. The method of example 15, wherein the target nucleic acid fragments are of different substantially known lengths. 17. The method of example 15, wherein the target nucleic acid fragments each comprise a genomic sequence of interest from one or more different locations in the genome. 18. The method of Example 15, wherein the target nucleic acid fragments each comprise a target sequence from a substantially known region in the nucleic acid material. 19. The method of any one of Examples 15-18, wherein isolating the target nucleic acid fragments substantially based on their known length comprises concentrating the target nucleic acid fragments by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI bead purification. 20. The method of Example 1, further comprising ligating at least one SMI and / or adapter sequence to at least one of the 5' or 3' ends of the cleaved target region of a predetermined length. 21. The method of example 1, wherein analyzing comprises quantification and / or sequencing of the target region. 22. The method of example 21, wherein the quantification comprises at least one of spectrophotometry, real-time PCR, and / or fluorescence-based quantification. 23. The method of example 21, wherein the sequencing comprises double-stranded sequencing, SPLiT double-stranded sequencing, Sanger sequencing, shotgun sequencing, bridge amplification / sequencing, nanopore sequencing, single-molecule real-time sequencing, ion torrent sequencing, pyrosequencing, digital sequencing (e.g., digital barcode-based sequencing), direct digital sequencing, sequencing by ligation, polony-based sequencing, current-based sequencing (e.g., tunneling current), mass spectrometry sequencing, microfluidics-based sequencing, and any combination thereof. 24. Sequencing is sequencing a first strand of the target region to generate first strand sequence reads; sequencing a second strand of the target region to generate second strand sequence reads; comparing the first strand sequence read with the second strand sequence read to generate an error-corrected sequence read. 25. The method of example 24, wherein the error-corrected sequence read comprises matching nucleotide bases between the first strand sequence read and the second strand sequence read. 26. The method of example 24 or example 25, wherein variations occurring at specific positions in error-corrected sequence reads are identified as true variants. 27. The method of any one of Examples 24-26, wherein variations occurring at specific positions in only one of the first strand sequence reads or the second strand sequence reads are identified as potential artifacts. 28. The method of any one of Examples 24-27, wherein the error-corrected sequence reads are used to identify or identify cancer, cancer risk, cancer mutation, cancer metabolic state, mutator gene phenotype, carcinogen exposure, toxin exposure, chronic inflammatory exposure, age, neurodegenerative disease, pathogen, drug resistance variant, fetal molecule, forensically relevant molecule, immunologically relevant molecule, mutated T cell receptor, mutated B cell receptor, mutated immunoglobulin locus, catabolic site in the genome, hypermutable site in the genome, low frequency variant, subclonal variant, minor molecular population, contaminant source, nucleic acid synthesis error, enzymatic modification error, chemical modification error, gene editing error, gene therapy error, part of nucleic acid information reservoir, microbial polymorphism, viral polymorphism, organ transplant, organ transplant rejection, cancer recurrence, residual cancer after treatment, pre-neoplastic state, dysplastic state, microchimerism state, stem cell transplant state, cell therapy state, nucleic acid label fixed to another molecule, or combinations thereof in the organism or subject from which the double-stranded target nucleic acid molecule is derived. 29. The method of any one of Examples 24-27, wherein the error-corrected sequence reads are used to identify a mutagenic compound or exposure. 30. The method of any one of Examples 24-27, wherein the error-corrected sequence reads are used to identify carcinogenic compounds or exposures. 31. The method of any one of Examples 24-27, wherein the nucleic acid material is derived from a forensic sample and the error-corrected sequence reads are used in forensic analysis. 32. The method of example 1, wherein the targeting endonuclease comprises at least one of a CRISPR-associated (Cas) enzyme, a ribonucleoprotein complex, a homing endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute nuclease, and / or a megaTAL nuclease. 33. The method of example 32, wherein the CRISPR-associated (Cas) enzyme is Cas9 or Cpf1. 34. The method of example 32, wherein the CRISPR-associated (Cas) enzyme is Cpf1 and the target region comprises a 5' overhang and a 3' overhang having a predetermined or known nucleotide sequence. 35. The method of example 1, wherein cleaving the nucleic acid material with a targeting endonuclease comprises cleaving the nucleic acid material with two or more targeting endonucleases. 36. The method of example 35, wherein the two or more targeting endonucleases comprise two or more Cas enzymes directed to two or more target regions. 37. The method of Example 35, wherein cleaving the nucleic acid material with a targeting endonuclease such that a target region of a predetermined length is separated from the remainder of the nucleic acid material comprises cleaving the target region with a pair of targeting endonucleases directed to cleave the nucleic acid material a predetermined distance apart to create a target region having a predetermined length. 38. The method of example 37, wherein the pair of targeting endonucleases comprises a pair of Cas enzymes. 39. The method of example 38, wherein the pair of Cas enzymes comprises Cas enzymes of the same type. 40. The method of example 38, wherein the pair of Cas enzymes comprises two different Cas enzymes. 