Target Enrichment

Abstract

The present disclosure, according to some embodiments, relates to methods and compositions for preparing polynucleotide libraries enriched for target sequences from a source material (e.g., a sample comprising or constituting a biological fluid, tissue, and / or specimen). The methods and compositions may provide efficient enrichment of target polynucleotides. Enrichment may include increasing the relative abundance of a target from a source material (where the target may be present in low abundance) to a generated library (where the target may be present in higher abundance). In some embodiments, the methods include attaching (e.g., ligating, linking, or otherwise fusing) adaptors to fragments of interest, nick translation, amplification (e.g., linear amplification), and target sequence selection, e.g., using affinity tagging.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 318,194, filed March 9, 2022, which is incorporated by reference in its entirety.

[0002] This disclosure includes a Sequence Listing, which was submitted electronically in .xml format under filename "DG-004-PCT.xml", created on March 1, 2023, and has a size of 15.6KB. This Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Next generation sequencing (NGS) has become a major tool in genomics research, providing a powerful avenue to study DNA samples. There are strong efforts to develop NGS-based methods for the analysis of genomic variations. As part of this effort, several methods have been developed to enrich for specific target sequences, e.g., subregions of the genome, to reduce costs and labor and improve outcomes. Target enrichment can be useful when only a portion of the genome needs to be enriched / analyzed, such as the "exome" (i.e., all transcribed sequences) or a smaller set of genes or genomic regions that are involved in a particular disease or pathway. For example, target enrichment can be used to select DNA representing a set of cancer genes prior to sequence analysis. Selective recovery of target sequences should theoretically reduce costs and increase sequencing depth compared to whole genome sequencing.

[0004] Target enrichment can be difficult to perform in low-input samples, not only because the amount of DNA in the sample is limited, but also because the enrichment method itself is inefficient.Therefore, there is still a need for new methods for target enrichment, especially those that can be used for low-input samples such as cell-free DNA (cfDNA) and DNA isolated from tissue sections. Summary of the Invention

[0005] Summary of the Invention The present disclosure provides a method for enriching polynucleotides comprising a sequence of interest ("target sequence") in a polynucleotide composition. In some embodiments, the polynucleotide composition may comprise, for example, genomic, cellular, organelle, acellular, or other polynucleotides (e.g., DNA and / or RNA) that have been fragmented to form a population of fragments, at least some (optionally all) of which have a size within a desired range of fragment sizes. The population of fragments may comprise one or more fragments having the target sequence. A method according to some embodiments may comprise attaching (e.g., ligating, linking, or otherwise fusing) a DNA adapter to the 5' end of a double-stranded polynucleotide fragment in a population of double-stranded polynucleotide fragments from a sample, the DNA adapter comprising a bottom strand and a top strand, forming a fusion product. The adapter top strand may comprise, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site, optionally a sample tag, optionally a unique molecular identifier (UMI), and a sequence that is complementary to the adapter bottom strand. For example, the adaptor top strand may be longer than the adaptor bottom strand if an optional sample tag and / or an optional UMI is included in the top strand. The adaptor bottom strand may include a 3' end that is not extendable (e.g., an end that includes an inverted deoxythymidine). The adaptor bottom strand may not include (may exclude) a 5' phosphate, a sample tag, a UMI, and / or a linear amplification annealing site. In some embodiments, the fusion product may include a nick that includes the 3' end of the adaptor top strand fused to the 5' end of the polynucleotide fragment and the unfused 5' end of the adaptor bottom strand and the 3' end of the polynucleotide fragment.

[0006] In some embodiments, the method may include attaching (e.g., ligating, linking, or otherwise fusing) a DNA adapter to the 5' end of a double-stranded polynucleotide fragment in a population of double-stranded polynucleotide fragments from a sample, the DNA adapter comprising a top strand and, optionally, a bottom strand, to form a fusion product. A single-stranded adapter may be attached to the double-stranded fragment, for example, using a single-stranded DNA ligase. The adapter top strand may comprise, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site, optionally a sample tag, optionally a unique molecular identifier (UMI), and a sequence that is optionally complementary to the adapter bottom strand. In cases where the top strand comprises a UMI (e.g., to reduce or prevent UMI hopping), it may be desirable to include an optional bottom strand. In some embodiments, the adapter top strand may lack a UMI and the DNA adapter may lack a bottom strand. For example, the adaptor top strand may be longer than the adaptor bottom strand if an optional sample tag and / or an optional UMI is included in the top strand. The adaptor bottom strand may include a 3' end that is not extendable (e.g., an end that includes an inverted deoxythymidine). The adaptor bottom strand may not include (may exclude) a 5' phosphate, a sample tag, a UMI, and / or a linear amplification annealing site. In some embodiments, the fusion product may include a nick that includes the 3' end of the adaptor top strand fused to the 5' end of the polynucleotide fragment and the unfused 5' end of the adaptor bottom strand and the 3' end of the polynucleotide fragment.

[0007] The method may further comprise contacting the fusion product with a nick translation polymerase for templated addition of nucleotides to the 3' end of the polynucleotide fragment to form a nick translation product comprising a 3' extension of the polynucleotide fragment, the extension being complementary to the adaptor top strand, according to some embodiments. The method may further comprise contacting the nick translation product with a primer having a sequence complementary to the linear amplification annealing site and a polymerase to generate an amplified (e.g., linearly amplified) nick translation product. The amplified (e.g., linearly amplified) nick translation product may comprise, for example, from 5' to 3', a sequence complementary to the linear amplification annealing site, optionally a sequence of the sample tag, optionally a sequence of the UMI, a sequence of a portion of the adaptor top strand, a sequence of one of the polynucleotide fragments, and optionally a sequence complementary to a portion (e.g., 1-25 nucleotides) of the adaptor top strand.

[0008] According to some embodiments, the method may further comprise contacting the nick translation product with a glycosylase and an endonuclease (e.g., sequentially, together as separate enzymes, or as a fusion protein) prior to or concurrently with the amplification (e.g., linear amplification), where the adaptor top strand template further comprises one or more modified nucleotides, the one or more modified nucleotides are optionally deoxyuridine, the glycosylase is optionally uracil-DNA glycosylase (UDG), and the endonuclease is optionally endonuclease VIII. The nick translation may be expected to displace the adaptor bottom strand, and such displaced adaptor bottom strand may be expected to be lost, for example, throughout subsequent steps without the need for any overt action. However, in some embodiments, the method may include removing the adapter bottom strand of the adapter template (after nick translation) by cleaving the adapter bottom strand with a reagent comprising a glycosylase and an endonuclease, wherein the adapter bottom strand comprises one or more modified nucleotides, optionally the one or more modified nucleotides are deoxyuridine, the glycosylase is optionally uracil-DNA glycosylase (UDG), and the endonuclease is optionally endonuclease VIII.

[0009] The method may, according to some embodiments, further comprise contacting the linearly amplified nick translation product with a target-specific oligonucleotide conjugated to an affinity domain (e.g., biotin) to form a target complex comprising the target sequence, the target-specific oligonucleotide, and a polynucleotide fragment having an affinity domain, wherein the target sequence is hybridized to the target-specific oligonucleotide. In some embodiments, the method may comprise binding the target complex to a solid support comprising an affinity capture domain (e.g., streptavidin) corresponding to the affinity domain.

[0010] In some embodiments, the method may include removing any overhanging polynucleotide sequence at the 3' end of the oligonucleotide hybridized complementary strand using a 3'-5' single-stranded exonuclease or multiple 3'-5' exonucleases to form a blunt-ended duplex at the 3' end of the complement and the 5' end of the oligonucleotide; attaching (e.g., ligating, linking or otherwise fusing) the blunt-ended duplex to a second adaptor comprising a second sample tag to form a second fusion product comprising, in the 5' to 3' direction, the first adaptor top strand, the oligonucleotide fragment, and the second adaptor; and / or amplifying the second fusion product to sequence one or both of the strands.

[0011] In some embodiments, it may be desirable to combine polynucleotide compositions for multiplex analysis. For example, polynucleotide compositions (e.g., including genomic, cellular, organelle, acellular, or other polynucleotides from the same or different samples, specimens, cells, body fluids, tissues, organisms, or other materials) may be combined prior to fragmentation or at any step of the methods disclosed herein, for example, after adapter fusion or after linear amplification. Thus, in some embodiments, the method may include combining adapter fusion products resulting from multiple separate polynucleotide compositions (e.g., including genomic, cellular, organelle, acellular, or other polynucleotides from the same or different samples, specimens, cells, body fluids, tissues, organisms, or other materials). For example, the method may include pooling a linear amplification product (e.g., resulting from a first adapter fusion, nick translation, and linear amplification) with further linear amplification products (e.g., resulting from a second adapter fusion, nick translation, and linear amplification), each of which comprises, in the 5' to 3' direction, a sequence complementary to a linear amplification annealing site, a sequence of a further sample tag, optionally a sequence of a UMI, a sequence of a portion of a further adapter top strand, and a sequence of a polynucleotide fragment of a further population of fragments.

[0012] In some embodiments, the method may include repeating the adapter fusion step with a second DNA adapter and the double-stranded polynucleotide fragments of the second fragment population to form additional fusion products, where the first adapter top strand includes a sample tag and the second adapter includes a bottom strand and a top strand (e.g., a longer top strand). The second adapter top strand may include, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site, a sample tag that is different from the first adapter sample tag, optionally a unique molecular identifier (UMI); and a sequence that is complementary to the second adapter bottom strand. The second adapter bottom strand may include a non-extendable 3' end and may optionally be free of a 5' phosphate, a sample tag, a UMI, and a linear amplification annealing site. In some embodiments, the polynucleotide fragments of the second population may include a target sequence. The further fusion product may comprise a 3' end of the second adaptor top strand fused to the 5' end of the second population of polynucleotide fragments and a nick comprising the unfused 5' end of the second adaptor bottom strand and the 3' end of the second population of polynucleotide fragments. The method may comprise pooling the fusion product and the further fusion product to form a pooled fusion product.

[0013] In some embodiments, contacting the fusion products with a nick translation polymerase may further comprise contacting the pooled fusion products with a nick translation polymerase for templated addition of nucleotides to the 3' ends of the polynucleotide fragments to form pooled nick translation products comprising 3' extensions of the polynucleotide fragments, the extensions being complementary to the respective adaptor top strands. Contacting the nick translation products with a primer having a sequence complementary to the linear amplification annealing site may further comprise contacting the pooled nick translation products with a primer and a polymerase to generate linear amplified pooled nick translation products, which may comprise, in the 5' to 3' direction, a sequence complementary to the linear amplification annealing site, a sequence of one of the respective sample tags, optionally a sequence of the UMI, a sequence of a portion of the respective adaptor top strands, a sequence of one of the polynucleotide fragments, and optionally a sequence complementary to a portion of the respective adaptor bottom strands.

[0014] According to some embodiments, a method for enriching target sequences may include contacting a population of fragmented polynucleotides with a ligase and a first adaptor, the first adaptor comprising a bottom strand and a top strand (e.g., a longer top strand), where (i) the bottom strand comprises, in a 5' to 3' direction, a 5'-OH, at least 10 nucleotides complementary to the top strand, and optionally, an inverted dT; and (ii) the top strand comprises, in a 5' to 3' direction, a sequence complementary to a linear amplification annealing site, optionally a sample tag, a unique molecular identifier (UMI), and a sequence complementary to the bottom strand, the top and bottom strands optionally, independently, comprising one or more deoxyuridines (e.g., distributed along the length of each strand), whereby the contacting creates a double-stranded fusion product comprising the 3' end of the adaptor top strand fused to the 5' end of the polynucleotide fragment and a nick comprising the unfused 5' end of the adaptor bottom strand and the 3' end of the polynucleotide fragment. In some embodiments, the method may further include contacting the double-stranded fusion product with a nick translation polymerase and nucleotide triphosphates to form a nick-translated duplex polynucleotide, the duplex including, in a 5' to 3' direction, a top strand including an adaptor top strand, a plus strand of one of the polynucleotide fragments, and a sequence complementary to the adaptor top strand, and a bottom strand including, in a 5' to 3' direction, a minus strand of one of the polynucleotide fragments, and a sequence complementary to the adaptor top strand; and, optionally, contacting the nick-translated duplex polynucleotide with a single nucleotide excision reagent (e.g., removes deoxyuridine) to form a polynucleotide duplex including a plus strand and a minus strand, each including a single nucleotide gap in the adaptor top strand of the nick-translated duplex polynucleotide, defining an oligonucleotide of varying length. The method may further include, in some embodiments, contacting the nick-translated duplex polynucleotide with a linear amplification primer having a sequence complementary to a linear amplification annealing site to form an annealed duplex.The method may, according to some embodiments, further comprise contacting the annealed duplex with a polymerase to form a primer extension product comprising, in a 5' to 3' direction, a sequence corresponding to at least a portion of the adaptor top strand and a sequence corresponding to at least a portion of the plus strand of one of the polynucleotide fragments. Amplification (e.g., linear amplification) may be achieved by repeating the annealing and polymerization steps at least once (e.g., 5-10 cycles, 5-20 cycles, 5-30 cycles, 10-50 cycles, or >50 cycles). In some embodiments, the method may further comprise contacting the primer extension product with a target isolation probe comprising an affinity domain and a target-specific oligonucleotide to form a target probe mixture comprising a target complex comprising the primer extension product, the target isolation probe, and, if one or more target sequences are present in the population of fragmented polynucleotides, the primer extension product having a target sequence hybridized to a target-specific oligonucleotide of the target isolation probe. The method may also include, according to some embodiments, contacting the target probe mixture with a solid support that includes an affinity capture domain that corresponds to the affinity domain of the target isolation probe to form an affinity complex that includes the affinity domain bound to the affinity capture domain. The method for enriching the target sequences may, in some embodiments, include separating the affinity complex from unbound primer extension products and unbound target isolation probe to form an enriched target sequence composition.

