Methods of polymerase free DNA end repair

Abstract

Provided herein are methods of preparing blunt-end DNA fragments for sequencing using single-strand-specific nucleases.

Description

[0001] METHODS OF POLYMERASE FREE DNA END REPAIR

[0002] RELATED APPLICATIONS

[0003] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 666,565, filed July 1, 2024, entitled “Methods of Polymerase Free DNA End Repair”, the entire contents of which is incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The content of the electronic sequence listing (W109470014WO00-SEQ-EMB.xml; Size: 4,194 bytes; and Date of Creation: June 25, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Preparation of DNA fragments for sequencing may include end-repair of double-stranded DNA fragments and subsequent A-tailing. Traditional methods of end-repair rely on DNA polymerases to fill in single-stranded overhangs prior to A-tailing.

[0008] SUMMARY

[0009] Provided herein are methods for producing a plurality of blunt-end DNA fragments.

[0010] Traditional methods of blunting DNA fragments rely on DNA polymerases, which can introduce artifacts such as loss of epigenetic markers, addition or substitution of incorrect nucleotides (e.g., errors), or formation of hairpins by single- stranded overhangs. The methods provided herein use single-strand-specific nucleases, which cleave single- stranded overhangs rather than adding nucleotides to fill them in, thereby retaining epigenetic markers and avoiding the introduction of errors while still resulting in high yield of DNA fragments for sequencing (e.g. blunt-end DNA fragments).

[0011] Accordingly, in some aspects, the disclosure provides a method of producing a plurality of blunt-end DNA fragments, the method comprising contacting a plurality of sticky-end DNA fragments with (a) a single-strand-specific nuclease; and (b) a DNA polymerase. In some embodiments, the sticky-end DNA fragments are derived from cell-free DNA. In some embodiments, the DNA polymerase lacks 5’ to 3’ exonuclease activity. In some embodiments, the DNA polymerase is a T4 polymerase. In some embodiments, the DNA polymerase is a Klenow fragment. In some embodiments, the DNA polymerase is KlenTaq. In some embodiments, the DNA polymerase is E. coli DNA polymerase I or B st pol large fragment.

[0012] In some embodiments, the single-strand-specific nuclease is nuclease Pl. In some embodiments, the single-strand-specific nuclease is nuclease SI. In some embodiments, the single-strand-specific nuclease is ExoT. In some embodiments, the single-strand-specific nuclease is mung bean nuclease (MBN).

[0013] In some embodiments, a method of producing a plurality of blunt-end DNA fragments provided herein further comprises isolating the plurality of blunt-end DNA fragments from the single-strand-specific nuclease. In some embodiments, the isolating comprises contacting the plurality of blunt-end DNA fragments with a DNA-binding reagent. In some embodiments, the DNA-binding reagent comprises paramagnetic beads.

[0014] In some embodiments, the method further comprises inactivating the single- strandspecific nuclease. In some embodiments, the inactivating comprises heating the single- strandspecific nuclease to a temperature of 75 °C to 90 °C. In some embodiments, the inactivating comprises contacting the single- strand- specific nuclease with a chelator. In some embodiments, the chelator is ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA).

[0015] In some embodiments, a method of producing a plurality of blunt-end DNA fragments provided herein further comprises A-tailing blunt-end DNA fragments of the plurality of blunt- end DNA fragments. In some embodiments, A-tailing the blunt-end DNA fragments comprises contacting the blunt-end DNA fragments with a poly-A terminal transferase. In some embodiments, the poly-A terminal transferase is a Klenow fragment polymerase.

[0016] In some embodiments, the method further comprises sequencing the plurality of blunt- end DNA fragments. In some embodiments, the method comprises contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase. In some embodiments, the method does not comprise contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase.

[0017] In some aspects, the disclosure provides a method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising: contacting a plurality of sticky-end DNA fragments with a single-strand-specific nuclease, thereby producing a plurality of single- strand- specific nuclease-treated DNA fragments; isolating the plurality of single- strand- specific nuclease-treated DNA fragments from the single- strand- specific nuclease or inactivating the single-strand-specific nuclease; and contacting the plurality of single- strand- specific nuclease- treated DNA fragments with a DNA polymerase and / or a poly-A terminal transferase, thereby producing a plurality of blunt-end DNA fragments.

[0018] In some aspects, the disclosure provides a method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising, in sequential order: (i) contacting a plurality of sticky-end DNA fragments with single- strand- specific nuclease Pl, thereby producing a plurality of single-strand-specific nuclease-treated DNA fragments; (ii) isolating the plurality of single-strand-specific nuclease-treated DNA fragments from the single-strand- specific nuclease Pl or inactivating the single-strand-specific nuclease Pl; (iii) contacting the plurality of single-strand-specific nuclease-treated DNA fragments with T4 polymerase, thereby producing a plurality of blunt-end DNA fragments; and (iv) contacting the plurality of blunt-end DNA fragments with a poly-A terminal transferase.