41. A method for concentrating a target nucleic acid material, comprising: providing a nucleic acid material; cleaving the nucleic acid material with one or more targeting endonucleases such that a target region of predetermined length is separated from the remainder of the nucleic acid material, wherein at least one targeting endonuclease comprises a capture label; capturing a predetermined length of the target region using an extraction moiety configured to bind to a capture label; Releasing a predetermined length of the target region from the targeting endonuclease; and analyzing the cleaved target region. 42. A method for concentrating a target nucleic acid material, comprising: providing a nucleic acid material; Binding a catalytically inactive CRISPR-associated (Cas) enzyme to a target region of a nucleic acid material; enzymatically treating the nucleic acid material with one or more nucleic acid digestive enzymes such that non-target nucleic acid material is destroyed and the target region is protected from the digestive enzymes by the bound catalytically inactive Cas enzyme; Releasing the target region from a catalytically inactive Cas enzyme; and and analyzing the target region. 43. The method of Example 42, wherein the binding step comprises binding a pair of catalytically inactive Cas enzymes to the target region such that the nucleic acid material between the bound Cas enzymes is enzymatically protected from digestive enzymes, thereby concentrating the target nucleic acid material in the target region. 44. The method of Example 42, wherein the catalytically inactive Cas enzyme comprises a capture label, and the method further comprises capturing the target region with an extraction moiety configured to bind to the capture label. 45. The method of example 42, further comprising enriching the target region by size selection. 46. ​​A method for concentrating a target nucleic acid material, comprising: providing a nucleic acid material; providing a pair of catalytically active target endonucleases and at least one catalytically inactive target endonuclease comprising a capture label, wherein the catalytically inactive target endonuclease is directed to bind to a target region of a nucleic acid material, and the pair of catalytically active target endonucleases are directed to bind to target regions on either side of the catalytically inactive target endonuclease; cleaving the nucleic acid material with a pair of catalytically active targeting endonucleases such that the target region is separated from the remainder of the nucleic acid material; capturing the target region using an extraction moiety configured to bind to a capture label; Releasing the target region from the targeting endonuclease; and analyzing the cleaved target region. 47. A method for concentrating a target nucleic acid material from a sample containing a plurality of nucleic acid fragments, comprising: providing one or more catalytically inactive CRISPR-associated (Cas) enzymes having capture labels to a sample containing target nucleic acid fragments and non-target nucleic acid fragments, wherein the one or more catalytically inactive Cas enzymes are configured to bind to the target nucleic acid fragments; providing a surface comprising an extraction moiety configured to bind to a capture label; and separating the target nucleic acid fragments from non-target nucleic acid fragments by capturing the target nucleic acid fragments through binding of a capture label by an extracting moiety. 48. The method of Example 47, further comprising attaching adapter molecules to the ends of the plurality of nucleic acid fragments prior to providing the one or more catalytically inactive CRISPR-associated (Cas) enzymes. 49. A method for concentrating target double-stranded nucleic acid material, comprising: providing a nucleic acid material; cleaving the nucleic acid material with one or more target endonucleases to generate double-stranded target nucleic acid fragments comprising 5' sticky ends having a 5' predetermined nucleotide sequence and / or 3' sticky ends having a 3' predetermined nucleotide sequence; and separating the double-stranded target nucleic acid molecule from the remainder of the nucleic acid material via at least one of the 5' sticky end and the 3' sticky end. 50. Providing at least one sequencing adaptor molecule comprising a ligatable end at least partially complementary to a 5' predetermined nucleotide sequence or a 3' predetermined nucleotide sequence; ligating at least one sequencing adaptor molecule to the double-stranded target nucleic acid molecule; 50. The method of Example 49, further comprising analyzing the double-stranded target nucleic acid fragments via sequencing. 51. The method of example 50, wherein at least one adapter molecule comprises a Y-shape or a U-shape. 52. The method of example 50, wherein at least one adapter molecule is a hairpin molecule. 53. The method of example 50, wherein at least one adapter molecule comprises a capture molecule configured to be bound by an extracting moiety. 54. The method of example 50, wherein a sequencing adapter molecule is ligated to each of the 5' and 3' sticky ends of the double-stranded target nucleic acid fragment. 55. The method of Example 49, wherein separating the double-stranded target nucleic acid molecule from the remainder of the nucleic acid material via at least one of a 5' sticky end and a 3' sticky end comprises providing an oligonucleotide having a 5' predetermined nucleotide sequence or a sequence at least partially complementary to a 3' predetermined nucleotide sequence. 56. The method of Example 55, wherein the oligonucleotide is bound to a surface. 57. The method of example 55, wherein the oligonucleotide comprises a capture label configured to bind to an extracting moiety. 58. The method of example 49, wherein the one or more targeting endonucleases include Cpf1. 59. The method of example 49, wherein the one or more targeting endonucleases comprise a Cas9 nickase. 