[0015] The method may in some embodiments include, for example, A-tailing the fragmented polynucleotides prior to fusion with a first adaptor, the top strand of which further comprises a 3'T or dU. The method may include repeating all method steps (e.g., for multiplex analysis), the population of fragmented polynucleotides of each repeated first adaptor fusion step being different from all preceding adaptor fusion steps, the sample tag of each first adaptor fusion step being different from all preceding first adaptor fusion steps, and optionally pooling the affinity complexes resulting from each repetition (or cycle). The method may include, for example, one or more optional wash steps after formation of the target complex and / or after formation of the affinity complex to remove (at least a portion of) the fragments lacking the target sequence. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 1A shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 1A illustrates a simple flow chart for an exemplary enrichment workflow. [Figure 1B-1] FIG. 1B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 1B illustrates an exemplary workflow. [Figure 1B-2] FIG. 1B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 1B illustrates an exemplary workflow. [Figure 1B-3] FIG. 1B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 1B illustrates an exemplary workflow. [Figure 2A] FIG. 2A shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 2A illustrates a simple flow chart for an exemplary enrichment workflow. [Figure 2B-1] FIG. 2B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 2B illustrates an exemplary workflow. [Figure 2B-2] FIG. 2B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 2B illustrates an exemplary workflow. [Figure 2B-3] FIG. 2B shows a schematic diagram of an exemplary method for forming an enriched library of target sequences from a complex (e.g., genomic) DNA sample. FIG. 2B illustrates an exemplary workflow. [Figure 3A] 3A illustrates sequencing results from three low-input samples, each enriched for a different target, one totaling 75 kb (FIG. 3A), one totaling 227 kb (FIG. 3B), and one totaling 1100 kb (FIG. 3C). [Figure 3B] 3A illustrates sequencing results from three low-input samples, each enriched for a different target, one totaling 75 kb (FIG. 3A), one totaling 227 kb (FIG. 3B), and one totaling 1100 kb (FIG. 3C). [Figure 3C] 3A illustrates sequencing results from three low-input samples, each enriched for a different target, one totaling 75 kb (FIG. 3A), one totaling 227 kb (FIG. 3B), and one totaling 1100 kb (FIG. 3C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure, in some embodiments, relates to methods and compositions for preparing polynucleotide libraries enriched for target sequences from a source material (e.g., a sample that includes or comprises a biological fluid, tissue, and / or specimen). The methods and compositions may provide efficient enrichment of target polynucleotides. Enrichment may include increasing the relative abundance of a target from the source material (where the target may be present in low abundance) to the generated library (where the target may be present in higher abundance). In some embodiments, the methods include attaching (e.g., ligating, linking, or otherwise fusing) adaptors to fragments of interest, nick translation, amplification (e.g., linear amplification), and target sequence selection, e.g., using affinity tagging.

[0018] Targeted genomic analysis applications may include, for example, the analysis of as few as one target of one or a few bases to identify clinically actionable mutations. Such applications may instead include the analysis of a million targets (or more) consisting of 50-100 megabases to sequence the whole exome or genome of a pathogen enriched from a host sample to discover variants of interest. One to a few targets are typically captured by PCR methods, and a much larger number of targets are efficiently captured by conventional hybridization capture of PCR-amplified whole genome libraries. Intermediate size panels for sensitive interrogation of hundreds to thousands of targets are insufficiently addressed by these methods. PCR has limited multiplexing capabilities, thereby requiring many separate reactions to cover a target set of this size. Conventional hybridization capture of targeted library fragments has reduced specificity at relatively small panel sizes due to non-specific capture of some off-target fragments. Additionally, biases can be introduced during PCR amplification prior to enrichment, amplifying polymerase errors introduced during PCR.

[0019] The present disclosure provides methods that, in some embodiments, capture the advantages of existing hybridization techniques while increasing specificity by performing hybridization capture of target fragments before they are fully converted into libraries with essential 3' and 5' fixed sequences and / or inhibiting conversion of off-target fragments into full-length library molecules. Additionally, the present disclosure provides methods that include amplification required with low input samples to increase sensitivity, but utilize linear amplification rather than PCR to avoid introducing significant amplification bias and / or amplifying polymerase-introduced errors.

[0020] General Considerations Aspects of the present disclosure can be further understood in light of the embodiments, section headings, figures, descriptions, and examples, none of which should be construed as limiting the overall scope of the disclosure in any way. Accordingly, the claims set forth below should be construed in light of the full breadth and spirit of the present disclosure.

[0021] Each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Unless otherwise noted, all reagents cited in this disclosure are available from New England Biolabs, Inc. (Ipswich, MA).

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Nonetheless, certain terms are defined herein with respect to the embodiments of the present disclosure and for clarity and ease of reference.

[0023] Numerical ranges are inclusive of the numbers that define the range. All numbers should be understood to include the midpoints of the integers above and below, i.e. the number 2 includes 1.5 to 2.5. The number 2.5 includes 2.45 to 2.55, etc. When sample numerical values ​​are given, each alone can represent an intermediate value in a range of values, and together can represent the ends of a range unless otherwise specified.

[0024] Sources for commonly understood terms and symbols include Kornberg and Baker, DNA Replication, 2nd ed. (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton et al. Dictionary of Microbiology and Molecular biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Markham, the Harper Collins Dictionary of Biology, Harper This may include standard articles and textbooks such as Perennial, NY (1991) and the like.

[0025] As used herein and in the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. For example, "a protein" refers to one or more proteins, i.e., a single protein as well as multiple proteins. It is further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0026] In the context of this disclosure, an "adapter" is a polynucleotide configured to be attached (e.g., ligated, linked or otherwise fused) to a desired polynucleotide (e.g., a fragmented polynucleotide). An adapter may be linear and have two ends. One end may be suitable for ligation to another polynucleotide. For example, the end may be blunt (e.g., for blunt end ligation), have a single nucleotide overhang ligation (e.g., a T or U overhang for ligation to an A-tailed polynucleotide), or have a longer overhang (e.g., 2-20 nucleotides for ligation to a polynucleotide with a complementary overhang). An adapter may include a 3' end that is not extendable. For example, each 3' end of an adapter may have an inverted dT (e.g., TriLink Biotechnologies) linked to the penultimate nucleotide by a 3'-3' linkage that is resistant to extension by a DNA polymerase. The other end of the polynucleotide (e.g., distal to the end selected or adapted for ligation) can be the same or different. If different, the end can contain any length and sequence of nucleotides with or without complementarity to the remainder of the adapter. For example, the end can contain a long overhang with no complementarity to the strand that is otherwise paired to the adapter. The adapter can contain a single strand capable of forming a loop (e.g., including a hairpin) in which at least a first portion of the adapter strand is hybridized to a second portion of the adapter strand.

[0027] A double-stranded adapter may include a top strand and a bottom strand, where each strand includes a separate oligonucleotide strand. A double-stranded adapter (e.g., a loop adapter) may include a top strand and a bottom strand, where a single polynucleotide strand folds back on itself to provide both the top and bottom strands (see, e.g., US 8,288,097 and US 2012 / 0244525A1).

[0028] In the context of this disclosure, an "affinity domain" refers to a domain that has high affinity (e.g., at least 10 -8 M) and a domain capable of binding with specificity. Exemplary materials with such properties include biotin, DBT, desthiobiotin, digoxigenin, glutathione, heparin, maltose, coenzyme A, protein A, brilliant blue FCF, azorubine, phytoestrogens, nickel, cobalt, zinc, polyhistidine, HA-tag, c-myc tag, FLAG-tag, S-tag, haptens for antibodies, mono- or oligosaccharide ligands for lectins, hormones, cytokines, toxins, pigments, and vitamins.

[0029] In the context of the present disclosure, an "affinity capture domain" refers to a domain that can bind to a corresponding affinity domain. Exemplary materials with such properties include avidin, streptavidin, neutravidin, maltose binding protein, GST, antibodies, lectins, nickel, cobalt, zinc, and polyhistidine. Further examples include groups that form irreversible bonds with protein tags, including benzylguanine or benzylchloropyrimidine (SNAP-tag); benzylcytosine (CLIP-tag); haloalkanes (HaloTags); CoA analogs (MCP-tags and ACP-tags); trimethoprim or methotrexate (TMP-tag); FlAsH or ReAsH (tetracysteine ​​tags); substrates for biotin ligase; substrates for phosphopantetheline transferase; and substrates for lipoic acid ligase. Affinity groups are used to selectively enrich samples by affinity purification methods, where the affinity binding partner is immobilized on a column, bead, microtiter plate, membrane or other solid support.

[0030] In the context of this disclosure, "linking", "fusing" and "ligating" may be used interchangeably and refer to any means of joining two (or more) nucleic acid ends, for example by ligases, transposases, topoisomerases, DNA-dependent protein kinases, and other end-joining or repair enzymes, where the ends may be in the same molecule (e.g., to form a circular nucleic acid molecule from a linear substrate) or in different molecules (e.g., to form a single linear nucleic acid product from two linear substrate molecules). Fusing two (or more) nucleic acid ends may include forming a covalent bond (e.g., a phosphodiester bond, a phosphorothioate bond, or any other linkage) between the ends. Fusing double-stranded nucleic acids may include fusing either or both of the 5' and 3' termini to either or both of the 3' and 5' termini of each of a second double-stranded nucleic acid. Fusing double-stranded nucleic acids may include fusing the 5' and 3' termini of a nick to each other. In some embodiments, fusing nucleic acid ends does not include nick translation.

[0031] In the context of this disclosure, "coverage" refers to the number of times a targeted subset of a population of polynucleotides (eg, sheared genomic DNA) is sequenced.

[0032] In the context of this disclosure, an "enriched library" refers to a library that has an increased amount of one or more targets compared to a reference library. For example, an enriched cell-free fetal DNA library may have a greater amount of one or more non-invasive prenatal testing targets than a maternal whole blood DNA library or a cell-free DNA library. The number of enriched targets in an enriched library may be 1, more than 1, more than 10, more than 100, more than 1000, more than 10,000, more than 100,000, or more than 500,000. An enriched library may include enriched targets corresponding to the whole exome of an organism.

[0033] In the context of this disclosure, "linear amplification" refers to an amplification reaction in which the amount of product increases linearly with time, rather than exponentially.

[0034] In the context of this disclosure, a "linear amplification annealing site" refers to a portion of a polynucleotide that has a sequence that is (a) complementary to an amplification primer and (b) unique within the polynucleotide. A linear amplification annealing site can be of sufficient complexity to be unique within a given polynucleotide in which it is present. For example, a linear amplification annealing site can be ≧15 nts, ≧20 nts, ≧25 nts, ≧30 nts, ≧35 nts, ≧40 nts, ≧45 nts, or ≧50 nts in length.

[0035] The term "high sensitivity" for determining the sequence of a read refers to the detection of rare variants that may be present in the genome. For example, in a cancer biopsy, only a small percentage, e.g., 0.1%, of the population of polynucleotides from a human sample may contain the sequence variant (e.g., SNP) of interest. Therefore, a method with high sensitivity is required to detect these rare events. The method related to linear amplification described herein and illustrated in Figures 1A-1B is a highly sensitive method.

[0036] In the context of this disclosure, "non-naturally occurring" refers to a polynucleotide, polypeptide, carbohydrate, lipid, or composition that does not exist in nature. Such a polynucleotide, polypeptide, carbohydrate, lipid, or composition may differ from a naturally occurring polynucleotide, polypeptide, carbohydrate, lipid, or composition in one or more ways. For example, a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) may differ in the type and arrangement of its component components (e.g., nucleotide sequence, amino acid sequence, or sugar molecule). A polymer may differ from a naturally occurring polymer with respect to the molecule(s) to which it is linked. For example, a "non-naturally occurring" protein may differ from a naturally occurring protein in its secondary, tertiary, or quaternary structure by having chemical bonds (e.g., covalent bonds including peptide bonds, phosphate bonds, disulfide bonds, ester bonds, and ether bonds, etc.) to a polypeptide (e.g., a fusion protein), lipid, carbohydrate, or any other molecule. Similarly, a "non-naturally occurring" polynucleotide or nucleic acid may contain one or more other modifications (e.g., added labels or other moieties) toward the 5'-terminus, 3'-terminus, and / or between the 5'- and 3'-terminus of the nucleic acid (e.g., methylation), one or more other modifications (e.g., added labels or other moieties). A "non-naturally occurring" composition may differ from a naturally occurring composition in one or more of the following ways: (a) having a component that is not associated with the naturally occurring component, (b) having a component at a concentration not found in nature, (c) otherwise excluding one or more components found in the naturally occurring composition, (d) having a form not found in nature, e.g., dry, lyophilized, crystalline, aqueous, and (e) having one or more additional components (e.g., buffers, surfactants, dyes, solvents, or preservatives) other than the components found in nature. All publications, patents, and patent applications mentioned herein are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0037] In the context of the present disclosure, "nick translation" of a double-stranded DNA molecule having at least one nick containing a 3'-OH refers to the templated addition of dNTPs beginning at the 3'-OH of the nick(s). Nick translation may also include 5' to 3' excision of nucleotides beginning at the nick(s). Nick translation may be catalyzed by DNA polymerases including, for example, E. coli DNA pol I, pol Ik, Taq DNA polymerase, OneTaq® DNA polymerase (New England Biolabs, Inc.), Bst DNA polymerase (e.g., large fragment), Bsu DNA polymerase, phi29 DNA polymerase, Therminator™ DNA polymerase, Klenow, Vent® DNA polymerase, and Deep Vent® DNA polymerase. DNA polymerases with nick translation and / or strand displacement activity may be referred to herein as "nick translation polymerases." A "nick" can include a break in the phosphate backbone with or without a short (eg, up to 15 nucleotides) gap.