[0019] In some embodiments, isolating the plurality of single-strand-specific nuclease-treated DNA fragments from the single- strand- specific nuclease Pl comprises contacting the plurality of single-strand-specific nuclease-treated DNA fragments with a DNA-binding reagent comprising paramagnetic beads.

[0020] In some embodiments, the plurality of sticky-end DNA fragments is derived from cell- free DNA. In some embodiments, the method further comprises sequencing blunt-end DNA fragments of the plurality of blunt-end DNA fragments. In some embodiments, sequencing comprises shotgun sequencing. In some embodiments, sequencing comprises long-read sequencing. In some embodiments, the sticky-end DNA fragments comprise methylated nucleotides.

[0021] In some aspects, the disclosure provides a method of A-tailing a DNA polynucleotide, the method comprising contacting the DNA polynucleotide with a DNA polymerase that lacks 5’ to 3’ exonuclease activity. In some embodiments, the DNA polynucleotide is double-stranded DNA. In some embodiments, the DNA polymerase is a T4 polymerase. In some embodiments, the DNA polymerase is a Klenow fragment. In some embodiments, the DNA polymerase is KlenTaq. In some embodiments, the DNA polymerase is E. coli DNA polymerase I or Bst pol large fragment. In some embodiments, the DNA polynucleotide comprises methylated nucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a schematic of T4 DNA polymerase end repair and A-tailing (ERAT) and single-strand-specific nuclease Pl ERAT (pERAT) methods.

[0023] FIGs. 2A-2B relate to post PCR final cell free DNA (cfDNA) libraries made with stubby or full length adapters using the pERAT method. FIG. 2A is the final yield and library size of 1 nanogram (ng) or 8 ng of cell-free DNA (cfDNA) input using pERAT methods provided herein with either 50 pM stubby adapter or 15 pM full length adapter. FIG. 2B shows library traces of 1 ng and 8 ng cfDNA libraries.

[0024] FIGs. 3A-3B relate to post-PCR final Covaris sheared Coriell DNA (NA12878) libraries made with stubby or full length adapters using pERAT methods provided herein. FIG. 3A shows the final yield and library size of 1 ng, 10 ng, or 100 ng of Covaris sheared Coriell DNA (NA12878) input using pERAT methods provided herein with either 50 nM stubby adapters or 5 pM - 15 pM full length adapter. FIG. 3B shows library traces of 1 ng, 10 ng, and 100 ng of Covaris sheared Coriell DNA (NA12878) libraries.

[0025] FIGs. 4A-4B show results from processing 10 ng of Covaris sheared Coriell DNA (NA12878) using pERAT methods provided herein, with and without end polishing with a DNA polymerase. FIG. 4A shows that addition of single-strand-specific nuclease Pl treated ends (pERAT) directly into end polishing results in substantially higher library yield than no end polishing treatment. FIG. 4B shows that library traces results in a substantial yield increase when single-strand-specific nuclease Pl treated DNA (pERAT) is subjected to an end polishing treatment.

[0026] FIG. 5 shows that ERAT polymerizes further into the 3’ end of DNA inserts and results in greater erasure of methylation (and thus likely other epigenetic markers) than pERAT methods provided herein.

[0027] FIGs. 6A-6D show DNA sequencing libraries prepared via pERAT methods provided herein or ERAT with sheared formalin-fixed paraffin-embedded (FFPE)-derived DNA of various qualities and Coriell DNA (NA12878) with stubby adapters. dCq values represent the FFPE quality, whereas a more negative dCq value represents worse quality of FFPE-derived DNA. FIG. 6A demonstrates that post library prep yields have similar performance between pERAT and ERAT. FIG. 6B demonstrates that post library prep yields show that pERAT has slightly shorter library sizes than ERAT. FIG. 6C shows chimera percentage of low quality FFPE- derived DNA is significantly lower with pERAT library prep than ERAT. FIG. 6D demonstrates that hairpin artifacts of all FFPE-derived DNA qualities are significantly lower with pERAT library prep than ERAT. Lower quality FFPE shows a more drastic difference between ERAT and pERAT, most likely due to DNA damage and single stranded DNA molecules within the FFPE DNA pool.

[0028] FIG. 7 shows reduction in C to A substitution errors using the pERAT method compared to ERAT method.