60. A kit for concentrating a target nucleic acid material, comprising: 1. A nucleic acid library comprising: nucleic acid material, and a plurality of catalytically inactive Cas enzymes, the Cas enzymes comprising a tag having a sequence code; a nucleic acid library in which a plurality of Cas enzymes bind to a plurality of site-specific target regions along a nucleic acid material; A plurality of probes, each probe comprising: an oligonucleotide sequence comprising the complement to the corresponding sequence code, and a plurality of probes comprising capture labels; A kit comprising: a lookup table that catalogs the relationship between site-specific target regions, wherein sequence codes are associated with the site-specific target regions and probes contain complements to the corresponding sequence codes. 61. The method of any one of the preceding examples, wherein the nucleic acid material is or comprises at least one of double-stranded DNA and double-stranded RNA. 62. The method according to any one of the preceding examples, wherein at least some of the nucleic acid material is damaged. 63. Damage can be caused by oxidation, alkylation, deamination, methylation, hydrolysis, hydroxylation, nicking, intrastrand crosslinks, interstrand crosslinks, blunt-end strand breaks, sticky-end double-strand breaks, phosphorylation, dephosphorylation, sumoylation, glycosylation, deglycosylation, putrescinylation, carboxylation, halogenation, formylation, single-strand gaps, damage from heat, damage from desiccation, damage from UV exposure, damage from gamma radiation, damage from X-rays, damage from ionizing radiation, damage from non-ionizing radiation, damage from heavy particle radiation, damage from nuclear decay, damage from beta radiation, damage from alpha radiation, damage from neutron radiation, damage from proton radiation, damage from cosmic radiation, damage from high pH, ​​damage from low pH, damage from reactive oxidative species, damage from free radicals, damage from peroxides, damage from hypochlorite, damage from tissue fixation such as formalin or formaldehyde, damage from reactive iron, damage from low ionic conditions, damage from high ionic conditions, damage from unbuffered conditions, 63. The method of Example 62, wherein the damage is or comprises at least one of damage from nucleases, damage from environmental exposure, damage from fire, damage from mechanical stress, damage from enzymatic degradation, damage from microorganisms, damage from mechanical shear in preparation, damage from enzymatic fragmentation in preparation, damage occurring spontaneously in vivo, damage occurring during nucleic acid extraction, damage occurring during sequencing library preparation, damage introduced by polymerases, damage introduced during nucleic acid repair, damage occurring during tailing of nucleic acid ends, damage occurring during nucleic acid ligation, damage occurring during sequencing, damage occurring from mechanical handling of DNA, damage occurring during passage through a nanopore, damage occurring as part of aging in an organism, damage occurring as a result of chemical exposure of an individual, damage caused by mutagens, damage caused by carcinogens, damage caused by clastogens, damage resulting from inflammatory damage in vivo due to oxygen exposure, damage resulting from one or more strand breaks, and any combination thereof. 64. The method of any one of the preceding examples, wherein the nucleic acid material is provided from a sample comprising one or more double-stranded nucleic acid molecules derived from a subject or organism. 65. Samples include body tissue, biopsy, skin sample, blood, serum, plasma, sweat, saliva, cerebrospinal fluid, mucus, uterine washing, vaginal swab, Pap smear, nasal swab, oral swab, tissue scraping, hair, fingerprint, urine, stool, vitreous fluid, peritoneal washing, sputum, bronchial washing, oral washing, pleural washing, gastric lavage, gastric juice, bile, pancreatic duct washing, bile duct washing, common bile duct washing, gallbladder fluid, synovial fluid, infected wound, non-infected wound, archaeological sample, forensic sample, water sample, tissue sample, food sample, bioreactor sample, plant sample, bacterial sample, protozoan sample, fungal sample, animal sample, viral sample, multi-organism sample, nail polish, semen, prostatic fluid, vaginal fluid, vaginal swab, fallopian tube washing 65. The method of Example 64, wherein the sample is or comprises a swabbin, a cell-free nucleic acid, an intracellular nucleic acid, a metagenomics sample, a wash or swab of an implanted foreign body, a nasal wash, an intestinal fluid, an epithelial brushing, an epithelial wash, a tissue biopsy, an autopsy sample, a necrosis sample, an organ sample, a human-identified sample, a non-human-identified sample, an artificially created nucleic acid sample, a synthetic gene sample, a deposited or preserved sample, tumor tissue, a fetal sample, an organ transplant sample, a microbial culture sample, a nuclear DNA sample, a mitochondrial DNA sample, a chloroplast DNA sample, an apicoplast DNA sample, an organelle sample, and any combination thereof. 66. The method of any one of the preceding examples, wherein the nucleic acid material comprises nucleic acid molecules of substantially or nearly uniform length. 67. The method of any one of the preceding Examples, wherein the target nucleic acid material is derived from a subject or organism. 68. The method of any one of any one of the preceding embodiments, wherein the target nucleic acid material is at least partially artificially synthesized. 69. The method of any one of the preceding examples, wherein up to 1000 ng of nucleic acid material is initially provided. 70. The method of any one of the preceding examples, wherein a maximum of 10 ng of nucleic acid material is initially provided. 71. The method of any one of the preceding examples, wherein the nucleic acid material comprises nucleic acid material from more than one source.