[0038] In the context of this disclosure, a "population of polynucleotides" or "library" refers to a composition that includes a plurality of polynucleotides having different properties (e.g., sequence, length). Each polynucleotide molecule in a population of polynucleotides can be unique relative to other polynucleotides in the population. A complex library can be more than 10 4 , 10 5 , 10 6 Or 10 7 The nucleic acid sequence may contain more than 10 distinct nucleic acid molecules or fewer but longer molecules, which fewer molecules may collectively contain up to 10 4 , 10 5 , 10 6 Or 10 7or more nucleotides. Examples of complex populations of polynucleotides include mammalian and human DNA and cDNA libraries. The population of polynucleotides may include or be derived from some or all of an organism's genomic DNA, organelle DNA, cDNA, mRNA, microRNA, long non-coding RNA, or other DNA or RNA. The population of polynucleotides may include or be derived from artificial DNA or artificial RNA. The population of polynucleotides may be prepared, for example, by chemically, enzymatically, or physically fragmenting some or all of an organism's DNA or RNA to generate fragments having a size or sizes within a desired range. The enriched library may have a complexity of less than 10%, less than 5%, less than 1%, less than 0.5%, or less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, less than 0.00001%, less than 0.000001%, or less than 0.0000001% compared to the corresponding non-enriched library.

[0039] In the context of this disclosure, a "polynucleotide" refers to any of DNA, RNA, and mixtures thereof. Polynucleotides may be single-stranded, double-stranded, or partially single-stranded and partially double-stranded. Polynucleotides may have blunt ends, 5' overhangs, and / or 3' overhangs. Polynucleotides may be naturally occurring (e.g., purified or otherwise derived from an organism's genome (nucleus or organelle) or cytoplasm (tRNA, mRNA)) or synthetic. Polynucleotides may include entire genomes, genes, fragments of DNA, or libraries of fragments. Polynucleotides may have and / or be depicted as having positive and negative strands or top and bottom strands. Polynucleotides include a wide range of sizes (e.g., from 2 nucleotides to 2 million nucleotides or even longer). Shorter polynucleotides (e.g., 2-250 nucleotides) may be referred to as polynucleotides or oligonucleotides.

[0040] In the context of this disclosure, a "sample" refers to a particular source of a particular population of polynucleotide fragments. Depending on the context, a sample may be a particular polynucleotide extract from a single cell, tissue, or individual biological entity, such as a plant, animal, or microorganism.

[0041] In the context of this disclosure, a "sample tag" is a molecular barcode that identifies the sample source of a population of polynucleotide fragments. Thus, the adapters attached (e.g., ligated, linked, or otherwise fused) to each strand in a duplex have the same sample tag as the other polynucleotide fragments in the population (e.g., FIG. 1B, i5 and i7 (Illumina)). The sample tag can be included in the first adapter, the second adapter, and / or the amplification primer (e.g., PCR primer) prior to sequencing.

[0042] In the context of this disclosure, a "strand" refers to a polynucleotide composed of nucleotides covalently linked to each other. A double-stranded or duplex polynucleotide (e.g., DNA) comprises two strands, a top or plus strand and a bottom or minus strand, each containing a sequence that is complementary to the other strand. Conventionally, the diagram of the strand designated as the top or plus strand is oriented with the 5' end to the left and the 3' end to the right, and the strand designated as the bottom or minus strand is oriented with the 3' end to the left and the 5' end to the right.

[0043] In the context of this disclosure, a "target sequence" refers to a portion of a polynucleotide fragment that constitutes or includes a sequence of interest. A target sequence may be of interest for any of a variety of reasons, for example, because it is diagnostic or otherwise relevant to a disease, infectious agent (e.g., yeast, bacteria, virus, phage), phenotype, genotype, allele, variant, or other state or condition. Examples of target sequences may include exons, introns, regulatory sequences, single nucleotide polymorphisms (SNPs), gene fusions, copy number variations, and indels. Analysis of target sequences may also be used to determine heterozygosity and homozygosity. Target sequences may be derived from contiguous or non-contiguous nucleotides in a genome. For example, target sequences may constitute or include expressed eukaryotic gene sequences (e.g., lacking some or all introns), gene fusions (e.g., a fusion of two genes that may not occur naturally and / or may otherwise be present in separate regions of the genome), and / or intron / exon boundaries. Target sequences include, for example, MTOR, DPYD, NRAS, NTRK1, DDR2, ALK, MSH2, MSH6, NFE2L2, IDH1, UGT1A1, VHL, RAF1, MLH1, CTNNB1, PIK3CA, FGFR3, PDGFRA, KIT, KDR, FBXW7, TERTpro, PIK3R1, APC, CSF1R, PDGFRB, FLT4, CCND3, ROS1, ESR1, PMS2, EGFR, CDK6, MET, SMO, BRAF, EZH2, FGFR1, JAK2, CD274, PDCD1LG2, CDKN2A, GNAQ, The target sequence may comprise or include all or a portion of a gene selected from PTCH1, ABL1, TSC1, GATA3, RET, PTEN, FGFR2, CCND1, CCND2, KRAS, CDK4, FLT3, FLT1, BRCA2, RB1, AKT1, MAP2K1, IDH2, TSC2, TP53, ERBB2, BRCA1, RNF43, SMAD4, STK11, GNA11, MAP2K2, KEAP1, JAK3, AKT2, GNAS, NF2, ARAF, AR, BRIP1, CHEK2, PALB2, RAD51C, and RAD51D. In some embodiments, the target sequence may comprise all or a portion of an exon.

[0044] In the context of this disclosure, a "target isolation probe" or "bait" refers to a molecule or complex that comprises an affinity domain and a target-specific oligonucleotide.

[0045] In the context of this disclosure, a "target-specific oligonucleotide" is an oligonucleotide that has a sequence that is complementary to at least a portion of a sequence of interest ("target sequence"). The specificity of a target-specific oligonucleotide is conferred by its nucleotide selection. Specificity can be further influenced by the stringency of the conditions under which the oligonucleotide is contacted with another polynucleotide, such as the temperature of contact, and the absence or concentration of salt(s), competitor(s), detergent(s), and especially crowding agent(s).

[0046] In the context of this disclosure, a "unique molecular identifier" (UMI) is a unique sequence of at least six nucleotides (6N) that has sufficient complexity to, taken together with other similar UMIs, uniquely label each polynucleotide molecule in a population of polynucleotide molecules. For example, the UMI of each molecule of adaptor 1033 can have a unique sequence. The UMI sequence can be random, haphazard, or otherwise varied to achieve a desired level of complexity. Longer random and unique sequences can be used, e.g., 2-15 nucleotides, 6-12 nucleotides, or 8-12 nucleotides.

[0047] The present disclosure provides methods for generating enriched libraries of target sequences. The methods according to some embodiments can efficiently generate libraries from low input samples that represent a majority (e.g., all or substantially all) of the nucleotides in the original sample.

[0048] Sample preparation In some embodiments, the population of polynucleotides may be generated from any source or sample, for example, any biological sample, including but not limited to plants (e.g., ornamental plants, vegetables, fruits, trees, woody shrubs, grasses, spore-forming plants, seed plants, and other plants), animals (e.g., vertebrates, invertebrates, reptiles, mammals, insects, worms, fish, birds, and other animals), bacteria, fungi (e.g., yeast), phages, viruses, cadaveric tissue, archaeological / ancient samples, any parts thereof (e.g., organs, tissues, cells, body fluids, solids, specimens), and any combination thereof. In some embodiments, the sample for fragmentation may include genomic DNA from a mammal (e.g., a human or non-human animal). Mammalian body fluids of interest may include blood, serum, plasma, saliva, mucus, sputum, cerebrospinal fluid, pleural fluid, tears, mammary ductal fluid, lymphatic fluid, sputum, synovial fluid, urine, amniotic fluid, and semen. In some embodiments, the sample may be obtained from a subject, e.g., a clinical specimen from a human subject. The sample may, in some embodiments, comprise cell-free polynucleotides, including, for example, circulating DNA in bodily fluids (e.g., peripheral blood from a subject, e.g., a cancer patient or a pregnant woman's blood) and DNA in vascular fluids (e.g., phloem) of plants. In some embodiments, the sample for fragmentation may comprise genomic DNA from plants (e.g., leaves, stems, roots, seeds, pistils, stamens, embryos).

[0049] According to some embodiments, the method may include extracting polynucleotides (e.g., genomic DNA) from a biological source and then fragmenting it mechanically (e.g., by sonication, nebulization, or shearing), enzymatically (e.g., using double-stranded DNA "dsDNA" Fragmentase® enzyme (New England Biolabs, Ipswich MA), transposase, nuclease), or chemically (e.g., exposure to heat in the presence of divalent metal ions, formalin fixation and paraffin embedding). In some embodiments, the sample may include polynucleotides having a desired size or size range without further fragmentation, for example, when a population of polynucleotides is formed synthetically (e.g., in vitro) or collected from circulating cell-free DNA (cfDNA), e.g., ctDNA.

[0050] A population of polynucleotide fragments may, according to some embodiments, have a median size that is below 1 kb. For example, the median or average size may be 50 bp to 500 bp, or 80 bp to 400 bp, 150 bp to 200 bp, or 200 bp to 300 bp, or 300 bp to 400 bp, or 400 bp to 500 bp, or 500 bp to 600 bp, or 600 bp to 700 bp. In some embodiments, a population of polynucleotide fragments may have a median size that is above 1 kb. For example, the median fragment size may be 1 kb to 5 kb, or 2 kb to 10 kb.

[0051] The method of the present disclosure may be used in some embodiments when the amount of polynucleotide is limited. For example, the method may be applied to a population of polypeptides that includes or contains less than 200 ng of fragmented DNA (e.g., 10 pg to 200 ng of fragmented DNA, 100 pg to 200 ng of fragmented DNA, 1 ng to 200 ng of fragmented DNA, 5 ng to 50 ng of fragmented DNA) or less than 10,000 haploid genome equivalents (e.g., <5,000, <1,000, <500, <100, <10). The method of the present disclosure may be used in some embodiments when the amount of polynucleotide is not so limited.

[0052] (First) Adapter ligation The method may, in some embodiments, include attaching (e.g., ligating, linking, or otherwise fusing) an adaptor (e.g., a double-stranded adaptor) to a polynucleotide fragment to generate a fusion product (e.g., a tagged fragment). The adaptor may comprise two separate strands hybridized together (e.g., as shown in Figures 1B or 2B) or a single strand having portions hybridized to each other and an intervening single-stranded region (e.g., a hairpin or loop adaptor). One or both strands of the adaptor may include one or more (e.g., one, two, or three) modified nucleotides, such as deoxyuridine, 8-oxoguanine, or deoxyinosine. The presence of modified nucleotides may confer desirable properties to the adapters, including, for example, limiting or preventing undesirable exonuclease digestion (e.g., phosphorothioates), reducing or eliminating undesirable secondary structures associated with G-rich regions (e.g., 8-aza-7-deazaG ("super-G")), or increasing the melting temperature of the duplex (e.g., with specific AT pairs using locked nucleic acids or by replacing T with 5-hydroxybutyl-2'-deoxyuridine ("super-T")). In some cases, the presence of modified nucleotides may provide the user with an efficient option for removal of the adapter in a subsequent step, for example, by enzymatic removal (e.g., using glycosylases) prior to linear amplification.

[0053] According to some embodiments, the method may include generating a population of A-tailed fragments from a population of polynucleotides (e.g., an unamplified, native population of polynucleotides). A-tailing may include, for example, contacting the polynucleotides with a polymerase and dATP under conditions that allow or support non-templated addition of a single dA to the 3' end(s). If the fragments are A-tailed, the adaptors may have corresponding 3' overhangs of T or U to facilitate ligation. The A-tailed polynucleotide fragments may facilitate ligation of the adaptors (e.g., by Watson-Crick base pairing of the 3' A with the 5' T or U), which can be efficiently accomplished using kits provided by New England Biolabs, Ipswich, MA (e.g., NEBNext® Ultra™ II End Repair / dA-Tailing Module). Alternatively, other means for attaching adaptors to double-stranded DNA in a specific orientation may be used, or similarly, A-tailing may be accomplished by using kits from other suppliers.