[0029] DETAILED DESCRIPTION

[0030] Provided herein are methods for producing a plurality of blunt-end DNA fragments. “Blunt-end” DNA fragments as used in the disclosure refers to fragments of double-stranded DNA that lack overhangs on either strand. Producing blunt-end DNA fragments is often a necessary step in preparing libraries for sequencing. Traditional methods of blunting DNA fragments use DNA polymerases (e.g. T4 DNA polymerase), which results in a loss of epigenetic markers at the ends of DNA fragments and can introduce errors (e.g. incorrect nucleotides or hairpin artifacts). Blunting single-stranded DNA overhangs with a polymerase biases downstream sequence analysis of the DNA by changing the epigenetic composition with of the base pairs as the polymerase adds non-epigenetically modified base pairs where epigenetically modified base pairs may have been prior to DNA fragmentation. Additionally, polymerases can introduce incorrect nucleotides (e.g., mutations, substitutions) during end repair, resulting in DNA fragments that do not reflect the original sequences. The methods provided herein use single- strand- specific nucleases (e.g., single-strand-specific nuclease Pl), which have the ability to blunt or nearly-blunt DNA fragments by cleaving single-stranded overhangs. Blunting overhangs with single- strand- specific nucleases rather than blunting them with a polymerase reduces the bias in sequencing analysis by minimizing and / or eliminating addition of non-epigenetically modified base pair where epigenetically modified base pairs may have been and can prevent the introduction of hairpin artifacts, resulting in a sequencing library with reduced errors that more accurately represents the genetic material of the samples from which the DNA was derived.

[0031] End-Repair

[0032] End-repair of DNA fragments is an important step in preparing DNA libraries for sequencing. End-repair involves modifying sticky-end (e.g., sheared) DNA fragments derived from samples (e.g., cell-free DNA or formalin-fixed paraffin-embedded tissues) to ensure they have blunt ends to prepare the fragments for subsequent steps, such as poly A-tailing (described in detail elsewhere herein) and adapter ligation.

[0033] Sticky-end DNA

[0034] A preliminary step in preparing DNA obtained from samples (e.g., cell-free DNA or formalin-fixed paraffin-embedded DNA) is shearing the DNA (e.g., using sonication, enzymatic fragmentation, or mechanical force) to obtain sticky-end DNA fragments, which are doublestranded DNA fragments of a sequenceable length (e.g., a few hundred base pairs for short-read sequencing and up to 20,000 base pairs for long-read sequencing) with single-stranded overhangs. Sticky-end DNA fragments subsequently undergo further processing to remove single-stranded overhangs to generate blunt-end DNA fragments. In some embodiments, the sticky-end DNA fragments comprise methylated nucleotides. “Methylated nucleotides” refers to nucleotides comprising a methyl group (-CH3).

[0035] In some aspects, the disclosure provides a method of producing a plurality of blunt-end DNA fragments, the method comprising contacting a plurality of sticky-end DNA fragments with a single-strand-specific nuclease and a DNA polymerase. As used herein, a “plurality” refers to at least two, and “contacting” refers to combining a plurality of DNA fragments with one or more additional reagents, e.g., combining a plurality of sticky-end DNA fragments with a single-strand specific single- strand- specific nuclease and / or a DNA polymerase.

[0036] DNA fragments (e.g., sticky-end DNA fragments) prepared according to the methods provided herein may be derived from any source of DNA. In some embodiments, the plurality of sticky-end DNA fragments is derived from cell-free DNA (cfDNA). Cell-free DNA can be obtained by non-invasive procedures, e.g., isolated from circulating peripheral blood, and can be used to screen for disease or disorders during fetal-development. In some embodiments, the plurality of sticky-end DNA fragments is derived from formalin-fixed paraffin-embedded (FFPE) samples. FFPE samples are particularly prone to DNA damage, and during traditional methods of end-repair that rely on DNA polymerases to produce blunt-end DNA fragments, DNA polymerase activity often results in the introduction of artifacts, such as hairpins or erroneous substitution of nucleotides where damage has occurred. Preparation of FFPE-derived DNA according to methods provided herein reduces the introduction of such artifacts.

[0037] Blunt-End DNA

[0038] In some aspects, the disclosure provides methods of preparing a plurality of blunt-end DNA fragments. Traditional methods of producing blunt-end DNA fragments (e.g., for sequencing) rely on DNA polymerases to fill in nucleotides complementary to single- stranded overhangs, which results in the loss of epigenetic markers and risks introducing hairpin artifacts when single- stranded overhangs fold over on themselves.

[0039] Provided herein are methods of producing a plurality of blunt-end DNA fragments, the method comprising contacting a plurality of sticky-end DNA fragments with (a) a single-strand- specific nuclease; and (b) a DNA polymerase. In some embodiments, the single-strand-specific nuclease is selected from Nuclease Pl, Nuclease SI, ExoT, and mung bean nuclease (MBN). In some embodiments, the single-strand-specific nuclease is Nuclease Pl. In some embodiments, the single- strand- specific nuclease is Nuclease SI. In some embodiments, the single- strandspecific nuclease is ExoT. In some embodiments, the single- strand- specific nuclease is MBN. In some embodiments, sticky-end fragments are treated with nuclease Pl in sodium acetate buffer at about pH 6.3. In some embodiments, the sodium acetate buffer comprises salts to facilitate nuclease Pl activity (e.g., MgCh, ZnCh, and / or NaCl).