[0223] Equivalents and Scope The above detailed description of embodiments of the present technology is not intended to be exhaustive or to limit the present technology to the precise form described above. Specific embodiments and examples of the present technology have been described above for illustrative purposes, but those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present technology. For example, while steps are presented in a given order, alternative embodiments may perform steps in a different order. Various embodiments described herein may also be combined to provide further embodiments. All references cited herein are incorporated by reference as if fully set forth herein.

[0224] From the above description, it should be understood that specific embodiments of the present technology are described herein for illustrative purposes, but well-known structures and functions have not been shown in detail to avoid unnecessarily obscuring the description of the embodiments of the present technology. Where the context permits, singular or plural terms may also include the plural or singular terms, respectively. Furthermore, while advantages associated with certain embodiments of the present technology are described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present technology. Thus, the present disclosure and related technology can encompass other embodiments not explicitly shown or described herein.

[0225] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosed technology described herein. The scope of the technology is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method for concentrating a target nucleic acid material, comprising: Providing a nucleic acid material; cleaving the nucleic acid material with one or more targeting endonucleases such that a predetermined length of a target region is separated from the remainder of the nucleic acid material; enzymatically destroying non-target nucleic acid material; Releasing the predetermined length of the target region from the targeting endonuclease; and analyzing the cleaved target region.

2. 10. The method of claim 1, wherein enzymatically destroying non-target nucleic acid material comprises providing an exonuclease enzyme.

3. 10. The method of claim 1, wherein enzymatically destroying non-target nucleic acid material comprises providing one or more of an exonuclease enzyme and an endonuclease enzyme.

4. The method of claim 1 , wherein the destroying comprises at least one of enzymatic digestion and enzymatic cleavage.

5. The method of any one of claims 1 to 4, wherein the one or more targeting endonucleases remain bound to the target region during the enzymatic destruction step.

6. 6. The method of any one of claims 1 to 5, wherein at least one targeting endonuclease is a ribonucleoprotein complex comprising a capture label, and wherein a length of the target region is physically separated from the remainder of the nucleic acid via the capture label, while the at least one targeting endonuclease remains bound to the target region.

7. 6. The method of claims 1-5, wherein at least one target endonuclease is a ribonucleoprotein complex comprising a capture label, the method further comprising capturing the target region with an extraction moiety configured to bind to the capture label.