[0054] In some embodiments, an adaptor comprising a top and a bottom strand may be attached (e.g., ligated, linked or otherwise fused) to a polynucleotide fragmentation product, with the top strand of the adaptor attached (e.g., ligated, linked or otherwise fused) to the plus or minus strand of the fragmented polynucleotide (e.g., ligated to the 3' end of the A-tailed fragmented polynucleotide). The top strand may comprise, in a 5' to 3' direction, a sequence complementary to a linear amplification annealing site (e.g., an NGS platform specific sequencing primer), optionally a sample tag, a unique molecular identifier (UMI), a sequence complementary to the bottom strand, and optionally a nucleotide complementary to A. In some embodiments, the top strand of the adaptor is longer than its bottom strand. The bottom strand of the adaptor may have at least 10 nucleotides (e.g., at least 15 nucleotides). Under some conditions, a shorter bottom strand may be appropriate. The 5' end of the bottom strand of the adaptor may include a 5'-OH, for example, to block ligation of the bottom strand to the bottom strand of the polynucleotide fragment. Adaptor ligation may be performed with T4 DNA ligase, circligase, PBCV-1 DNA ligase, or another ligase. The ligation product may optionally be separated from other components of the ligation mixture.

[0055] In some embodiments, after ligation, a region of the hairpin or loop adaptor can be cleaved to create a duplex where the top and bottom strands are on different molecules. In some cases, the cleaved region of the hairpin adaptor can contain modified residues such as deoxyuridine, a base that can be cleaved using a glycosylase (e.g., UDG), although other methods are known.

[0056] Strand displacement / nick translation The fusion product (tagged fragment) containing a nick (e.g., a nick defined by the unligated end of the bottom strand of the adapter and the A-tail of the fragmented polynucleotide) may be contacted with a strand displacement polymerase or a nick translation polymerase for templated addition of nucleotides. The product of either approach may be referred to as a nick translation product. For example, nucleotides may be added starting with the A-tail of the fragmented polynucleotide, with the template being the top strand of the adapter. The nascent strand of the formed double-stranded polynucleotide may be complementary to the top strand of the adapter, and may optionally be free of modified nucleotides (e.g., by performing strand displacement / nick translation in the absence of modified nucleotides). Each strand of the formed double-stranded polynucleotide may comprise, in the 5' to 3' direction, the top strand of the adapter, the polynucleotide fragment, and the nascent strand (which is complementary to the top strand of the adapter). If the top strand of the adapter contains one or more modified nucleotides, the product of the strand displacement / nick translation may be contacted with a single nucleotide excision reagent (e.g., glycosylase and endonuclease) to remove the modified nucleotides. For example, a single nucleotide excision reagent may remove the base of a modified nucleotide and cleave the phosphodiester backbone at the 3' and 5' sides of the modified base (or at the abasic site resulting from removal of the modified base). For example, the top strand of an adapter containing deoxyuridine may be contacted with UDG (e.g., Antarctic Thermolabile UDG (New England Biolabs, Ipswich, MA)) for, e.g., 10 minutes at 37°C. UDG catalyzes the release of free uracil from the adapter sequence fused to the 5' end of the DNA fragment, generating an abasic site in the adapter sequence. The abasic site is susceptible to hydrolytic cleavage and decomposes upon increasing the reaction temperature in the following thermal cycling reaction. Alternatively, UDG may be used in combination with EndoVIII (e.g., USER®, New England Biolabs, Inc.). Other modified nucleotide / reagent combinations may be used.The site of the removed single nucleotide (e.g., deoxyuridine or other modified nucleotide) may define an end of the oligonucleotide of variable length, depending, for example, on the position of the removed nucleotide in the initial strand, but in any case, shorter than the initial strand. Contiguous modified nucleotides, when excised, may be referred to as a single nucleotide gap, consecutive single nucleotide gap, or nucleotide gap. Excision of two modified nucleotides adjacent to an unmodified nucleotide may release the single nucleotide. The single nucleotide and oligonucleotide formed by the action of the single nucleotide excision reagent at the working reaction temperature (e.g., 10°C to 40°C (or higher)) may remain unbase-paired to the other strand. Thus, the single nucleotide and oligonucleotide may be lost in subsequent steps. In some embodiments, the nucleotide and oligonucleotide formed by the action of the single nucleotide excision reagent may be removed / separated from the remaining strand containing the polynucleotide fragment and the nascent strand derived from the nick translation by optionally including a specific purification step.

[0057] Linear Amplification According to some embodiments, the nick translation products (e.g., remaining after removal of the modified nucleotides) may be linearly amplified. Linear amplification may be performed by contacting the fusion products (e.g., simultaneously with or subsequent to removal of the modified nucleotides) with a polymerase, dNTPs, and linear amplification primers to generate a reaction mixture. In some embodiments, the polymerase may be a thermostable polymerase, and the reaction mixture may be thermally cycled at least once (e.g., at least 5 times, or at least 10 times, or at least 20 times) to generate several copies of each tagged fragment, with the copy number matching the cycle number. Linear amplification may be performed using NEBNext Ultra II Q5® Master Mix (a master mix containing Q5 DNA polymerase (New England Biolabs, Ipswich, MA)), although other polymerases can be used. The products of linear amplification may have a copy of the molecular barcode of the top strand of the adapter on the 3' side of the polynucleotide fragment, and may be referred to as 5'-tagged amplicons.

[0058] In some embodiments, polymerases active at lower temperatures (e.g., Klenow exo-, Bsu large fragment, and phi29 for moderate temperature reactions (25-40° C.) and large fragment of Bst DNA polymerase for higher temperature (50-65° C.) reactions) may be used, and the reaction mixture may be incubated with or without thermal cycling to generate several copies of each tagged fragment. For example, linear amplification may be performed using isothermal amplification (e.g., instead of thermal cycling). Examples of isothermal amplification may include ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), Qβ replicase-based amplification or rolling circle amplification, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR), boomerang DNA amplification (BDA), helicase-dependent amplification (HDA).

[0059] Linear amplification may be performed, according to some embodiments, by denaturing the polynucleotide to be amplified (e.g., at 98°C for 30 seconds), contacting the denatured polynucleotide with a polymerase, dNTPs, and linear amplification primers to form a reaction mixture, and thermally cycling the reaction mixture at least once (e.g., at least 5 times, at least 10 times, at least 15 times, or at least 20 times). Thermal cycling may be performed in the following steps: (1) at a temperature above 90°C (e.g., 98°C) for at least 5 seconds, (2) at a temperature below 60°C (e.g., 55°C) for at least 5 seconds, and (3) at a temperature in the range of 65°C to 80°C (e.g., 70°C to 75°C) for at least 10 seconds. At the first temperature, the polynucleotide fragments are denatured. At the next temperature, the linear amplification primer anneals to the 3' end of the adapter sequence. At the third temperature, a polymerase (e.g., Q5 polymerase) extends the linear amplification primer. Other thermal cycling conditions are known and may be readily used.

[0060] In some embodiments, it may be desirable to use a polymerase for linear amplification that has a low error rate (e.g., 8-4 times less errors than Taq polymerase). Linear amplification may be performed with a proofreading DNA polymerase, which may have 3' to 5' exonuclease activity. Examples of proofreading thermostable polymerases include Pfu (Agilent Technologies, Santa Clara, CA), Pwo (Roche, Basel, Switzerland), Tgo (Roche, Basel Switzerland), VENT® (New England Biolabs, Ipswich, MA), DEEP VENT® (New England Biolabs, Ipswich, MA), KOD HiFi (Novagen, Madison, WI), PFX50™ (Invitrogen, Waltham, MA), HERCULASE II™ (Agilent Technologies, Santa Clara, CA), PLATINUM PFX™ (Life Technologies, Waltham, MA), and ProofStart™ (Qiagen, Hilden, Germany). Further examples of proofreading thermostable polymerases include, but are not limited to, PHUSION® (Thermo Fisher Scientific, Waltham, MA), PFUULTRA™ (Agilent Technologies, Santa Clara, CA), PFUULTRA™ II (Agilent Technologies, Santa Clara, CA), IPROOF™ (Bio-Rad, Hercules, CA), Q5 polymerase, and KAPAHIFI™ (Kapa Biosystems, Wilmington, MA). On average, these polymerases may produce 20 times fewer errors than Taq polymerase, and the polymerases can be readily used herein. Linear amplification may be performed using non-proofreading DNA polymerases.Examples of non-proofreading thermostable polymerases (ie, thermostable polymerases that lack 3' to 5' exonuclease activity) include, but are not limited to, Taq and Tth.

[0061] In some embodiments, linear amplification reduces or avoids amplification bias and / or amplification of sequence errors (e.g., nucleotide incorporation errors that may occur in early rounds of amplification). Linear amplification may provide a more streamlined process (e.g., compared to non-linear amplification) by avoiding the need for additional steps or measures to safeguard product quality. For example, non-linear amplification methods may omit the USER® digestion step to retain adapters at both ends of the fragments, but would then benefit from including a blocking oligonucleotide to preserve the specificity of the subsequent bait hybridization. Such an oligo would be required for the bait to hybridize to a target fragment and prevent the adapter sequence of that target fragment from hybridizing to the complementary adapter sequence of a random non-target fragment. Such hybridization may form a "daisy chain" amplification product that includes target and off-target molecules. Nevertheless, non-linear amplification may be an option in some embodiments, for example, where loss of quality may be acceptable and / or where additional precautions are included to offset loss of quality.

[0062] Hybridization The linear amplification product (e.g., 5'-tagged amplification product) may be contacted (e.g., hybridized to) a target-specific oligonucleotide to create a complex. In some embodiments, the target isolation probe may include a target-specific oligonucleotide linked (e.g., covalently) to an affinity domain (e.g., biotin). The target-specific oligonucleotide may be designed to hybridize to any target, including targets of diagnostic and / or therapeutic interest (e.g., target sequences in cancer-associated genes) and targets of breeding interest (e.g., traits, genotypes, phenotypes and markers thereof). Contacting the linear amplification product with the target isolation probe may include contacting the linear amplification product with a target isolation probe having at least 1, at least 5, at least 10, at least 100, at least 1,000, or at least 10,000 target-specific oligonucleotides, each with a different specificity, to isolate and enrich fragments having different elements of the target region or genome. Each target-specific oligonucleotide may have a length of 30 to 100 nucleotides. Shorter and longer target-specific oligonucleotides may be used under some conditions / circumstances. For example, smaller targets may be desired when the polynucleotide fragments to be evaluated are small, such as fragments resulting from urinary cfDNA. Smaller targets may also be desired or acceptable when evaluating small genomes (where shorter sequences retain uniqueness / specificity). In some embodiments, target-specific oligonucleotide pairs may be used, with one member of the pair hybridizing to the top strand of the target and the other member of the pair hybridizing to the bottom strand of the target.

[0063] The target sequence may be at the 3' end of the region of interest. In some embodiments, a biotinylated target-specific oligonucleotide is added to a hybridization solution containing a pool of DNA fragments. The hybridization solution containing the target-specific oligonucleotide and the DNA fragments is then incubated to allow the target-specific oligonucleotide to hybridize to the 5'-tagged amplification products containing the target sequence. This hybridization may be performed at relatively high stringency or relatively low stringency. When low stringency conditions are used, a significant amount of non-specific binding to the target-specific oligonucleotide may occur. The non-specifically bound sequences may be removed in a subsequent step by treatment with a 3'-5' single-stranded exonuclease and a double-stranded exonuclease (e.g., exonuclease III).

[0064] Complexes (e.g., including target isolation probes hybridized to target regions of tagged fragments) can, in some embodiments, be captured on the support by an affinity capture domain (e.g., streptavidin) linked (e.g., covalently) to the support. Target-containing tagged fragments can thereby be enriched or separated from target-free tagged fragments. In some embodiments, streptavidin-coated magnetic beads can be added to the reaction mixture after hybridization of 5'-tagged amplification products to target-specific oligonucleotides. The magnetic beads can be magnetically isolated and then washed, thereby enriching for complexes containing 5'-tagged amplification products. Alternatives to biotin include SNAP-tag® (New England Biolabs, Ipswich, Mass.), a protein that reacts with benzylguanine and can be modified to bind to affinity capture domains.

[0065] The solid support may comprise a matrix formed from or coated with the capture domains. The solid support may be, for example, a bead, including magnetic beads, a column, a porous matrix, or a flat surface formed, for example, from plastic or paper.

[0066] Creating a 3' blunt end The affinity captured complex may, in some embodiments, have a 3' overhang relative to the 5' end of the hybridized target-specific oligonucleotide. The 3' overhang may include one or more 3' nucleotides of the fragmented polynucleotide and / or one or more nucleotides corresponding to the top strand of the adaptor. The 3' overhang may be removed by contacting the affinity captured complex with a single-stranded 3'-5' exonuclease to create a blunt end with the same length as the 3' end of the target sequence. One or more single-stranded 3'-5' exonucleases may be used individually or in combination to catalyze the stepwise removal of mononucleotides from the 3' end of the single-stranded DNA to create a blunt end. For example, exonuclease I and exonuclease T may be used individually or in combination. The 3'-5' single-stranded exonuclease may trim the 3' end of the captured 5'-tagged amplification product until it is the same length as the target sequence. The blunt end may include a 5' overhang (e.g., a 1-5 nucleotide 5' overhang), for example, if the 3'-5' single-stranded exonuclease trims the 3' end of the captured 5'-tagged amplicon just beyond the target sequence. A 3' nuclease reaction buffer containing one or more single-stranded 3' exonucleases may, in some embodiments, be added to the captured complex (still containing the 5'-tagged amplicon) to create a reaction mixture. After the reaction mixture is incubated (e.g., for approximately 10 minutes at 25°C, then for 5 minutes at 37°C), the captured complex may be separated from the remaining mixture (e.g., magnetically in the case of affinity capture domains bound to magnetic beads, or by decanting, pipetting, or otherwise removing the liquid if the affinity capture domain is bound to the surface of a tube or plate) and washed. The mixture can then be discarded, as the exonuclease-treated complexes are still tethered to the support (e.g., beads, tube, plate).