[0040] In some embodiments, the DNA polymerase lacks 5’ to 3’ exonuclease activity. DNA polymerases that lack 5’ to 3’ exonuclease activity are not expected to digest nucleotides in the 5’ to 3’ direction. In some embodiments, the DNA polymerase is selected from a T4 polymerase, a Klenow fragment, KlenTaq, and E. coli DNA polymerase I or Bst pol large fragment. In some embodiments, the DNA polymerase is a T4 polymerase. In some embodiments, the DNA polymerase is a Klenow fragment. In some embodiments, the DNA polymerase is KlenTaq. In some embodiments, the DNA polymerase is E. coli DNA polymerase I or Bst pol large fragment. In some embodiments, a method of producing a plurality of blunt-end fragments is performed in a buffer that is suitable for DNA polymerase and single-strand-specific nuclease activity.

[0041] In some embodiments, the method comprises contacting a plurality of sticky-end DNA fragments with a single-strand-specific nuclease in a first reaction, incubating the reaction to produce a plurality of single-strand-specific nuclease treated DNA fragments, and then contacting the plurality of single- strand- specific nuclease treated DNA fragments with a DNA polymerase in a second reaction to produce a plurality of blunt-end fragments. In some embodiments, incubating the first reaction comprises incubating in conditions that are suitable for single- strand- specific nuclease activity. In some embodiments, incubating the first reaction comprises incubating for about 5-15 minutes. In some embodiments, incubating the first reaction comprises incubating for about 10 minutes. In some embodiments, incubating the first reaction comprises incubating for at least 5 minutes or at least 10 minutes. In some embodiments, incubating the first reaction comprises incubating at about 22-37 °C. In some embodiments, incubating the first reaction comprises incubating at about 30-37 °C. In some embodiments, incubating the first reaction comprises incubating at about 37 °C.

[0042] In some embodiments, the method comprises isolating and / or inactivating the single- strand- specific nuclease treated DNA fragments from the single- strand- specific nuclease prior to contacting with the DNA polymerase. In some embodiments, isolating the single- strand- specific nuclease treated DNA fragments from the single- strand- specific nuclease comprises isolating using any suitable method including a method described herein (e.g., isolating using SPRI clean up). In some embodiments, isolating the plurality of nuclease-treated DNA fragments from the Nuclease Pl comprises contacting the plurality of nuclease-treated DNA fragments with a DNA- binding reagent comprising paramagnetic beads.

[0043] In some embodiments, the method comprises inactivating the single-strand-specific nuclease treated DNA fragments from the single- strand- specific nuclease prior to contacting with the DNA polymerase. Inactivating may be performed in any suitable way including using a method described herein (e.g., heat deactivation or chemical denaturation).

[0044] In some embodiments, the method comprises incubating the second reaction. In some embodiments, incubating the second reaction comprises incubating in conditions that are suitable for DNA polymerase activity and / or enzymatic a-tailing of the single-strand-specific nuclease treated DNA fragments. In some embodiments, the method comprises incubating the second reaction for 10-60 minutes. In some embodiments, the method comprises incubating the second reaction for about 20-40 minutes. In some embodiments, the method comprises incubating the second reaction for about 10-30 minutes at about 15-30 °C. In some embodiments, the method comprises incubating the second reaction for about 20 minutes at about 20 °C. In some embodiments, the method comprises incubating the second reaction for about 10-30 minutes at about 15-30 °C and then incubating the second reaction for at least 5 minutes or about 5-15 minutes (e.g., about 10 minutes) at about 60-70 °C (e.g., at about 65 °C). In some embodiments, the method comprises incubating the second reaction for about 20 minutes at about 20 °C and then incubating the second reaction for about 10 min at about 65 °C.

[0045] Once a plurality of blunt-end DNA fragments is prepared, they may be isolated from the single-strand-specific nuclease used to produce the blunt-end DNA fragments, or the single- strand- specific nuclease may be inactivated by physical or chemical means.

[0046] In some embodiments, the disclosure provides a method comprising isolating a plurality of blunt-end DNA fragments from a single- strand- specific nuclease. “Isolating” a plurality of blunt-end DNA fragments from a single- strand- specific nuclease refers to separating the plurality of blunt-end DNA fragments from the single-strand specific nuclease. The skilled person will understand that separating is not always a perfect separation and there may be a small amount of residual single- strand- specific nuclease with the blunt-end DNA fragments after the separating step. In some embodiments, the isolating comprises contacting the plurality of blunt-end DNA fragments with a DNA-binding reagent. In some embodiments, the DNA-binding reagent comprises silica. In some embodiments, the DNA-binding reagent comprises paramagnetic beads. In some embodiments, the paramagnetic beads are carboxylated. In some embodiments, the paramagnetic beads are coated with silicane. In some embodiments, the paramagnetic beads are SPRI beads.

[0047] In some embodiments, a cleanup step is performed after contacting sticky-end DNA fragments with a single-strand-specific nuclease. In some embodiments, the cleanup step comprises a 2x SPRI cleanup which is performed to remove reaction enzymes and buffer from the reaction. In some embodiments, the cleanup step comprises adding 500 mM of DTT and briefly (e.g., for 1 minute, for 2 minutes, for 3 minutes, for 4 minutes, or for 5 minutes) incubating the fragments on ice. In some embodiments, the cleanup step comprises using SPRI beads to isolate blunt-end DNA fragments (e.g., prior to A-tailing, A-tailed blunt-end DNA fragments, and / or adapter-ligated blunt-end DNA fragments). Any suitable DNA-binding reagent may be used.