8. 8. The method of claim 6 or 7, wherein the capture label is or comprises at least one of an acrydite, azide, azide (NHS ester), digoxigenin (NHS ester), I Linker, Amino modifier C6, Amino modifier C12, Amino modifier C6 dT, Unilink amino modifier, hexynyl, 5-octadiynyl dU, biotin, biotin (azide), biotin dT, biotin TEG, dual biotin, PC biotin, desthiobiotin TEG, thiol modifier C3, dithiol, thiol modifier C6 S-S, succinyl group.

9. 8. The method of claim 7, wherein the extraction moiety is or comprises at least one of the following: aminosilane, epoxysilane, isothiocyanate, aminophenylsilane, aminopropylsilane, mercaptosilane, aldehyde, epoxide, phosphonate, streptavidin, avidin, hapten that recognizes an antibody, a specific nucleic acid sequence, magnetically attractive particles (Dynabeads), photosensitive resin.

10. The method of claim 7 , wherein the extraction moiety is bound to a surface.

11. The method of claim 7, wherein the target regions are physically separated after enzymatic destruction of the non-target nucleic acid material.

12. 12. The method of any one of claims 1 to 11, wherein the one or more targeting endonucleases are selected from the group consisting of ribonucleoproteins, Cas enzymes, Cas9-like enzymes, Cpf1 enzymes, meganucleases, transcription activator-like effector-based nucleases (TALENs), zinc finger nucleases, Argonaute nucleases, or combinations thereof.

13. The method of any one of claims 1 to 12, wherein the one or more targeting endonucleases comprise Cas9, or CPF1, or a derivative thereof.

14. 14. The method of any one of claims 1 to 13, wherein cleaving the nucleic acid material comprises cleaving the nucleic acid material with one or more target endonucleases such that two or more target nucleic acid fragments of substantially known length are formed.

15. 15. The method of claim 14, further comprising isolating the two or more target nucleic acid fragments based on the predetermined length.

16. 16. The method of claim 15, wherein the target nucleic acid fragments are of different substantially known lengths.

17. 16. The method of claim 15, wherein the target nucleic acid fragments each comprise a genomic sequence of interest from one or more different locations in the genome.

18. 16. The method of claim 15, wherein the target nucleic acid fragments each comprise a target sequence from a substantially known region in the nucleic acid material.

19. 19. The method of any one of claims 15-18, wherein isolating the target nucleic acid fragments based on the substantially known length comprises concentrating the target nucleic acid fragments by gel electrophoresis, gel purification, liquid chromatography, size exclusion purification, filtration, or SPRI bead purification.

20. 2. The method of claim 1, further comprising ligating at least one SMI and / or adapter sequence to at least one of the 5' or 3' ends of the cleaved target region of a predetermined length.

21. The method of claim 1 , wherein analyzing comprises quantification and / or sequencing of the target region.

22. 22. The method of claim 21, wherein the quantification comprises at least one of spectrophotometry, real-time PCR, and / or fluorescence-based quantification.

23. 22. The method of claim 21, wherein the sequencing comprises double-stranded sequencing, SPLiT double-stranded sequencing, Sanger sequencing, shotgun sequencing, bridge amplification / sequencing, nanopore sequencing, single molecule real-time sequencing, ion torrent sequencing, pyrosequencing, digital sequencing (e.g., digital barcode-based sequencing), direct digital sequencing, sequencing by ligation, polony-based sequencing, current-based sequencing (e.g., tunneling current), mass spectrometry sequencing, microfluidic-based sequencing, and any combination thereof.

24. Sequencing sequencing a first strand of the target region to generate first strand sequence reads; sequencing a second strand of the target region to generate second strand sequence reads; and comparing the first strand sequence read with the second strand sequence read to generate an error-corrected sequence read.

25. 25. The method of Claim 24, wherein the error-corrected sequence reads comprise matching nucleotide bases between the first strand sequence read and the second strand sequence read.

26. 26. The method of Claim 24 or Claim 25, wherein variations occurring at specific positions in the error-corrected sequence reads are identified as true variants.

27. 27. The method of any one of claims 24-26, wherein variations occurring at specific positions in only one of the first strand sequence reads or the second strand sequence reads are identified as potential artifacts.

