SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 21, 2020, is named 2020-10-23_GH0054US_Sequence_Listing_ST25.txt and is 970 bytes in size.

INTRODUCTION AND SUMMARY

Repair of overhangs that occur in certain types of DNA molecules is an important step in preparing the molecules for further analysis, such as sequencing and/or amplification. For example, 3′ overhangs, in which a number of nucleotides near the 3′ end of a molecule are single-stranded, can occur in sheared DNA and cell-free DNA, e.g., obtained from blood samples. It can be desirable to convert 3′ overhangs to blunt ends or to a single-base overhang for compatibility with subsequent steps such as ligation of a tag, barcode, or adapter.

Existing methods for overhang repair use a 3′ to 5′ exonuclease to resect the 3′ overhang. This approach results in loss of information in that nucleotides are removed and the location of the end of the original molecule cannot be determined. Thus, 3′ overhang repair by exonucleolysis can prevent one from obtaining information regarding, e.g., the sequence of the bases between the 5′ and 3′ ends as well as nucleosome positioning. Accordingly, there is a need for improved methods of 3′ overhang repair. The present disclosure aims to meet this need, provide other benefits, or at least provide the public with a useful choice.

Accordingly, the following embodiments are provided. Embodiment 1 is a method of repairing a partially double-stranded DNA fragment, the method comprising:

(a) contacting the partially double-stranded DNA fragment with one or more primers of a primer population, wherein the partially double-stranded DNA fragment comprises a 3′ overhang and the primer population comprises a random target-hybridizing sequence;

(b) extending one or more primers of the primer population along the DNA fragment using a DNA polymerase, thereby producing one or more extended primers annealed to the DNA fragment; and

(c) ligating the 3′ end of one or more extended primers to the 5′ end of an extended primer or a strand of the partially double-stranded DNA fragment, thereby providing a repaired DNA fragment.

Embodiment 2 is the method of any one of the preceding embodiments, wherein the DNA polymerase lacks 3′ to 5′ exonuclease activity.

Embodiment 3 is the method of any one of the preceding embodiments, wherein the DNA polymerase lacks 5′ to 3′ exonuclease activity.

Embodiment 4 is the method of any one of the preceding embodiments, wherein the DNA polymerase lacks strand displacement activity.

Embodiment 5 is the method of any one of the preceding embodiments, wherein the DNA polymerase is a Klenow fragment.

Embodiment 6 is the method of the immediately preceding embodiment, wherein the DNA polymerase is an exo-Klenow fragment.

Embodiment 7 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment has 3′ overhangs at each end.

Embodiment 8 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment has a 3′ overhang and (i) a blunt end or (ii) a 5′ overhang.

Embodiment 9 is the method of the immediately preceding embodiment, wherein the 5′ overhang is repaired by extending the 3′ end along the 5′ overhang.

Embodiment 10 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment is from a bodily fluid sample.

Embodiment 11 is the method of the immediately preceding embodiment, wherein the bodily fluid is whole blood, serum, plasma, or urine.

Embodiment 12 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment is a cfDNA fragment.

Embodiment 13 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment is mammalian.

Embodiment 14 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment is human.

Embodiment 15 is the method of any one of the preceding embodiments, wherein the partially double-stranded DNA fragment is part of a population of DNA fragments in a composition.

Embodiment 16 is the method of the immediately preceding embodiment, wherein the population of DNA fragments comprise sheared DNA.

Embodiment 17 is the method of embodiment 15 or 16, wherein the population of DNA fragments comprise epigenetically modified DNA.

Embodiment 18 is the method of any one of embodiments 15-17, wherein the population of DNA fragments comprises fragments from a plurality of genomic loci.

Embodiment 19 is the method of any one of embodiments 15-18, wherein the population of DNA fragments is non-enriched.

Embodiment 20 is the method of any one of embodiments 15-19, wherein the population of DNA fragments is unamplified.

Embodiment 21 is the method of any one of the preceding embodiments, wherein the random target-hybridizing sequence has a length of at least 4, 5, 6, 7, 8, 9, or 10 nucleotides.

Embodiment 22 is the method of any one of the preceding embodiments, wherein the random target-hybridizing sequence has a length of about 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides.

Embodiment 23 is the method of any one of the preceding embodiments, wherein the primers of the primer population are single-stranded.

Embodiment 24 is the method of any one of embodiments 1-20, wherein the primers of the primer population are double-stranded with a 3′ overhang and the random target-hybridizing sequence is in the 3′ overhang.

Embodiment 25 is the method of any one of embodiments 1-20, wherein the primers of the primer population are hairpins with a 3′ overhang and the random target-hybridizing sequence is in the 3′ overhang.