[0048] In some embodiments, the disclosure provides a method comprising inactivating the single-strand-specific nuclease. “Inactivating” refers to decreasing and inhibiting the nuclease activity of the single-strand-specific nuclease. In some embodiments, the inactivating comprises physical means. In some embodiments, the inactivating comprises heating the single-strand- specific nuclease to a temperature of 75 °C to 90 °C. In some embodiments, the inactivating comprises chemical means. In some embodiments, the inactivation comprises contacting the single-strand-specific nuclease with a chelator. In some embodiments, the chelator is tetrakis-(2- pyridylmethyl)ethylenediamine (TPEN), diethylenetriaminepentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), or ethylene glycol tetraacetic acid (EGTA). In some embodiments, the chelator is EDTA or EGTA. In some embodiments, the inactivation comprises contacting the single-strand-specific nuclease with denaturing or chaotropic agent.

[0049] Poly A-Tailing of Blunt-End DNA fragments

[0050] In some embodiments, the disclosure provides a method comprising A-tailing blunt-end DNA fragments of the plurality of blunt-end DNA fragments. A-tailing is a process wherein adenines (As) are terminally added to double-stranded DNA fragments to facilitate subsequent ligation with sequencing adapters. In some embodiments, A-tailing the blunt-end DNA fragments comprises contacting the blunt-end DNA fragments with a poly-A terminal transferase. In some embodiments, the poly-A terminal transferase is terminal deoxynucleotidyl transferase (TdT). In some embodiments, the poly-A terminal transferase is polymerase 9. In some embodiments, the poly-A terminal transferase is a Klenow fragment. In some embodiments, the poly-A terminal transferase is Taq polymerase. The poly-A terminal transferase can be any suitable poly-A DNA polymerase.

[0051] In some aspects, the disclosure provides a method of A-tailing a DNA polynucleotide, the method comprising contacting the DNA polynucleotide with a DNA polymerase that lacks 5’ to 3’ exonuclease activity. A “polynucleotide” refers to a polymer of nucleotides. In some embodiments, the polynucleotide is double-stranded DNA. In some embodiments, the DNA polymerase is selected from a T4 polymerase, a Klenow fragment, KlenTaq, and E. coli DNA polymerase I or B st pol large fragment. In some embodiments, the DNA polymerase is a T4 polymerase. In some embodiments, the DNA polymerase is a Klenow fragment. In some embodiments, the DNA polymerase is KlenTaq. In some embodiments, the DNA polymerase is E. coli DNA polymerase I or B st pol large fragment. In some embodiments, the DNA polynucleotide comprises epigenetically-modified nucleotides (e.g., methylation). In some embodiments, the DNA polynucleotide comprises methylated nucleotides. In some embodiments, A-tailing is performed in a tris acetate buffered solution at about pH 8.3. In some embodiments, the buffer comprises additional deoxynucleotides (e.g., dATP and / or dNTP). In some embodiments, the buffer comprises salts to facilitate A-tailing (e.g., MgCh). In some embodiments, A-tailed blunt-end DNA fragments may be subjected to methylation to facilitate adapter ligation. In some embodiments, a method provided herein comprises contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase. In some embodiments, a method provided herein does not comprise contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase.

[0052] Ligation of Sequencing Adapters

[0053] DNA preparation for sequencing (e.g., next-generation sequencing) often involves adding adapter polynucleotide sequences to the DNA. Certain technologies, like ILLUMINA® sequencing, use the adapter sequences to perform sequencing of the polynucleotides. Adapters are ligated to blunt-end DNA fragments, a process that involves attaching short, known sequences (the adapters) to the ends of DNA fragments. The adapters enable the fragments to bind to sequencing primers and provide a way to identify and amplify the sequences during the sequencing process.

[0054] In some embodiments, blunt-end DNA fragments of the plurality of blunt-end DNA fragments are ligated with sequencing adapters. In some embodiments, the sequencing adapters are full-length adapters. In some embodiments, the sequencing adapters are T-tailed adapters. In some embodiments, the sequencing adapters are Y-adapters. In some embodiments, the sequencing adapters are stubby adapters. In some embodiments, the sequencing adapters are ILLUMINA® sequencing adapters.

[0055] In some embodiments, the blunt-end DNA fragments that were adapter- ligated are amplified via polymerase chain reaction (PCR). In some embodiments, following PCR amplification, a cleanup step is performed prior to sequencing or DNA quantification. In some embodiments, the cleanup step comprises a IX SPRI cleanup to remove reaction enzymes and buffers.