[0067] (Second) Adapter Fusion According to some embodiments, a 3' adaptor (e.g., double-stranded adaptor) may be attached (e.g., ligated, linked or otherwise fused) to the 3' end of the captured polynucleotide fragment of the exonuclease-treated complex to generate a strand of 3'-tagged polynucleotide fragments that contain the target sequence, which may also remain tagged at their 5' end from the preceding (first) adaptor fusion. For example, the captured complex (e.g., following washing) may be contacted with the 3' adaptor, ligase and / or ligation buffer and incubated for an appropriate time and at an appropriate temperature to allow ligation. In some embodiments, the 3' adaptor may include a blocking moiety at its 3' end(s), for example, where it may be desirable for the resulting ligation product to be resistant to 3'-5' double-stranded DNA exonuclease. The blocking moiety may comprise one or more phosphorothioates at the 3' end, with a single-stranded portion at the 3' end or with a hairpin structure that becomes single-stranded after it is partially digested. In some embodiments, the 3' adaptor may have a long (e.g., ≧3 nt, ≧4 nt, ≧5 nt, ≧10 nt, ≧20 nt, ≧25 nt, ≧50 nt, or ≧100 nt) 3' single-stranded overhang that makes it resistant to digestion by double-stranded exonucleases.

[0068] As an example, a 3' adapter sequence and T4 DNA ligase may be added to the ligation mixture to blunt-end ligate the 3' adapter to the target sequence. A-tailing the 3' end of the target sequence for ligation with an adapter having a complementary T-overhang may be an alternative. Other ligation methods known in the art may be used, including, for example, linking the 3' adapter sequence to the target sequence by ligation using, for example, circ ligase or Taq ligase. In this example, as described above, the 3' adapter is double-stranded, with its top strand ligated to the 3' end of the polynucleotide sequence fragment and its bottom strand containing several deoxyuridine residues or other modified nucleotides. The positive strand of the 3' adapter may include the 3' adapter sequence (e.g., NGS platform specific sequencing primer site and library amplification primer site). After incubation, e.g., for 15 minutes at 20°C, the ligation product (e.g., a complex in which the ligated 3' adapter is still bound to the affinity capture domain and support) can be isolated from the ligation mixture (e.g., using magnetism). The separated ligation mixture (now devoid of ligation product) can be discarded, as the ligation product, including the complex containing the 5'- and 3'-tagged strands that contain the target sequence, remains tethered to the support.

[0069] Sample purification and amplification In some embodiments, the second adaptor fusion product, which includes the strand tagged at both the 5' and 3' ends and includes the target sequence, can be purified. For example, the strand tagged at both the 5' and 3' ends and includes the target sequence can be separated or isolated from the unligated target isolation probe, the target isolation probe ligated to the strand below the 3'-adaptor, and off-target polynucleotides (e.g., polynucleotides lacking the target sequence) by contacting the second adaptor ligation product with a double-stranded 3'-5' exonuclease that catalyzes the stepwise removal of mononucleotides from the 3' end of the double-stranded DNA and also cleaves the abasic site. The double-stranded 3'-5' exonuclease can remove any remaining double-stranded off-target DNA molecules.

[0070] As an example, the magnetic beads may be washed and resuspended in 3'-double stranded exonuclease buffer. A double stranded 3'-5' exonuclease, e.g., exonuclease III, may be added to form a mixture, and the exonuclease mixture may be incubated (e.g., 15 minutes at 37°C), thereby removing any remaining non-target sequences with unprotected or accessible double stranded 3' ends. UDG or another deglycosylase may also be added to remove bases from modified nucleotides (e.g., deoxyuridine) from the 3' adapter minus strand. In this example, the target specific oligonucleotide may remain hybridized to the target sequence, and the magnetic beads may be isolated from the remaining reaction mixture using magnetism and washed. The remaining reaction mixture may be discarded, as the exonuclease treated ligation products include complexes containing 5' and 3' tagged strands that contain the target sequence, but are still tethered to the beads. The tagged strand can be rendered 3'-5' exonuclease insensitive to protect the strand from degradation during this exonuclease treatment.

[0071] Optionally, the strands tagged at both the 5' and 3' ends containing the target sequence may be amplified by PCR, for example, by contacting the strands with a first primer that hybridizes to a portion of the 3' adaptor and a second primer that hybridizes to a linear amplification primer sequence of the first adaptor to generate a PCR product. The contacting may further include contacting the strands with a PCR mixture comprising water and a PCR master mix (e.g., a master mix comprising a polymerase, dNTPs, metal ions, salts, and / or buffers). The PCR reaction conditions may include (a) a temperature above 90°C (e.g., 98°C) for at least 30 seconds, (b) (1) a temperature above 90°C (e.g., 98°C) for at least 10 seconds, (2) a temperature in the range of 60°C to 70°C (e.g., 62°C) for at least 15 seconds, and (3) 18 cycles of a temperature higher than step (2) (e.g., 72°C) for at least 20 seconds, and (c) a temperature of 72°C for at least 3 minutes. According to some embodiments, the strand that contains the target sequence and is tagged at both the 5' and 3' ends is not amplified.

[0072] Sequencing The strands containing the target sequence and tagged at both the 5' and 3' ends (e.g., with suitable sequencing compatible sequences), whether amplified or not, may be sequenced (e.g., by any desired sequencing method) according to some embodiments. For example, the strands may be sequenced by next generation sequencing methods, and using suitable primers (e.g., primer extension using Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (SOLiD platform), Life Technologies' Ion Torrent platform, MGI's CoolMPS, Oxford Nanopore sequencing, and Pacific Biosciences' fluorescence-based cleavage method, among others), at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1M, at least 10M, at least 100M, or at least 1B sequence reads may be obtained. The reads may be paired end reads. The sequences required for a selected sequencing method can be included in the primer containing any desired adaptor and / or second adaptor, in the PCR primer used after ligation of the second adaptor, or both.

[0073] Examples of sequencing methods are described in the following references: Margulies et al. (Nature 2005 437:376-80); Ronaghi et al. (Analytical Biochemistry 1996 242:84-9); Shendure (Science 2005 309:1728); Imelfort et al. (Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009;553:79-108); Appleby et al. (Methods Mol Biol. 2009;513:19-39); English (PLoS One. 2012 7:e47768); and Morozova (Genomics. 2008 92:255-64), which is incorporated by reference for a general description of the method and the specific steps of the method, including all starting products, reagents, and final products for each of the steps. The sequence reads may be analyzed by a computer to identify sequence variations in the sample, such as point mutations, deletions, insertions, and rearrangements.

[0074] multiplexing Advantages of multiplexing include (a) the ability to analyze multiple samples in one sequencing reaction while maintaining a means to track the source of each polynucleotide and each sample from which it was derived, and (b) pooling samples can increase the efficiency and reduce the cost of workflows used to enrich targets and sequence samples. Multiplexing as described herein can include pooling two, tens, hundreds, or thousands of samples.

[0075] In some embodiments, the adaptors fused onto the fragments in the first step of the method may carry a sample tag, and the reaction may be multiplexed by combining samples from different sources (e.g., each with its own tag) into a pool. The pool may be distinguished (e.g., from other samples or pools) by its sample tag. In some embodiments, the method may include attaching (e.g., ligating, linking, or otherwise fusing) a double-stranded adaptor to individual polynucleotide fragments from a single sample with a first sample tag, and then pooling the polynucleotide fragments fused with polynucleotides with one or more other sample tags from one or more other samples. When the sample undergoes linear amplification, an adaptor may be attached (e.g., ligated, linked, or otherwise fused) to the 5' end of the sample DNA, the adaptor having, in the 5' to 3' direction, a sequence complementary to the linear amplification annealing site, optionally a sample tag, a UMI, a sequence complementary to the bottom strand, and optionally a top strand containing a nucleotide complementary to A. During linear amplification, primer extension creates a 5'-tagged amplification product. This reaction can be cycled multiple times to generate many copies of tagged polynucleotide fragments, making this strategy useful for high-depth coverage sequencing needs, such as the analysis of cfDNA.Alternatively, if the sample does not undergo linear amplification, the adapter sequence with the sample tag and, optionally, UMI can be attached (e.g., ligated, linked, or otherwise fused) to the 5' end of the sample DNA.This strategy is suitable for low-depth coverage sequencing needs of non-restrictive samples, such as genotyping.

[0076] The sample preparation, fragmentation, repair, and A-tailing steps may be performed as described herein using multiple samples in parallel in some multiplexing embodiments. The double-stranded adapter may include a longer top strand that includes a sample tag disposed on the bottom strand and a single-stranded extension located between the duplex region containing the first single-stranded adapter sequence and a sequence that is complementary to the bottom strand. Each sample may be prepared to include a distinct sample tag, where each tagged polynucleotide fragment can be traced back to the sample from which it originated by its sample tag.

[0077] As shown, after all samples are bound to the adaptors with sample tags and, optionally, UMI sequences, the samples may be pooled in a single vessel and proceed together through the remainder of the enrichment steps. Optionally, the samples may be pooled after linear amplification. After pooling, the method may include hybridizing the pool of adaptor-tagged samples with a target-specific oligonucleotide to create a complex. The complex may be bound to a solid support, thereby enriching for 5'-tagged molecules that contain the target sequence. If the target-specific oligonucleotide is biotinylated, enrichment may be performed using a support that contains streptavidin (e.g., magnetic streptavidin beads). Next, in some embodiments, the method may include treating the complex with a 3'-5' single-stranded exonuclease to remove any overhanging 3' ends from the product molecules and create a blunt end at the 3' end of the target sequence. However, this step is optional and the second adaptor may be added by another means. The second double-stranded adapter may be attached (e.g., ligated, linked or otherwise fused) to the blunt end of the exonuclease-treated complex, thereby adding a 3' adapter sequence onto the 3' end of the target sequence to create a 5'- and 3'-tagged strand that includes the target sequence. Other fusion strategies may be used in this step. For example, the exonuclease-treated complex may be A-tailed, and it is contemplated that a T-tailed adapter may be used. The strand (e.g., the top strand) of the second double-stranded adapter may be 3'-5' double-stranded DNA exonuclease resistant due to the 3' blocking moiety. In these embodiments, the method may include treating the ligation product with a 3'-5' double-stranded exonuclease. The 5'- and 3'-tagged strands may then be amplified by PCR using a first primer that hybridizes to the 3' adapter sequence and a second primer that hybridizes to the complement of the first adapter sequence to create an amplification product. In this step, sample tags may be added to identify pools of samples in a multiplex reaction.

[0078] The amplification products may be sequenced by any convenient method to generate sequence reads that include the sequence, at least a portion of the target sequence, and a sample tag or its complement. During analysis, the sequence reads may be assigned to samples based on the sample tag present in the sequence read. This method may be performed in a high-throughput format. As few as one and as many as 96 samples, or as many as 384 or more samples, each with a different sample tag, may be pooled together, where the pool is labeled with a single sample tag on the 3' adaptor. These pooled samples may then be pooled into larger pools, each with a single sample tag, but containing multiple sample tag sequences for analysis in a single sequencing reaction. A single sequencing reaction may include multiple concentrated preparations of 3' and 5' adapter fused polynucleotide target sequences from one or more samples, two or more samples, three or more samples, five or more samples, ten or more samples, fifty or more samples, one hundred or more samples, five hundred or more samples, one thousand or more samples, five thousand or more samples, up to and including about 10,000 or more samples, which may be obtained from the same source or different sources. For example, the sample may be a plant extract, and the source may be various plants. In this example, the original individual polynucleotide fragments containing the target sequence may be tracked by UMI, each sample from which the polynucleotide originates may be tracked by sample tag, and each plant from which the seed originates may be tracked by sample tag sequence.

[0079] The above samples may include further multiplexing in relation to hybridization. Hybridization reactions may be performed with a single or pair of target isolation probes, or may be multiplexed by performing each reaction with many hybridization probes (e.g., 3 or more, 5 or more, 10 or more, 100 or more, or 1,000 or more target isolation probes).

[0080] kit The present disclosure relates to kits for carrying out the methods described herein. The kits may include, for example, any system for delivering materials or reagents for carrying out the methods described herein. In some embodiments, the kits may include a system that allows for the storage, transport, or delivery of reaction reagents (e.g., probes, enzymes, adapters, primers, reaction reagents, reaction vessels and / or surfaces in suitable containers) and / or supporting materials (e.g., written instructions for carrying out the assay, instructions for use) from one location to another. For example, in some embodiments, the kits include one or more enclosures (e.g., boxes) that contain one or more reaction reagents and / or supporting materials. Such contents may be delivered together or separately to the intended recipient. For example, a first container may contain an enzyme for use in an enrichment method, and a second container may contain an adapter. The kits may be formulated alone or in combination to select and enrich target templates from a nucleic acid sample containing non-target and target sequences. The kits may include one or more adapters, primers; exonuclease; ligase, polymerase(s), buffer; and nucleotides as described herein. The kit may further include one or more buffer solutions and standards for the generation of DNA libraries. These components may be present in a single reaction vessel or in multiple tubes and may be packaged separately or together.