Embodiment 26 is the method of any one of embodiments 21-22, wherein the hairpin or double-stranded region of the primers comprises an adapter.

Embodiment 27 is the method of any one of embodiments 21-23, wherein the hairpin or double-stranded region of the primers comprises a tag.

Embodiment 28 is the method of embodiment 24, wherein the tag comprises a barcode.

Embodiment 29 is the method of embodiment 25, wherein the primer population comprises a plurality of different barcodes.

Embodiment 30 is the method of any one of the preceding embodiments, wherein at least steps (a)-(c) are performed in a single tube.

Embodiment 31 is the method of any one of the preceding embodiments, wherein the double-stranded DNA fragment is in a composition and for at least steps (a)-(c) no component is removed from the composition.

Embodiment 32 is the method of any one of the preceding embodiments, wherein the repaired DNA fragment comprises one or two blunt ends, or the method further comprises blunting one or two ends of the repaired DNA fragment.

Embodiment 33 is the method of the immediately preceding embodiment, further comprising end-tailing the repaired DNA fragment using a polymerase that performs a non-template directed addition of a nucleotide to the 3′ ends of blunt-ended nucleic acids, optionally wherein A is added preferentially to G preferentially to C or T.

Embodiment 34 is the method of any one of the preceding embodiments, further comprising ligating a tag to the repaired DNA fragment (optionally at both ends), optionally wherein the tag comprises a barcode and/or adapter.

Embodiment 35 is the method of any one of the preceding embodiments, further comprising purifying the repaired DNA fragment.

Embodiment 36 is the method of any one of the preceding embodiments, further comprising denaturing one or more enzymes used in step (b) and/or (c) after step (b) and/or (c).

Embodiment 37 is the method of any one of the preceding embodiments, further comprising amplifying the repaired DNA fragment.

Embodiment 38 is the method of the immediately preceding embodiment, wherein the repaired DNA fragment comprises one or more adapters (e.g., two adapters) and amplifying the repaired DNA fragment uses one or more (e.g., two) amplification oligomers that anneal to the one or more adapters.

Embodiment 39 is the method of any one of the preceding embodiments, further comprising enriching the repaired DNA fragments for fragments of interest, thereby providing enriched DNA fragments, optionally wherein the enriching step is performed after an amplification step.

Embodiment 40 is the method of the immediately preceding embodiment, wherein the fragments of interest comprise loci that vary in a disease or disorder-associated manner, optionally wherein the disease or disorder is a cancer.

Embodiment 41 is the method of the immediately preceding embodiment, wherein the variation is one or more of single nucleotide variation, copy number variation, gene fusion, or indels.

Embodiment 42 is the method of the immediately preceding embodiment, wherein the fragments of interest comprise one, two, three, or four of fragments that show single nucleotide disease or disorder-associated variation; copy number disease or disorder-associated variation; disease or disorder-associated gene fusion; or disease or disorder-associated indels, optionally wherein the disease or disorder is cancer.

Embodiment 43 is the method of any one of embodiments 36-39, further comprising amplifying the enriched DNA fragments.

Embodiment 44 is the method of any one of the preceding embodiments, further comprising sequencing the repaired DNA fragment.

Embodiment 45 is the method of the immediately preceding embodiment wherein the sequencing sequences a nucleotide that formed a 3′ overhang in the partially double-stranded DNA fragment.

Embodiment 46 is the method of embodiment 41 or 42, wherein the sequencing is high-throughput sequencing.

Embodiment 47 is the method of any one of embodiments 44-46, wherein a plurality of repaired DNA fragments are generated and at least a portion of the repaired DNA fragments comprise tags, and the sequencing generates a plurality of sequence reads from a plurality of repaired DNA fragments.

Embodiment 48 is the method of embodiment 47, wherein the sequence reads comprise sequence of nucleotides that formed a 3′ overhang in the partially double-stranded DNA fragment and sequence of a tag.

In some embodiments of each and every aspect of the invention, the results of the systems and/or methods disclosed herein are used as an input to generate a report. The report may be in a paper or electronic format. For example, information on the presence or absence of cancer, as determined by the methods or systems disclosed herein, can be displayed in such a report. Alternatively or additionally, the report may comprise information relating to or derived from the identity of nucleobases in the sample. The methods or systems disclosed herein may further comprise a step of communicating the report to a third party, such as the subject from whom the sample is derived or a health care practitioner.

The various steps of the methods disclosed herein, or the steps carried out by the systems disclosed herein, may be carried out at the same time or different times, and/or in the same geographical location or different geographical locations, e.g. countries. The various steps of the methods disclosed herein can be performed by the same person or different people.