[0056] Sequencing

[0057] In some embodiments, the disclosure provides a method comprising sequencing a plurality of blunt-end DNA fragments. In some embodiments, sequencing comprises nextgeneration sequencing. In some embodiments, sequencing comprises shotgun sequencing. In some embodiments, sequencing comprises amplicon sequencing. In some embodiments, sequencing comprises short-read sequencing. In some embodiments, sequencing comprises long-read sequencing. In some embodiments, sequencing comprises ILLUMINA® sequencing, SOLiD™ sequencing, PACBIO® sequencing, or nanopore sequencing. In some embodiments, sequencing comprises ILLUMINA® sequencing. In some embodiments, sequencing comprises PACBIO® or nanopore sequencing. In some embodiments, sequencing comprises Sanger sequencing.

[0058] In some aspects, the disclosure provides a method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising: contacting a plurality of sticky-end DNA fragments with a single-strand-specific nuclease, thereby producing a plurality of single- strand- specific nuclease-treated DNA fragments; isolating the plurality of single- strand- specific nuclease-treated DNA fragments from the single- strand- specific nuclease or inactivating the single-strand-specific nuclease; and contacting the plurality of single- strand- specific nuclease- treated DNA fragments with a DNA polymerase and / or a poly-A terminal transferase, thereby producing a plurality of blunt-end DNA fragments.

[0059] In some aspects, the disclosure provides a method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising, in sequential order (i) contacting a plurality of sticky-end DNA fragments with Single-strand-specific nuclease Pl, thereby producing a plurality of single-strand-specific nuclease-treated DNA fragments; (ii) isolating the plurality of single-strand-specific nuclease-treated DNA fragments from the Single- strandspecific nuclease Pl or inactivating the Single- strand- specific nuclease Pl; (iii) contacting the plurality of single-strand-specific nuclease-treated DNA fragments with T4 polymerase, thereby producing a plurality of blunt-end DNA fragments; and (iv) contacting the plurality of blunt-end DNA fragments with a poly-A terminal transferase.

[0060] In some embodiments, a method provided herein further comprises sequencing blunt-end DNA fragments of the plurality of blunt-end DNA fragments. In some embodiments, sequencing comprises shotgun sequencing. In some embodiments, sequencing comprises long-read sequencing.

[0061] EXAMPLES

[0062] Example 1. End-repair with single-strand-specific nuclease Pl results in high library yield, reduced substitution error, and preservation of epigenetic markers.

[0063] Traditional end repair / A-tailing DNA library preparation (ERAT) with sticky end DNA fragments (e.g., sheared DNA) utilizes DNA polymerase (e.g., T4 DNA polymerase) to end repair via polymerization to blunt-end DNA. Polymerization results in a loss of epigenetic markers at DNA ends, and introduces errors (e.g., additions, substitutions). Hairpin formation can also be exacerbated by polymerization when a single stranded overhang folds over on itself, priming DNA polymerase activity.

[0064] Presented herein are end repair methods that utilize single- strand- specific nucleases that have the ability to blunt sticky-end DNA fragments by cleaving single stranded overhangs. Single- strand- specific nucleases leave either blunt ends or small overhangs that can be polished by a quick polymerase treatment. Single-strand-specific nuclease-generated blunt-end DNA can be subsequently A-tailed for adapter ligation and sequencing (FIG. 1). Methods

[0065] DNA (NA 12878) from the Coriell Institute was obtained and sheared via Covaris shearing to generate sticky-end DNA fragments. Sticky-end fragments were treated with single- strand- specific nuclease Pl for 10 minutes at 37 degrees Celsius in a sodium acetate buffer with a pH of about 6.3, to cleave single-stranded overhangs and generate blunt-end DNA fragments. Amino acid sequences of single- strand- specific nucleases used are shown in Table 1.

[0066] To halt single- strand- specific nuclease Pl activation, samples were either treated with paramagnetic beads to isolate the blunt-end DNA fragments or with heat or a chelating agent to inactivate single- strand- specific nuclease Pl. A cleanup step was performed prior to proceeding to A-tailing. In some instances, a 2X SPRI cleanup was used to remove reaction enzymes and buffers from the reaction, after which samples were eluted in water. In other instances, 500 mM DTT was added and the samples were incubated on ice for 2 minutes before proceeding to A- tailing.

[0067] Blunt-end DNA fragments were treated with T4 DNA polymerase KlenTaq for 10 minutes at 60 degrees Celsius to A-tail DNA fragments, with or without a brief initial treatment (e.g., 30 minutes) at 20 degrees Celsius to polish blunt-end DNA fragments prior to A-tailing. Treating sticky-end DNA fragments with nuclease Pl results in the production of blunt-end DNA fragments and, occasionally, nearly-blunt DNA fragments (e.g., DNA fragments with single-stranded overhangs of no more than 4 nucleotides). In some experiments, polishing and / or A-tailing was performed in a tris acetate buffered solution at about pH 8.3 comprising additional deoxynucleotides (e.g. dATP and / or dNTP).