[0081] Automated Workflows The methods disclosed herein may be performed with at least some automation. Systems for processing multiple samples in parallel may be adapted for use with the disclosed methods. For example, systems for processing samples in test tube racks, multi-well plates, on droplets on surfaces, and / or through microfluidics (including variants that use pressure, electrical potential, acoustic forces, and / or other forces to manipulate liquids and contact materials). The methods disclosed herein may be performed, for example, using an Echo® 525 Liquid Handler (Labcyte, Inc. San Jose, CA) or by a microfluidic device or lab-on-a-chip (Aqua Drop, Sharp). In the method shown in FIG. 1A, steps 1010, 1020, and 1030 may be performed in a single buffer, which is then replaced by a different buffer in step 1040. Steps 1050-1090 may be performed in a single reaction tube by adding reagents sequentially or all at once. In some embodiments, the steps from sample to sequencing may be automated on a single device.

[0082] In some embodiments, it may be desirable to combine an enriched library with one or more other enriched libraries, for example, for sequencing. For example, multiple environmental specimens may have collected enriched libraries (e.g., enriched for possible pathogen DNA present) prepared from each. This may be accomplished, for example, in a platform utilizing a 96-well dish, where 5' adaptor addition, hybridization, capture, enrichment, and 3' adaptor addition are performed in individual wells of a 96-well plate. Following accomplishment of 5' adaptor fusion of polynucleotide fragments from a single sample (single environmental specimen), polynucleotide fragments from multiple samples (samples from multiple locations or from a single location over time) from all 96 wells may be combined in a single well in a second 96-well plate for capture enrichment and 3' adaptor fusion. The multiplexed samples (representing multiple specimens) from multiple wells in the second plate may then be pooled for sequencing. Sequence data may be deconvoluted using sample tags and / or UMIs to associate specific sequences with specific specimens. EXAMPLES

[0083] Some particular example embodiments may be illustrated by one or more of the examples provided herein.

[0084] Example 1: Library preparation including fragmentation and adaptor ligation In some embodiments, the enrichment method may be performed according to the example shown in Figures 1A and 1B. As shown, the method 1000 may include a step 1010 of fragmenting a polynucleotide (e.g., genomic DNA) to form fragmented polynucleotides. The fragmented polynucleotides may include hybridized plus 1011 and minus 1012 strands. The fragmentation 1010 may include physically, mechanically, chemically, and / or enzymatically fragmenting the polynucleotides.

[0085] In some embodiments, the method may include a step 1020 of preparing the ends of the fragmented polynucleotides for adaptor ligation. The step 1020 of preparing the ends of the fragmented polynucleotides for adaptor ligation may include (a) repairing the fragmented polynucleotide ends 1020a to form blunt 5' ends and blunt 3' ends, (b) A-tailing the 3' ends 1020b, or (c) both (a) and (b) (e.g., in order) to form single base A overhangs at each 3' end. The prepared fragmented polynucleotides include plus 1021 and minus 1022 strands hybridized to each other and include blunt or A-tailed ends.

[0086] The method may include contacting 1030 (e.g., ligating) the fragmented polynucleotides having A-tailed 3' ends to a first adaptor (e.g., double stranded adaptor, single stranded hairpin adaptor) having a blunt end or a 5' nucleotide complementary to the 3' A-tail. In some embodiments, the first adaptor, adaptor 1033, may include a top strand 1034 and a bottom strand 1035. In some embodiments, the double stranded adaptor may include multiple deoxyuridines on the top strand, the bottom strand, or both the top and bottom strands. The top strand 1034 of adaptor 1033 may include, in the 5' to 3' direction, a sequence complementary to a linear amplification annealing site ("LAAS" in FIG. 1B), optionally a sample tag (e.g., i5), a unique molecular identifier (UMI), a sequence complementary to the bottom strand 1034, and a nucleotide complementary to A. The bottom strand 1035 of the adaptor 1033 may include, in a 5' to 3' direction, a 5'-OH (e.g., a 5' terminus lacking a phosphate), a sequence complementary to the top strand, and a modified nucleotide that is not extendable (e.g., an inverted dT). In some embodiments, the contacting step 1030 may include ligating the 3' end of the adaptor top strand to the 5' end of the fragmented polynucleotide without ligating the 5' end of the adaptor bottom strand to the 3' (A-tailed) end of the fragmented polynucleotide to form a double-stranded ligation product that includes a plus strand 1031 that includes a single-stranded break or nick and a minus strand 1032 that includes a single-stranded break or nick.

[0087] According to some embodiments, the method 1000 may include a step 1040 of contacting the double-stranded ligation product comprising nicks 1046 and 1047 with a nick translation or strand displacement polymerase to form a nick-translated double-stranded polynucleotide comprising plus strand 1041 and minus strand 1042. Examples of nick translation polymerases include E. coli DNA polymerase I, pol Ik, Taq DNA polymerase, OneTaq® DNA polymerase (New England Biolabs, Inc.), Bst DNA polymerase (e.g., large fragment), Bsu DNA polymerase, phi29 DNA polymerase, Therminator™ DNA polymerase, Klenow, Vent® DNA polymerase, and Deep Vent® DNA polymerase.

[0088] Method 1000 may, in some embodiments, include contacting 1050 the nick-translated duplex polynucleotide (e.g., including one or more modified nucleotides) with a single nucleotide excision reagent to form a polynucleotide duplex including a plus strand 1051 and a minus strand 1052, each including a gap (e.g., pre-occupied with a modified nucleotide) that defines an oligonucleotide of varying (e.g., generally short) length. For example, the plus strand 1051 of the formed polynucleotide duplex may include, in the 5' to 3' direction, a portion having a gap (e.g., a single nucleotide pre-occupied with a U), the plus strand of the double-stranded fragmented polynucleotide, and a portion including a sequence complementary to the first adaptor, a UMI, a sample tag, and a linear amplification annealing site.

[0089] An example of a single nucleotide excision reagent may include an enzyme preparation (e.g., USER® enzyme M5505 or M5508 (New England Biolabs, Inc.)) that achieves uracil removal and phosphodiester bond cleavage, or may include a combination of enzymes (e.g., uracil DNA glycosylase in combination with endonuclease VIII). It is recognized that some polynucleotide sequences may contain consecutive sequences of deoxyuridines. Treatment with the single nucleotide excision reagent may result in corresponding consecutive single nucleotide gaps that are gaps of two or more nucleotides according to the number of consecutive deoxyuridines originally present. The method may optionally include a step of removing the oligonucleotide formed by treatment with the single nucleotide excision reagent.

[0090] The method 1000 may include a step 1060 of contacting each strand (1051, 1052) of the tagging fragment (a polynucleotide duplex comprising a double-stranded fragmented polynucleotide and a 3' overhang (e.g., a 3' overhang comprising, in a 5' to 3' direction, a sequence complementary to a first adaptor, a sample tag, a UMI, and a linear amplification annealing site)) with a linear amplification primer 1061, the linear amplification primer having a sequence complementary to the linear amplification annealing site to form annealed complexes 1065 and 1066. In some embodiments, the method may include a step 1070 of contacting the annealed complexes 1065 and 1066 with a polymerase to form a primer extension product 1071 complementary to the plus strand 1051 and a primer extension product 1072 complementary to the minus strand 1052. Such annealing 1060 and primer extension 1070 may be repeated as desired (e.g., 2-200 cycles total) to amplify each strand (1051, 1052) of the polynucleotide duplex including the double-stranded fragmented polynucleotide and the 3' overhang, in which case the primer extension products may collectively be referred to as amplification products. The amplification here will be linear since each strand of the polynucleotide duplex has only one linear annealing site. The resulting primer extension products constitute a linear amplified population of polynucleotides corresponding to the initial fragmentation products. The primer extension products may include, in the 5' to 3' direction, a copy of the primer, the sample tag, the UMI, a sequence complementary to a portion of the adapter, and a copy of the plus strand of the double-stranded fragmented polynucleotide or a copy of the minus strand of the double-stranded fragmented polynucleotide. Method 1000 may optionally include a step 1075 of pooling the linear amplification post-primer extension products (e.g., primer extension products resulting from a workflow without a prior pooling step).

[0091] In some embodiments, the primer extension products may be examined for the presence of a polynucleotide having a sequence of interest (a "target sequence"). For example, the method may include a step 1080 of contacting the primer extension products with a target isolation probe (1081, 1082) to form a mixture comprising the primer extension products and the target isolation probe. The mixture may further include a target complex 1085, 1086 comprising the primer extension products having the target sequence hybridized (e.g., specifically hybridized) to a target-specific oligonucleotide of the target isolation probe, if the target sequence is present. The target isolation probe (bait) may comprise an affinity domain (e.g., biotin) and a polynucleotide having a polynucleotide sequence complementary to the plus strand of a sequence of interest (e.g., a sequence of interest somewhere within the fragmented polynucleotide, e.g., a sequence of interest at or near the 5' or 3' end of the plus strand of the fragmented polynucleotide) or a polynucleotide sequence complementary to the minus strand of a sequence of interest (e.g., a sequence of interest somewhere within the fragmented polynucleotide, e.g., a sequence of interest at or near the 5' or 3' end of the minus strand of the fragmented polynucleotide). The conditions of the contacting step 1080 may be adjusted as desired, e.g., to increase or decrease the stringency of bait to target sequence hybridization.

[0092] Optionally, method 1000 may include a step 1090 of contacting the mixture potentially including the target complex with a 3' single-stranded exonuclease to remove any 3' overhangs and form an end defined by the 3' end of the target sequence and the 5' end of the bait (e.g., a blunt end).

[0093] In some embodiments, the method 1000 may include a step 1100 of contacting the mixture potentially containing the target complexes 1085, 1086 with a solid support 1101 (e.g., magnetic beads), the solid support including an affinity capture domain (e.g., streptavidin) corresponding to the affinity domain (e.g., biotin) of the target isolation probe. The contacting step 1100 may include binding the affinity domain (e.g., biotin) to its corresponding affinity capture domain (e.g., streptavidin) to form an affinity capture complex 1105 including the affinity domain bound to the affinity capture domain. The contacting step 1100 may be performed to enrich primer extension products having a target sequence. For example, if the affinity domain is biotin, the target sequence may be enriched by binding the affinity domain (biotin) to a support (e.g., beads) including a streptavidin affinity capture domain. In some embodiments, streptavidin-coated magnetic beads may be added to the reaction mixture after hybridization of the primer extension products to the target-specific oligonucleotides.

[0094] In some embodiments, method 1000 may optionally include a step 1110 of concentrating affinity capture complexes 1105, e.g., by blocking the solid support with a magnet for magnetic beads and / or by washing to remove unbound affinity capture domains. The magnetic beads may be magnetically isolated and then washed, thereby enriching for complexes containing primer extension products with target sequences.

[0095] The method 1000 may include a step 1120 of contacting the blunt-ended target complex with a second adaptor, e.g., blunt-ended adaptor 1121. The blunt-ended adaptor 1121 may have a top strand 1121a and a bottom strand 1121b. The top strand 1121a may include, in a 5' to 3' direction, a 5' phosphate, a sequence complementary to the adaptor bottom strand, and a nuclease-resistant 3' end. The blunt-ended adaptor nuclease-resistant 3' end may include one or more phosphorothioate internucleotide linkages and / or a 3' C3 spacer (Integrated DNA Technologies, Inc.). The top strand 1121a may optionally include a sample tag (optionally the same or different from the sample tag of the adaptor 1033). The blunt-ended adaptor bottom strand 1121b is shorter than the top strand and may include deoxyuridines without a 5' phosphate, without a sample tag, without a UMI, and without a linear amplification annealing site. The blunt-ended adaptor bottom strand 1121b may comprise a sequence complementary to at least a portion of the 5' end of the top strand 1121a. The contacting step 1120 may further comprise contacting the blunt-ended target complex and the blunt-ended adaptor 1121 with a ligase to form a target complex adaptor ligation product. The ligation product may be washed, for example, to remove unligated adaptors from the target complex adaptor ligation product.

[0096] Method 1000 may include a step 1130 of contacting the target complex-adapter ligation product with an amplification primer that includes, in a 5' to 3' direction, a sequence complementary to the blunt-end adapter and a sample tag (optionally the same or different from the sample tag of adapter 1033) denoted as index 1 (i7) sequence. The contacting step 1130 may further include amplifying (e.g., by PCR, LAMP) to form a population of polynucleotides enriched with the target sequence (e.g., compared to the initial population of fragmented polynucleotides), which may be referred to as a final amplified library. The final amplified library may be sequenced, for example, using available next generation sequencing protocols (e.g., Illumina).

