ERROR REMOVAL USING IMPROVED LIBRARY PREPARATION METHODS
Methods for preparing sequencing libraries from a DNA-containing test sample, as well as methods for reducing the occurrence of edge errors prior to sequencing, are provided.
1 . A method for preparing a sequencing library from a test sample comprising a plurality of double-stranded DNA (dsDNA) molecules, the method comprising:
(a) obtaining a test sample comprising a plurality of dsDNA molecules, wherein the dsDNA molecules comprise one or more free single-stranded DNA (ssDNA) overhangs at one or both ends of the dsDNA molecules;
(b) treating the dsDNA molecules to remove the free ssDNA overhangs, thereby generating a plurality of blunt ended dsDNA molecules;
(c) modifying the blunt ended dsDNA molecules for adapter ligation;
(d) ligating a plurality of dsDNA adapters to the plurality of blunt ended dsDNA molecules obtained from step (c) to generate a plurality of dsDNA adapter-molecule constructs; and
(e) amplifying the dsDNA adapter-molecule constructs to generate a sequencing library.
2 . The method according to claim 1 , wherein treating the dsDNA molecules to remove the free ssDNA overhangs comprises an exonuclease pretreatment step, a DNA template repair pretreatment step, a heat inactivation step, or a combination thereof.
3 . The method according to claim 1 , further comprising:
(f) sequencing the sequencing library to obtain a plurality of sequence reads; and
(g) detecting the presence or absence of cancer, determining cancer status, monitoring cancer progression and/or determining a cancer classification from the plurality of sequence reads.
4 . The method according to claim 1 , wherein the dsDNA molecules are cell-free DNA (cfDNA) fragments.
5 . The method according to claim 4 , wherein the cfDNA fragments originate from healthy cells and from cancer cells.
6 . (canceled)
7 . The method according to claim 1 , wherein the free single-stranded overhang comprises a free 5-end.
8 . The method according to claim 1 , wherein the free single-stranded DNA overhang comprises a free 3′-end.
9 . The method according to claim 2 , wherein the exonuclease pretreatment step comprises a single strand DNA nuclease.
10 . (canceled)
11 . (canceled)
12 . The method according to claim 9 , wherein removal of the free single-stranded DNA using the single-strand DNA nuclease results in a plurality of blunt ended dsDNA molecules.
13 . The method according to claim 1 , wherein modification of the plurality of dsDNA fragments comprises end-repairing and A-tailing prior to ligation step (d).
14 . The method according to claim 1 , wherein the adapters further comprise a sample-specific index sequence.
15 . The method according to claim 1 , wherein the adapters further comprise a universal priming site.
16 . The method according to claim 1 , wherein the adapters further comprise one or more sequencing oligonucleotides for use in cluster generation and/or sequencing.
17 . (canceled)
18 . (canceled)
19 . (canceled)
20 . The method according to claim 3 , wherein monitoring cancer progression further comprises monitoring disease progression, monitoring therapy, or monitoring cancer growth.
21 . The method according to claim 3 , wherein the cancer classification further comprises determining a cancer type and/or a cancer tissue of origin.
22 . The method according to claim 3 , wherein monitoring cancer progression further comprises monitoring disease progression, monitoring therapy, or monitoring cancer growth.
23 . (canceled)
24 . A method for preparing a sequencing library from a test sample comprising a plurality of double-stranded DNA (dsDNA) molecules, the method comprising:
(a) obtaining a test sample comprising a plurality of dsDNA molecules;
(b) treating the dsDNA molecules to remove and/or repair one or more uracil residues within the dsDNA molecules;
(c) modifying the plurality of dsDNA fragments for adapter ligation;
(d) ligating a plurality of dsDNA adapters to the plurality of dsDNA molecules obtained from step (c) to generate a plurality of dsDNA adapter-molecule constructs; and
(e) amplifying the dsDNA adapter-molecule constructs to generate a sequencing library.
25 . The method according to claim 24 , further comprising:
(f) sequencing the sequencing library to obtain a plurality of sequence reads; and
(g) detecting the presence or absence of cancer, determining cancer status, monitoring cancer progression and/or determining a cancer classification from the plurality of sequence reads.
26 . (canceled)
27 . (canceled)
28 . (canceled)
29 . The method according to claim 24 , wherein a uracil-specific excision reagent is used to remove one or more uracil residues from the dsDNA molecules.
30 . The method according to claim 29 , wherein the removed uracil residue is replaced with a cytosine residue using a DNA polymerase and/or a DNA ligase.
31 .- 49 . (canceled)