28. 28. The method of any one of claims 24-27, wherein the error-corrected sequence reads are used to identify or identify cancer, cancer risk, cancer mutations, cancer metabolic states, mutator gene phenotypes, carcinogen exposure, toxin exposure, chronic inflammatory exposure, age, neurodegenerative disease, pathogens, drug resistance variants, fetal molecules, forensically relevant molecules, immunologically relevant molecules, mutated T cell receptors, mutated B cell receptors, mutated immunoglobulin loci, catabolism sites in the genome, hypermutable sites in the genome, low frequency variants, subclonal variants, low abundance molecular populations, contaminant sources, nucleic acid synthesis errors, enzymatic modification errors, chemical modification errors, gene editing errors, gene therapy errors, portions of nucleic acid information stores, microbial polymorphism, viral polymorphism, organ transplants, organ transplant rejections, cancer recurrence, residual cancer after treatment, pre-neoplastic states, dysplastic states, microchimerism states, stem cell transplant states, cell therapy states, nucleic acid labels fixed to another molecule, or combinations thereof in the organism or subject from which the double-stranded target nucleic acid molecule is derived.

29. 28. The method of any one of claims 24 to 27, wherein the error-corrected sequence reads are used to identify a mutagenic compound or exposure.

30. 28. The method of any one of claims 24 to 27, wherein the error-corrected sequence reads are used to identify carcinogenic compounds or exposures.

31. 28. The method of any one of claims 24 to 27, wherein the nucleic acid material is derived from a forensic sample and the error-corrected sequence reads are used in forensic analysis.

32. 2. The method of claim 1, wherein the targeting endonuclease comprises at least one of a CRISPR-associated (Cas) enzyme, a ribonucleoprotein complex, a homing endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute nuclease, and / or a MegaTAL nuclease.

33. 33. The method of claim 32, wherein the CRISPR-associated (Cas) enzyme is Cas9 or Cpf1.

34. 33. The method of Claim 32, wherein the CRISPR-associated (Cas) enzyme is Cpf1 and the target region comprises a 5' overhang and a 3' overhang having a predetermined or known nucleotide sequence.

35. 10. The method of claim 1, wherein cleaving the nucleic acid material with a targeting endonuclease comprises cleaving the nucleic acid material with two or more targeting endonucleases.

36. 36. The method of claim 35, wherein the two or more targeting endonucleases comprise two or more Cas enzymes directed to two or more target regions.

37. 36. The method of claim 35, wherein cleaving the nucleic acid material with a targeting endonuclease such that a target region of a predetermined length is separated from the remainder of the nucleic acid material comprises cleaving the target region with a pair of targeting endonucleases directed to cleave the nucleic acid material a predetermined distance apart to create the target region having the predetermined length.

38. 38. The method of claim 37, wherein the pair of targeting endonucleases comprises a pair of Cas enzymes.

39. 39. The method of claim 38, wherein the pair of Cas enzymes comprises Cas enzymes of the same type.

40. 39. The method of claim 38, wherein the pair of Cas enzymes comprises two different Cas enzymes.

41. 1. A method for concentrating a target nucleic acid material, comprising: Providing a nucleic acid material; cleaving the nucleic acid material with one or more targeting endonucleases such that a target region of predetermined length is separated from the remainder of the nucleic acid material, at least one targeting endonuclease comprising a capture label; capturing the predetermined length of the target region with an extraction moiety configured to bind to the capture label; Releasing the predetermined length of the target region from the targeting endonuclease; and analyzing the cleaved target region.

42. 1. A method for concentrating a target nucleic acid material, comprising: Providing a nucleic acid material; Binding a catalytically inactive CRISPR-associated (Cas) enzyme to a target region of the nucleic acid material; enzymatically treating the nucleic acid material with one or more nucleic acid digestive enzymes such that non-target nucleic acid material is destroyed and the target region is protected from digestive enzymes by the bound catalytically inactive Cas enzyme; Releasing the target region from the catalytically inactive Cas enzyme; and and analyzing the target region.

43. 43. The method of claim 42, wherein the binding step comprises binding a pair of catalytically inactive Cas enzymes to the target region such that nucleic acid material between the bound Cas enzymes is enzymatically protected from the digestive enzymes, thereby concentrating the target nucleic acid material in the target region.

44. 43. The method of claim 42, wherein the catalytically inactive Cas enzyme comprises a capture label, the method further comprising capturing the target region with an extraction moiety configured to bind to the capture label.