[0097] [Example 2] In some embodiments, the enrichment method may be performed according to the examples shown in Figures 2A and 2B. As shown, the method 2000 may include a step 2030 (e.g., by bead ligation tagmentation or in-solution tagmentation) of attaching a polynucleotide (e.g., an unfragmented polynucleotide) to a first adaptor (e.g., a double-stranded adaptor, a single-stranded hairpin adaptor). In some embodiments, the first adaptor, adaptor 2033, may include a top strand 2034 and a bottom strand 2035. In some embodiments, the double-stranded adaptor may include multiple deoxyuridines on the top strand, the bottom strand, or both the top and bottom strands. The top strand 2034 of adaptor 2033 may include, in the 5' to 3' direction, a sequence complementary to a linear amplification annealing site ("LAAS" in Figure 2B), optionally a sample tag (e.g., i5), a unique molecular identifier (UMI), and a sequence complementary to the bottom strand 2034 (e.g., including a transposase binding sequence). The bottom strand 2035 of the adaptor 2033 may include, in a 5' to 3' direction, a 5'-OH (e.g., a 5' terminus lacking a phosphate), a sequence complementary to the top strand, and a modified nucleotide that is not extendable (e.g., an inverted dT). In some embodiments, the contacting step 2030 may include ligating the 3' end of the adaptor top strand to the 5' end of the fragmented polynucleotide without ligating the 5' end of the adaptor bottom strand to the 3' end of the fragmented polynucleotide to form a double-stranded ligation product including a plus strand 2031 that includes a single-stranded break or nick and a minus strand 2032 that includes a single-stranded break or nick.

[0098] According to some embodiments, the method 2000 may include a step 2040 of contacting the double-stranded ligation product comprising nicks 2046 and 2047 with a nick translation or strand displacement polymerase to form a nick-translated double-stranded polynucleotide comprising plus strand 2041 and minus strand 2042. Examples of nick translation polymerases include E. coli DNA polymerase I, pol Ik, Taq DNA polymerase, OneTaq® DNA polymerase (New England Biolabs, Inc.), Bst DNA polymerase (e.g., large fragment), Bsu DNA polymerase, phi29 DNA polymerase, Therminator™ DNA polymerase, Klenow, Vent® DNA polymerase, and Deep Vent® DNA polymerase.

[0099] Method 2000 may, in some embodiments, include contacting 2050 a nick-translated duplex polynucleotide (e.g., including one or more modified nucleotides) with a single nucleotide excision reagent to form a polynucleotide duplex including a plus strand 2051 and a minus strand 2052, each including a gap (e.g., previously occupied by a modified nucleotide) that defines an oligonucleotide of varying (e.g., generally short) length. For example, the plus strand 2051 of the formed polynucleotide duplex may include, in the 5' to 3' direction, a portion having a gap (e.g., a single nucleotide previously occupied by a U), the plus strand of the double-stranded fragmented polynucleotide, and a portion containing a sequence complementary to the first adaptor, a UMI, a sample tag, and a linear amplification annealing site.

[0100] An example of a single nucleotide excision reagent may include an enzyme preparation (e.g., USER® enzyme M5505 or M5508 (New England Biolabs, Inc.)) that achieves uracil removal and phosphodiester bond cleavage, or may include a combination of enzymes (e.g., uracil DNA glycosylase in combination with endonuclease VIII). It is recognized that some polynucleotide sequences may contain consecutive sequences of deoxyuridines. Treatment with the single nucleotide excision reagent may result in corresponding consecutive single nucleotide gaps that are gaps of two or more nucleotides according to the number of consecutive deoxyuridines originally present. The method may optionally include a step of removing the oligonucleotide formed by treatment with the single nucleotide excision reagent.

[0101] Method 2000 may include a step 2060 of contacting each strand (2051, 2052) of the tagging fragment (a polynucleotide duplex comprising a double-stranded fragmented polynucleotide and a 3' overhang (e.g., a 3' overhang comprising, in a 5' to 3' direction, a sequence complementary to a first adaptor, a sample tag, a UMI, and a linear amplification annealing site)) with a linear amplification primer 2061, the linear amplification primer having a sequence complementary to the linear amplification annealing site to form annealed complexes 2065 and 2066. In some embodiments, the method may include a step 2070 of contacting annealed complexes 2065 and 2066 with a polymerase to form a primer extension product 2071 complementary to the plus strand 2051 and a primer extension product 2072 complementary to the minus strand 2052. Such annealing 2060 and primer extension 2070 may be repeated as desired (e.g., 2-200 cycles total) to amplify each strand (2051, 2052) of the polynucleotide duplex including the double-stranded fragmented polynucleotide and the 3' overhang, in which case the primer extension products may collectively be referred to as amplification products. The amplification here will be linear since each strand of the polynucleotide duplex has only one linear annealing site. The resulting primer extension products constitute a linear amplified population of polynucleotides corresponding to the initial fragmentation products. The primer extension products may include, in the 5' to 3' direction, a copy of the primer, the sample tag, the UMI, a sequence complementary to a portion of the adapter, and a copy of the plus strand of the double-stranded fragmented polynucleotide or a copy of the minus strand of the double-stranded fragmented polynucleotide. Method 2000 may optionally include a step 2075 of pooling the linear amplification post-primer extension products (e.g., primer extension products resulting from a workflow without a prior pooling step).

[0102] In some embodiments, the primer extension products may be examined for the presence of a polynucleotide having a sequence of interest (a "target sequence"). For example, the method may include a step 2080 of contacting the primer extension products with a target isolation probe (2081, 2082) to form a mixture comprising the primer extension products and the target isolation probe. The mixture may further include a target complex 2085, 2086 comprising the primer extension products having the target sequence hybridized (e.g., specifically hybridized) to a target-specific oligonucleotide of the target isolation probe, if the target sequence is present. The target isolation probe (bait) may comprise an affinity domain (e.g., biotin) and a polynucleotide having a polynucleotide sequence complementary to the plus strand of a sequence of interest (e.g., a sequence of interest somewhere within the fragmented polynucleotide, e.g., a sequence of interest at or near the 5' or 3' end of the plus strand of the fragmented polynucleotide) or a polynucleotide sequence complementary to the minus strand of a sequence of interest (e.g., a sequence of interest somewhere within the fragmented polynucleotide, e.g., a sequence of interest at or near the 5' or 3' end of the minus strand of the fragmented polynucleotide). The conditions of the contacting step 2080 may be adjusted as desired, e.g., to increase or decrease the stringency of bait to target sequence hybridization.

[0103] Optionally, method 2000 may include a step 2090 of contacting the mixture potentially including the target complex with a 3' single-stranded exonuclease to remove any 3' overhangs and form an end defined by the 3' end of the target sequence and the 5' end of the bait (e.g., a blunt end).

[0104] In some embodiments, the method 2000 may include a step 2100 of contacting the mixture potentially containing the target complexes 2085, 2086 with a solid support 2101 (e.g., magnetic beads), the solid support including an affinity capture domain (e.g., streptavidin) that corresponds to the affinity domain (e.g., biotin) of the target isolation probe. The contacting step 2100 may include binding the affinity domain (e.g., biotin) to its corresponding affinity capture domain (e.g., streptavidin) to form an affinity capture complex 2105 including the affinity domain bound to the affinity capture domain. The contacting step 2100 may be performed to enrich primer extension products having a target sequence. For example, if the affinity domain is biotin, the target sequence may be enriched by binding the affinity domain (biotin) to a support (e.g., beads) that includes a streptavidin affinity capture domain. In some embodiments, streptavidin-coated magnetic beads may be added to the reaction mixture after hybridization of the primer extension products to the target-specific oligonucleotides.

[0105] In some embodiments, method 2000 may optionally include a step 2110 of concentrating affinity capture complexes 2105, e.g., by blocking the solid support with a magnet for magnetic beads and / or by washing to remove unbound affinity capture domains. The magnetic beads may be magnetically isolated and then washed, thereby enriching for complexes containing primer extension products with target sequences.

[0106] The method 2000 may include a step 2120 of contacting the blunt-ended target complex with a second adaptor, e.g., blunt-ended adaptor 2121. The blunt-ended adaptor 2121 may have a top strand 2121a and a bottom strand 2121b. The top strand 2121a may include, in a 5' to 3' direction, a 5' phosphate, a sequence that is complementary to the adaptor bottom strand, and a nuclease resistant 3' end. The nuclease resistant 3' end of the blunt-ended adaptor may include one or more phosphorothioate internucleotide linkages and / or a 3' C3 spacer (Integrated DNA Technologies, Inc.). The top strand 2121a may optionally include a sample tag (optionally the same or different from the sample tag of the adaptor 2033). The bottom strand 2121b of the blunt adapter may be shorter than the top strand and may include deoxyuridines with no 5' phosphate, no sample tag, no UMI, and no linear amplification annealing site. The bottom strand 2121b of the blunt adapter may include a sequence complementary to at least a portion of the 5' end of the top strand 2121a. The contacting step 2120 may further include contacting the blunt target complex and the blunt adapter 2121 with a ligase to form a target complex adapter ligation product. The ligation product may be washed, for example, to remove unligated adapters from the target complex adapter ligation product.

[0107] Method 2000 may include a step 2130 of contacting the target complex-adapter ligation product with an amplification primer that includes, in a 5' to 3' direction, a sequence complementary to the blunt-end adapter and a sample tag (optionally the same or different from the sample tag of adapter 2033) denoted as index 1 (i7) sequence. The contacting step 2130 may further include amplifying (e.g., by PCR, LAMP) to form a population of polynucleotides enriched with the target sequence (e.g., compared to the initial population of fragmented polynucleotides), which may be referred to as a final amplified library. The final amplified library may be sequenced, for example, using available next generation sequencing protocols (e.g., Illumina).

[0108] Example 3: Enriched libraries of target sequences from human DNA using linear amplification Human gDNA was added to the NEBNext Ultra II End Repair / dA tailing enzyme mix and buffer according to the manufacturer's instructions (NEB). The mix was cooled to 4°C and double-stranded adapter template (first adapter) was added, with the top strand being 5'A. * A * T * GAU ACG GCG ACC ACC GAG AUC UAC ACT ATA GCC TNN NNN NNN NNN N AC ACU CTT TCC CUA CAC GAC GCU CTT CCG AUC * T (SEQ ID NO: 1), where the asterisks represent phosphorothioate linkages and the bold underlined letters represent example sample tags.

[0109] A 12N (random sequence of 12 nucleotides) UMI, a sample tag (8 bold underlined letters) and several deoxyuridine residues were present in the adapter. NEBNext Ultra II Ligation Master Mix was added to the reaction mixture and incubated at 20°C for 15 min, followed by 65°C for 15 min. Nick translation polymerase and dNTPs were added and incubated at 65°C for an additional 45 min, followed by 80°C for 20 min. After cooling to room temperature, USER enzyme was added, followed by incubation at 37°C for 15 min. DNA fragments were purified using NEB sample purification beads and eluted in nuclease-free water.

[0110] The aqueous DNA fragments were added to NEBNext Hot Start HiFi 2X Master Mix and a linear amplification primer with the sequence 5'AATGATACGGCGACCACC3' (SEQ ID NO:2), where the 3'OH is replaced by a 3'C3 spacer (Integrated DNA Technologies, Inc.). The reaction was incubated at 98°C for 30 seconds, then underwent 20 cycles of 98°C for 10 seconds, 55°C for 45 seconds, and 72°C for 20 seconds, followed by a final incubation at 72°C for 5 minutes (step 3 in Figures 1A-1B).

[0111] The reaction was transferred to a hybridization mix containing a target-specific oligonucleotide (bait) and a target isolation probe, each of which contains an affinity domain (i.e., biotin), and incubated at 95 °C for 10 min and then at 58 °C for 16 h (step 4 in Figure 1A-1B). The target-specific oligonucleotide was designed to bind to the 3' end of the target region. After hybridization, the target isolation probe / target DNA complex was bound to NEB hydrophilic streptavidin magnetic beads at 48 °C for 10 min and then washed twice in wash buffer at 62 °C for 5 min (step 5 in Figure 1A-1B).

[0112] The beads were resuspended in 3'-5' single stranded exonuclease buffer with enzyme and incubated at 25°C for 10 minutes and 37°C for 5 minutes (step 5 in Figure 1A-1B). The beads were then washed and resuspended in 1x NEB Quick ligation buffer. NEB Quick ligase and a 3' adapter for ligation to the 3' end of the target sequence were added to the buffer and incubated at 20°C for 15 minutes (step 6 in Figure 1A-1B). This 3' adapter had a protective modification on the 3' end of the top strand and a bottom complementary strand with a modified base. The 3' adapter has the top strand sequence 5'5Phos / AGATC GGAAG AGCAC ACGTC TGAAC TCC * A * G * T * C * A * C / 3SpC3 3' (SEQ ID NO: 3) and bottom strand sequence 5' ACGNG TGCNC TNCCG ANC * T 3' and N is ideoxy U (SEQ ID NO: 4). The beads were then washed, resuspended in 100 μl of 1×NEBuffer1 containing endonuclease VIII and UDG, and incubated at 37° C. for 15 minutes (1090 in FIGS. 1A-1B).

[0113] The magnetic beads were then washed and resuspended in 1× NEBNext Q5 Hot Start HiFi PCR Master Mix containing NEBNext Direct® PCR primers (New England Biolabs, Ipswich, MA) for PCR amplification.