[0068] Stubby adapters or Y-adapters were next ligated onto A-tailed blunt-end DNA fragments to prepare the fragments for sequencing. Prior to amplifying the libraries with polymerase chain reaction (PCR) to increase the amount of material, a IX SPRI cleanup step was performed to remove reaction enzymes and buffers, after which libraries were eluted in water. Sequencing libraries are able to be visualized and quantified on an Agilent TapeStation. Prior to amplifying the libraries and / or after amplifying the libraries, a quantitative PCR (qPCR) step was optionally performed to determine post-ligation yield and post-amplification yield, respectively.

[0069] Table 1. Exemplary single-strand-specific nuclease sequences

[0070] Results

[0071] PCR product of cell-free DNA (cfDNA) blunt-end DNA fragments prepared from either 1 ng of cfDNA or 8 ng of cfDNA as described herein was visualized post-ligation with either 50 pm of stubby or 15 pm of Y-adapters on a D1000 Tape. Libraries ligated with stubby adapters resulted in slightly higher yield than those ligated with Y-adapters, and yield was markedly increased when the amount of starting cfDNA was increased (FIGs. 2A-2B).

[0072] To further interrogate the relationship between amount of starting DNA and amount of adapters used for ligation, 10 ng and 100 ng of sheared, sticky-end DNA fragments derived from cfDNA were prepared as described herein and were ligated with either 50 pm of stubby adapters or 15 pm of Y-adapters, while 1 ng of sticky-end DNA fragments were ligated with 5 pm of Y- adapters as a control. Ligated DNA fragments were amplified via PCR. Increasing the starting amount of DNA material to 100 ng resulted in markedly higher library yield, which was approximately the same between samples ligated with stubby adapters versus Y-adapters (FIGs. 3A-3B).

[0073] Library yield of sticky-end DNA fragments from cfDNA subjected to pERAT methods described herein with or without end-polishing with T4 DNA polymerase was compared. Library traces showed a substantial yield increase when pERAT-treated DNA was also subjected to an end-polishing treatment (FIGs. 4A-4B).

[0074] The retention of epigenetic markers in libraries with methylated cytosine nucleotides prepared with pERAT versus ERAT was compared. End repair (pERAT) was performed using a mixture of 5mdCTP (methylated dCTP) and dATP, dTTP, dGTP. All 5mdCTP’s which were inserted by the polymerase during end repair were subsequently converted to T’s. The approximate length of 3’ recesses remaining after Pl treatment was identified by assessing the conversion rate of C bases to T’s. Treatment of sheared, sticky-end DNA fragments with ERAT, which polymerizes further into the 3’ end of DNA fragments, resulted in a greater percentage of methylated dCTPs converted compared to treatment with pERAT. The results demonstrate that pERAT preserves the native state of original input DNA in the final adapter-ligated sequencing library (FIG. 5).

[0075] Library yields from ERAT and pERAT treated DNA fragments of varying quality (as indicated by dCq values, where a lower dCq indicates poorer quality) derived from formalin- fixed paraffin-embedded (FFPE) were tested next. FFPE-derived DNA has a high number of damaged bases, which are converted to inaccurate double-stand nucleotide substitutions during polymerase-mediated end repair. Library yield was found to be similar among samples treated with pERAT or ERAT (FIGs. 6A-6B). However, there was a profound reduction in hairpin artifacts in samples subjected to pERAT compared to those subjected to ERAT (FIGs. 6C-6D), indicating that single-strand-specific nuclease Pl-mediated end repair decreases the DNA errors associated with FFPE-derived samples.

[0076] The effect of pERAT on reduction in substitution errors was next evaluated. PCR-free libraries were prepared from E.coli either using the pERAT method described in this disclosure, or the traditional ERAT method, and sequenced using the AVITI sequencer (Element Biosciences). The C to A errors along the length of read 2 were measured by counting the number of mismatches to the reference genome. The pERAT method of library preparation resulted in drastically reduced C to A substitution rate compared to ERAT (FIG. 7), indicating that it will likely be useful for increasing sensitivity and specificity in high-stringency applications such as Minimal Residual Disease (MRD) detection.

[0077] In sum, the results demonstrate that the methods described herein using end repair and A- tailing with single-strand-specific nuclease Pl (pERAT) result in higher yield and conversion efficiency, substantially reduces epigenetic marker overwriting at DNA ends, and reduces hairpin artifacts and errors within DNA. Accordingly, such methods are useful for generating libraries that accurately reflect the genetic material of the samples from which they are derived. EQUIVALENTS AND SCOPE

[0078] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0079] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0080] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0081] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0082] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0083] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A method of producing a plurality of blunt-end DNA fragments, the method comprising contacting a plurality of sticky-end DNA fragments with:(a) a single-strand-specific nuclease; and(b) a DNA polymerase.