45. 43. The method of claim 42, further comprising enriching the target region by size selection.

46. 1. A method for concentrating a target nucleic acid material, comprising: Providing a nucleic acid material; providing a pair of catalytically active target endonucleases and at least one catalytically inactive target endonuclease comprising a capture label, wherein the catalytically inactive target endonuclease is directed to bind to the target region of the nucleic acid material, and the pair of catalytically active target endonucleases are directed to bind to the target region on either side of the catalytically inactive target endonuclease; cleaving the nucleic acid material with the pair of catalytically active targeting endonucleases such that the target region is separated from the remainder of the nucleic acid material; capturing the target region with an extraction moiety configured to bind to the capture label; Releasing the target region from the targeting endonuclease; and and analyzing the cleaved target region.

47. 1. A method for concentrating a target nucleic acid material from a sample containing a plurality of nucleic acid fragments, comprising: providing one or more catalytically inactive CRISPR-associated (Cas) enzymes having a capture label to the sample comprising target and non-target nucleic acid fragments, wherein the one or more catalytically inactive Cas enzymes are configured to bind to the target nucleic acid fragments; providing a surface comprising an extraction moiety configured to bind to said capture label; and separating the target nucleic acid fragments from the non-target nucleic acid fragments by capturing the target nucleic acid fragments via binding of the capture label by the extraction moiety.

48. 48. The method of Claim 47, further comprising attaching adapter molecules to the ends of the plurality of nucleic acid fragments prior to providing the one or more catalytically inactive CRISPR-associated (Cas) enzymes.

49. 1. A method for concentrating a target double-stranded nucleic acid material, comprising: Providing a nucleic acid material; cleaving the nucleic acid material with one or more target endonucleases to generate double stranded target nucleic acid fragments comprising 5' sticky ends having a 5' predetermined nucleotide sequence and / or 3' sticky ends having a 3' predetermined nucleotide sequence; and separating the double-stranded target nucleic acid molecule from the remainder of the nucleic acid material via at least one of the 5' sticky end and the 3' sticky end.

50. providing at least one sequencing adaptor molecule comprising a ligatable end at least partially complementary to said 5' predetermined nucleotide sequence or said 3' predetermined nucleotide sequence; ligating said at least one sequencing adaptor molecule to said double-stranded target nucleic acid molecule; 50. The method of claim 49, further comprising analyzing the double-stranded target nucleic acid fragments via sequencing.

51. 51. The method of claim 50, wherein the at least one adaptor molecule comprises a Y-shape or a U-shape.

52. 51. The method of claim 50, wherein the at least one adaptor molecule is a hairpin molecule.

53. 51. The method of claim 50, wherein the at least one adaptor molecule comprises a capture molecule configured to be bound by an extraction moiety.

54. 51. The method of claim 50, wherein a sequencing adaptor molecule is ligated to each of the 5' and 3' sticky ends of the double-stranded target nucleic acid fragment.

55. 50. The method of claim 49, wherein separating the double-stranded target nucleic acid molecule from the remainder of the nucleic acid material via at least one of the 5' sticky end and the 3' sticky end comprises providing an oligonucleotide having a sequence at least partially complementary to the 5' predetermined nucleotide sequence or the 3' predetermined nucleotide sequence.

56. 56. The method of claim 55, wherein the oligonucleotide is bound to a surface.

57. 56. The method of claim 55, wherein the oligonucleotide comprises a capture label configured to bind to an extracting moiety.

58. 50. The method of claim 49, wherein the one or more targeting endonucleases comprises Cpf1.

59. 50. The method of Claim 49, wherein the one or more targeting endonucleases comprises a Cas9 nickase.

60. 1. A kit for concentrating a target nucleic acid material, comprising:

1. A nucleic acid library comprising: Nucleic acid material, and a plurality of catalytically inactive Cas enzymes, the Cas enzymes comprising a tag having a sequence code; a nucleic acid library, wherein the plurality of Cas enzymes bind to a plurality of site-specific target regions along the nucleic acid material; A plurality of probes, each probe comprising: an oligonucleotide sequence comprising a complement to the corresponding sequence code; and a plurality of probes comprising capture labels; a lookup table cataloging the relationship between the site-specific target regions, wherein the sequence codes are associated with the site-specific target regions and the probes include the complements to the corresponding sequence codes.

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