[0114] Example 4: Analysis of enriched libraries Enriched libraries were prepared according to Example 1, starting with 15, 30, and 100 ng of fragmented human DNA input and sequenced with 75 bp paired-end reads. Raw sequencing files were demultiplexed using the Picard tool ExtractIlluminaBarcodes to assign reads to sample indexes followed by Picard's IlluminaBasecallsToSam, which creates unmapped BAM files containing the reads for each sample ("Picard Toolkit" 2019, Broad Institute, GitHub Repository). Unaligned BAMs were aligned to the human reference genome (build GRCh38 / hg38) using bwa mem (Li H. 2013, Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM.arXiv:1303.3997v2[q-bio.GN]). Sequencing duplicates were then identified with Picard's MarkDuplicates, which uses both position and unique molecular identifiers (UMIs) to identify duplicates, and the duplicate-labeled BAMs are then used in variant calling.Germline calling is performed using GATK's HaplotypeCaller and somatic calling is performed using both GATK's MuTect2 and AstraZeneca's VarDict (Poplin R et al., 2017, Scaling accurate genetic variant discovery to tens of thousands of samples bioRxiv, 201178. DOI:10.1101 / 201178; "GATK Mutect2" 2019, Broad Institute, GitHub Repository; David Benjamin et al., 2019, Calling Somatic SNVs and Indels with Mutect2. bioRxiv 861054; doi:https: / / doi.org / 10.1101 / 861054; Zhongwu Lai et al., VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research, Nucleic Acids Research, Vol. 44, Issue 1). 11, 20 June 2016, Page e108, https: / / doi.org / 10.1093 / nar / gkw227). QC metrics are calculated from duplicate-labeled BAMs using a combination of Picard and custom tools. Sequencing depth of coverage across number of reads for these target-enriched libraries is shown in Figure 3A (75 kb target), Figure 3B (227 kb target), and Figure 3c (1100 kb target).

Claims

1. (a) fusing a DNA adaptor to a 5' end of a double-stranded polynucleotide fragment of a population of fragments, wherein the DNA adaptor comprises a bottom strand and a top strand; (i) the adapter top strand is, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site; Optionally, a sample tag; Optionally, a unique molecular identifier (UMI), and A sequence complementary to the adapter bottom strand Including, (ii) the adapter bottom strand comprises a non-extendable 3' end, and one or more of the polynucleotide fragments comprises a target sequence, and a fusion product is formed that comprises the 3' end of the adapter top strand fused to the 5' end of the polynucleotide fragment, and a nick comprising the unfused 5' end of the adapter bottom strand and the 3' end of the polynucleotide fragment; (b) contacting the fusion product with a nick translation polymerase for templated addition of nucleotides to the 3' end of the polynucleotide fragment to form a nick translation product that includes a 3' extension of the polynucleotide fragment, the extension being complementary to the adapter top strand; and (c) in the 5' to 3' direction: a sequence complementary to the linear amplification annealing site; Optionally, a sequence of the sample tag; Optionally, a sequence of a UMI: A partial sequence of the top strand of the adaptor, the sequence of one of the polynucleotide fragments, and Optionally, a sequence complementary to a portion of the adapter bottom strand contacting the nick translation product with a primer having a sequence complementary to the linear amplification annealing site and a polymerase to generate a linear amplified nick translation product comprising: The method includes:

2. 2. The method of claim 1, further comprising contacting the nick translation product with a glycosylase and an endonuclease prior to or concurrently with (c), wherein the adapter top strand template further comprises one or more modified nucleotides, the one or more modified nucleotides optionally being deoxyuridine, the glycosylase optionally being uracil-DNA glycosylase (UDG), and the endonuclease optionally being endonuclease VIII.

3. 3. The method of claim 1 or 2, further comprising, after (1b), removing the adapter bottom strand of the adapter template by cleaving the adapter bottom strand with a reagent comprising a glycosylase and an endonuclease, wherein the adapter bottom strand comprises one or more modified nucleotides, optionally the one or more modified nucleotides are deoxyuridine, the glycosylase is optionally uracil-DNA glycosylase (UDG), and the endonuclease is optionally endonuclease VIII.

4. The method of any one of claims 1 to 3, wherein the non-extendable 3' end of the adapter bottom strand comprises an inverted deoxythymidine.

5. 3. The method of claim 1 or 2, further comprising the step of: (d) contacting the linearly amplified nick translation product with a target-specific oligonucleotide bound to an affinity domain to form a target complex comprising a target sequence, the target-specific oligonucleotide, and a polynucleotide fragment having an affinity domain, wherein the target sequence is hybridized to the target-specific oligonucleotide.

6. 6. The method of claim 5, further comprising the step of: (e) binding the target complex to a solid support that contains an affinity capture domain that corresponds to the affinity domain.

7. The method of claim 6 , wherein the affinity domain is biotin and the affinity capture domain comprises streptavidin.

8. (f) removing any overhanging polynucleotide sequence at the 3' end of the complementary strand hybridized to the oligonucleotide using a 3'-5' single-stranded exonuclease or multiple 3'-5' exonucleases to form a blunt-ended duplex at the 3' end of the complement and the 5' end of the oligonucleotide; (g) fusing the blunt-ended duplex to a second adaptor comprising a second sample tag to form a second fusion product comprising, in a 5' to 3' direction, the first adaptor top strand, the oligonucleotide fragment, and the second adaptor; and (h) amplifying the second fusion product for sequencing of one or both of the strands; The method of claim 5 or claim 6, further comprising:

9. The method further comprises the step of pooling the nick translation product with additional nick translation products, wherein the additional nick translation products are in the 5' to 3' direction: a sequence complementary to the linear amplification annealing site; a sequence of a further sample tag; Optionally, a sequence of a UMI: a further sequence of part of the top strand of the adaptor; and the sequences of the polynucleotide fragments of the further population of fragments; The method of claim 1 , each comprising:

10. repeating step (a) with a second DNA adaptor and double-stranded polynucleotide fragments of a second fragment population; wherein the first adaptor top strand comprises the sample tag and the second adaptor comprises a bottom strand and a top strand; (i) a second adaptor top strand, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site; a sample tag different from the first adapter sample tag; Optionally, a unique molecular identifier (UMI); and A sequence that is complementary to the bottom strand of the second adapter, Including, (ii) the second adaptor bottom strand comprises a non-extendable 3' end, one or more of the polynucleotide fragments of the second population comprise the target sequence, and a further fusion product is formed that comprises a 3' end of the second adaptor top strand fused to a 5' end of the polynucleotide fragment of the second population and a nick comprising the unfused 5' end of the second adaptor bottom strand and the 3' end of the polynucleotide fragment of the second population; and Pooling the fusion product and the additional fusion product to form a pooled fusion product. The method of claim 1 further comprising:

11. contacting the fusion products with a nick translation polymerase further comprises contacting the pooled fusion products with a nick translation polymerase for templated addition of nucleotides to the 3' ends of the polynucleotide fragments to form pooled nick translation products comprising 3' extensions of the polynucleotide fragments, the extensions being complementary to the top strands of the respective adapters; Contacting the nick translation products with a primer having a sequence complementary to the linear amplification annealing site results in linearly amplified pooled nick translation products, in a 5' to 3' direction, a sequence complementary to the linear amplification annealing site; one sequence for each sample tag; Optionally, a sequence of a UMI: A partial sequence of each of the top strands of the adapters, the sequence of one of the polynucleotide fragments, and Optionally, a sequence complementary to a portion of each adapter bottom strand contacting the pooled nick translation products with a primer and a polymerase to generate a linear amplified pooled nick translation product comprising: The method of claim 10.

12. The method of any one of claims 1 to 11, wherein the fusing in step (a) comprises ligating or tagmenting a DNA adapter to the 5' end of the double-stranded polynucleotide fragment.

13. The method of any one of claims 1 to 12, wherein the top strand is longer than the adapter bottom strand.

14. 14. The method of claim 13, wherein the adapter top strand comprises at least one of the optional sample tags and an optional UMI.

15. 15. The method of claim 13 or 14, wherein the adapter bottom strand does not contain a 5' phosphate, a sample tag, a UMI, or a linear amplification annealing site.

16. 1. A method for enriching a target sequence, comprising: (a) contacting the population of fragmented polynucleotides with a ligase and a first adaptor, where the first adaptor comprises a bottom strand and a longer top strand; (i) the bottom strand comprises, in a 5' to 3' direction, a 5'-OH, at least 10 nucleotides complementary to the top strand, and, optionally, an inverted dT; (ii) the top strand comprises, in a 5' to 3' direction, a sequence complementary to a linear amplification annealing site, optionally a sample tag, a unique molecular identifier (UMI), and a sequence complementary to the bottom strand; the top and bottom strands optionally, independently, comprise one or more deoxyuridines, and the contacting of (a) produces a double-stranded fusion product comprising a 3' end of the adaptor top strand fused to the 5' end of the polynucleotide fragment and a nick comprising the unfused 5' end of the adaptor bottom strand and the 3' end of the polynucleotide fragment; (b) contacting the double-stranded fusion product with a nick translation polymerase and nucleotide triphosphates to form a nick-translated double-stranded polynucleotide, where the duplex comprises, in a 5' to 3' direction, a top strand that comprises the adapter top strand, the plus strand of one of the polynucleotide fragments, and a sequence complementary to the adapter top strand, and a bottom strand that comprises, in a 5' to 3' direction, the adapter top strand, the minus strand of one of the polynucleotide fragments, and a sequence complementary to the adapter top strand; (c) optionally contacting the nick-translated duplex polynucleotide with a single nucleotide excision reagent to form a polynucleotide duplex comprising a plus strand and a minus strand, each comprising a single nucleotide gap in an adaptor top strand of the nick-translated duplex polynucleotide, the gap defining an oligonucleotide of varying length; (d) contacting the nick-translated duplex polynucleotide with a linear amplification primer having a sequence that is complementary to the linear amplification annealing site to form an annealed duplex; (e) in the 5' to 3' direction, a sequence corresponding to at least a portion of the adaptor top strand, and A sequence corresponding to at least a portion of the positive strand of one of the polynucleotide fragments. contacting the annealed duplex with a polymerase to form a primer extension product comprising: (f) repeating the contacting of (d) and the contacting of (e) at least once; (g) contacting the primer extension products with a target isolation probe comprising an affinity domain and a target-specific oligonucleotide to form a target probe mixture comprising the primer extension products, the target isolation probe, and, if one or more target sequences are present in the population of fragmented polynucleotides, a target complex comprising the primer extension products having target sequences hybridized to the target-specific oligonucleotides of the target isolation probe; (h) contacting the target probe mixture with a solid support containing an affinity capture domain that corresponds to the affinity domain of the target isolation probe to form an affinity complex containing the affinity capture domain bound to the affinity capture domain; and (i) separating the affinity complex from unbound primer extension products and unbound target isolation probes to form an enriched target sequence composition. The method includes:

17. 17. The method of claim 16, wherein the adapter top strand and the adapter bottom strand each comprise a plurality of deoxyuridines, and the single nucleotide excision reagent removes the deoxyuridines.

18. 17. The method of claim 16, further comprising A-tailing the fragmented polynucleotides prior to the contacting in (a), and wherein the top strand of the adapter further comprises a 3' T or dU.

19. 17. The method of claim 16, wherein the first adaptor comprises an optional sample tag.

20. 20. The method of claim 19, further comprising repeating steps (a) through (i), wherein the population of fragmented polynucleotides of each repeated step (a) is different from all prior executions of step (a), and wherein the optional sample tag of each repeated step (a) is different from all prior executions of step (a).

21. 21. The method of claim 20, further comprising pooling affinity complexes resulting from a first performance of steps (a) through (i) together with affinity complexes resulting from each repetition of steps (a) through (i).

22. The method according to any one of claims 16 to 21, wherein the fusion in step (a) comprises ligation or tagmentation.

23. (a) fusing a DNA adaptor to a 5' end of a double-stranded polynucleotide fragment of a population of fragments, wherein the DNA adaptor comprises a top strand and, optionally, a bottom strand, the adaptor top strand having, from 5' to 3', a sequence of at least 8 nucleotides that is complementary to a linear amplification annealing site; Optionally, a sample tag; Optionally, a unique molecular identifier (UMI); and A sequence that is optionally complementary to the adapter bottom strand; Including, one or more of the polynucleotide fragments comprises the target sequence, and a fusion product is formed that comprises a nick comprising the 3' end of the adaptor top strand fused to the 5' end of the polynucleotide fragment and the 3' end of the polynucleotide fragment and, optionally, the unfused 5' end of the adaptor bottom strand; (b) contacting the fusion product with a nick translation polymerase for templated addition of nucleotides to the 3' end of the polynucleotide fragment to form a nick translation product that includes a 3' extension of the polynucleotide fragment, the extension being complementary to the adapter top strand; and (c) contacting the nick translation product with a primer having a sequence complementary to the linear amplification annealing site and a polymerase to generate a linearly amplified nick translation product, where the linearly amplified nick translation product has, in the 5' to 3' direction: a sequence complementary to the linear amplification annealing site; Optionally, a sequence of the sample tag; Optionally, a sequence of a UMI: A partial sequence of the top strand of the adaptor, the sequence of one of the polynucleotide fragments, and Optionally, a sequence complementary to a portion of the adapter bottom strand Includes The method includes:

24. 24. The method of claim 23, wherein the adaptor top strand lacks a UMI and the DNA adaptor lacks a bottom strand.

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