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 .- 26 . (canceled)
27 . A method for preparing a sequencing library from a test sample comprising a plurality of double-stranded DNA (dsDNA) fragments, the method comprising:
(a) obtaining a test sample comprising a plurality of dsDNA fragments, wherein the dsDNA fragments each comprise a forward strand and a reverse strand;
(b) providing a reaction mixture comprising one or more modified bases having a melting temperature that is higher than a melting temperature of a non-modified DNA base, wherein the one or more modified bases 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, 2′-Omethyl, or any combination thereof;
(c) adding the one or more modified bases to the dsDNA fragments through a polymerase reaction to create a plurality of modified-dsDNA constructs; and
(d) amplifying the modified-dsDNA constructs to generate a sequencing library.
28 . The method according to claim 27 , wherein the dsDNA fragments are cell-free DNA (cfDNA) fragments.
29 . The method according to claim 27 , wherein a plurality of dsDNA adapters are ligated to both ends of the modified-dsDNA constructs with a ligase.
30 . The method according to claim 29 , wherein the dsDNA fragments are modified prior to ligation of the dsDNA adapters.
31 . The method according to claim 30 , wherein the modification comprises end-repairing, A-tailing, phosphorylation, or any combination thereof.
32 . The method according to claim 27 , 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.
33 . The method according to claim 32 , wherein the test sample is a plasma sample.
34 . The method according to claim 32 , wherein the test sample comprises dsDNA originating from healthy cells and from cancer cells.
35 . The method according to claim 29 , wherein the dsDNA adapters comprise 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 two complementary regions, and a first single-stranded region.
36 . The method according to claim 35 , wherein the fork-shaped adapters further comprise one or more modified bases located at each 3′ end of the fork-shaped adapters.
37 . The method according to claim 35 , wherein the fork-shaped adapters further comprise one or more modified bases located upstream of a 3′ end or upstream of a 5′ end.
38 . (canceled)
39 . The method according to claim 29 , wherein the plurality of dsDNA adapters are hairpin adapters.
40 . The method according to claim 29 , wherein the plurality of dsDNA adapters are linear adapters.
41 . (canceled)
42 . The method according to claim 29 , wherein the ligase is a T4 DNA ligase or a T7 DNA ligase.
43 . (canceled)
44 . The method according to claim 27 , further comprising sequencing the sequencing library to obtain a plurality of sequence reads, and analyzing the sequence reads to detect a presence or absence of cancer, determine cancer status, monitor cancer progression and/or determine a cancer classification.
45 . The method according to claim 44 , 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.
46 . The method according to claim 44 , 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.
47 .- 48 . (canceled)
49 . The method according to claim 44 , wherein monitoring cancer progression further comprises monitoring disease progression, monitoring therapy, or monitoring cancer growth.
50 . The method according to claim 44 , wherein the cancer classification comprises determining a cancer type and/or a cancer tissue of origin.
51 . The method according to claim 44 , wherein the cancer comprises a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, a blastoma, a germ cell tumor, or any combination thereof.