Enhanced ligation in sequencing library preparation
Methods for preparing a sequencing library from a DNA-containing test sample are provided. In some embodiments, the methods involve rescuing a partially ligated DNA fragment to enhance library preparation conversion efficiencies. In some embodiments, the methods involve improving recovery of duplex sequence information from double-stranded DNA.
1. A method for preparing a sequencing library from a test sample comprising a plurality of double-stranded DNA fragments, the method comprising the steps:
(a) obtaining a test sample comprising a plurality of double-stranded DNA (dsDNA) fragments, wherein the dsDNA fragments each comprise a forward strand and a reverse strand;
(b) contacting the test sample with a plurality of dsDNA adapters, wherein the dsDNA adapters comprise a single stranded 3′ T base overhang, wherein the single stranded 3′ T base overhang, or one or more bases located immediately upstream of the single stranded 3′ T base overhang, has a melting temperature that is higher than a melting temperature of a non-modified DNA base, wherein the single stranded 3′ T base overhang, or the one or more bases located immediately upstream of the single stranded 3′ T base overhang, are selected from the group consisting of: locked nucleic acid (LNA) bases, bridged nucleic acid (BNA) bases, super T (5-hydroxybutynl-2′-deoxyuridine), C-5 propynyl-U, or any combination thereof;
(c) ligating the dsDNA adapters to the dsDNA fragments to create a plurality of dsDNA adapter-fragment constructs; and
(d) amplifying the dsDNA adapter-fragment constructs to generate a sequencing library.
2. The method according to claim 1 , wherein the method further comprises:
(a) sequencing the library to obtain a plurality of sequence reads; and
(b) analyzing the plurality of sequence reads to detect the presence or absence of cancer, determine cancer status, monitor cancer progression and/or determine a cancer classification in the subject.
3. The method according to claim 1 , wherein the dsDNA fragments are cell-free DNA (cfDNA) fragments.
4. The method according to claim 1 , wherein a dsDNA fragment is modified prior to ligation of the double-stranded DNA adapters and wherein the modification comprises end-repairing, A-tailing, phosphorylation, or any combination thereof.
5. The method according to claim 1 , wherein the test sample comprises whole blood, a blood fraction, plasma, serum, urine, fecal matter, saliva, a tissue biopsy, pleural fluid, pericardial fluid, cerebrospinal fluid, peritoneal fluid, or any combination thereof.
6. The method according to claim 1 , wherein the test sample is a plasma sample.
7. The method according to claim 1 , wherein the test sample comprises dsDNA fragments originating from healthy cells and from cancer cells.
8. The method according to claim 1 , wherein the dsDNA adapters are ligated to a first end and a second end of the dsDNA fragments.
9. The method according to claim 1 , wherein the dsDNA adapter comprises a fork-shaped sequencing adapter formed by annealing a pair of partially complementary oligonucleotides to one another, wherein the fork-shaped sequencing adapter comprises a first double-stranded region, formed from hybridization between the complementary regions, and a second single-stranded region.
10. The method according to claim 9 , wherein the one or more modified bases are located at the 3′ end of the double-stranded region of the fork-shaped sequencing adapter.
11. The method according to claim 9 , wherein the one or more modified bases are located upstream of a 3′ end of the fork-shaped sequencing adapter.
12. The method according to claim 1 , wherein the dsDNA adapters are hairpin adapters or linear adapters.
13. The method according to claim 1 , wherein the ligase is a T4 DNA ligase or a T7 DNA ligase.
14. The method according to claim 2 , further comprising enriching the sequencing library for one or more target dsDNA fragments that are known to be, or suspected of being, indicative of cancer.
15. The method according to claim 2 , wherein the sequence reads are obtained from a next-generation sequencing (NGS) procedure, massively parallel sequencing using a sequencing-by-synthesis procedure, or a paired-end sequencing procedure.