2. The method of claim 1, wherein the sticky-end DNA fragments are derived from cell-free DNA.

3. The method of claim 1 or claim 2, wherein the DNA polymerase lacks 5’ to 3’ exonuclease activity.

4. The method of claim 3, wherein the DNA polymerase is a T4 polymerase.

5. The method of claim 3, wherein the DNA polymerase is a Klenow fragment.

6. The method of claim 3, wherein the DNA polymerase is KlenTaq.

7. The method of claim 3, wherein the DNA polymerase is E. coli DNA polymerase I or B st pol large fragment.

8. The method of any one of claims 1-7, wherein the single- strand- specific nuclease is nuclease Pl.

9. The method of any one of claims 1-7, wherein the single- strand- specific nuclease is nuclease S 1.

10. The method of any one of claims 1-7, wherein the single- strand- specific nuclease is ExoT.

11. The method of any one of claims 1-7, wherein the single- strange specific nuclease is mung bean nuclease (MBN).

12. The method of any one of claims 1-11, wherein the method further comprises isolating the plurality of blunt-end DNA fragments from the single- strand- specific nuclease.

13. The method of claim 12, wherein the isolating comprises contacting the plurality of blunt-end DNA fragments with a DNA-binding reagent.

14. The method of claim 13, wherein the DNA-binding reagent comprises paramagnetic beads.

15. The method of any one of claims 1-14, wherein the method further comprises inactivating the single-strand-specific nuclease.

16. The method of claim 15, wherein the inactivating comprises heating the single- strandspecific nuclease to a temperature of 75 °C to 90 °C.

17. The method of claim 15, wherein the inactivating comprises contacting the single- strandspecific nuclease with a chelator.

18. The method of claim 17, wherein the chelator is ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA).

19. The method of any one of claims 1-18, further comprising A-tailing blunt-end DNA fragments of the plurality of blunt-end DNA fragments.

20. The method of any one of claims 19, wherein A-tailing the blunt-end DNA fragments comprises contacting the blunt-end DNA fragments with a poly-A terminal transferase.

21. The method of claim 20, wherein the poly-A terminal transferase is a Klenow fragment polymerase.

22. The method of any one of claims 1-21, wherein the method further comprises sequencing the plurality of blunt-end DNA fragments.

23. The method of any one of claims 1-22, wherein the method comprises contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase.

24. The method of any one of claims 1-22, wherein the method does not comprise contacting the plurality of blunt-end DNA fragments with a T4 polynucleotide kinase.

25. A method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising: contacting a plurality of sticky-end DNA fragments with a single-strand-specific nuclease, thereby producing a plurality of single- strand- specific nuclease-treated DNA fragments; isolating the plurality of single- strand- specific nuclease-treated DNA fragments from the single-strand-specific nuclease or inactivating the single-strand-specific nuclease; and contacting the plurality of single- strand- specific nuclease-treated DNA fragments with a DNA polymerase and / or a poly-A terminal transferase, thereby producing a plurality of blunt-end DNA fragments.

26. A method of preparing a plurality of sticky-end DNA fragments for sequencing, the method comprising, in sequential order:(i) contacting a plurality of sticky-end DNA fragments with single- strand- specific nuclease Pl, thereby producing a plurality of single- strand- specific nuclease-treated DNA fragments;(ii) isolating the plurality of single- strand- specific nuclease-treated DNA fragments from the single-strand-specific nuclease Pl or inactivating the single- strand- specific nuclease Pl;(iii) contacting the plurality of single- strand- specific nuclease-treated DNA fragments with T4 polymerase, thereby producing a plurality of blunt-end DNA fragments; and(iv) contacting the plurality of blunt-end DNA fragments with a poly-A terminal transferase.

27. The method of claim 26, wherein isolating the plurality of single- strand- specific nuclease-treated DNA fragments from the single-strand-specific nuclease Pl comprises contacting the plurality of single- strand- specific nuclease-treated DNA fragments with a DNA- binding reagent comprising paramagnetic beads.

28. The method of claim 26 or 27, wherein the plurality of sticky-end DNA fragments is derived from cell-free DNA.

29. The method of any one of claims 25-28, further comprising sequencing blunt-end DNA fragments of the plurality of blunt-end DNA fragments.

30. The method of any one of claims 29, wherein sequencing comprises shotgun sequencing.

31. The method of any one of claims 29, wherein sequencing comprises long-read sequencing.

32. The method of any one of claims 1-31, wherein the sticky-end DNA fragments comprise methylated nucleotides.

33. A method of A-tailing a DNA polynucleotide, the method comprising contacting the DNA polynucleotide with a DNA polymerase that lacks 5’ to 3’ exonuclease activity.

34. The method of claim 33, wherein the DNA polynucleotide is double- stranded DNA.

35. The method of claim 33 or claim 34, wherein the DNA polymerase is a T4 polymerase.

36. The method of claim 33 or claim 34, wherein the DNA polymerase is a Klenow fragment.

37. The method of claim 33 or claim 34, wherein the DNA polymerase is KlenTaq.

38. The method of claim 33 or claim 34, wherein the DNA polymerase is E. coli DNA polymerase I or B st pol large fragment.

39. The method of any one of claims 33-38, wherein the DNA polynucleotide comprises methylated nucleotides.

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