Methods for high efficiency library preparation using double-stranded adapters
Methods for preparing a sequencing library from a DNA-containing test sample are provided, including methods for rescuing one or more partially ligated DNA fragments to enhance library preparation conversion efficiencies. The subject methods can further be used to improve 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:
(a) obtaining a test sample comprising a plurality of double-stranded DNA (dsDNA) fragments, the dsDNA fragments comprising a forward strand and a reverse strand;
(b) adding double-stranded adapters to the dsDNA sample and ligating the double-stranded adapters to both ends of the dsDNA fragments;
(c) extending unligated 3′-ends of the dsDNA fragments with a DNA polymerase to create dsDNA fragment-adapter templates, wherein the polymerase further comprises strand displacement activity;
(d) adding a poly-adenine tail to the 3′-ends of the dsDNA fragment-adapter templates;
(e) adding a set of single-stranded DNA (ssDNA) oligonucleotides and hybridizing the ssDNA oligonucleotides to the dsDNA fragment-adapter templates; and
(f) extending the set of ssDNA oligonucleotides to create a dsDNA sequencing library.
2. The method of claim 1 , wherein the dsDNA sequencing library is sequenced to obtain sequence reads.
3. The method of claim 2 , wherein the sequence reads are obtained from next-generation sequencing (NGS).
4. The method of claim 2 , wherein the sequence reads are obtained from massively parallel sequencing using sequencing-by-synthesis.
5. The method of claim 1 , wherein the test sample comprises a plurality of cell-free DNA molecules.
6. The method of 5 , wherein the test sample is from a whole blood, a blood fraction, plasma, serum, urine, fecal, saliva, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, or peritoneal fluid test sample.
7. The method of claim 5 , wherein the test sample includes nucleic acids originating from healthy cells and from cancer cells.
8. The method of claim 1 , wherein the DNA fragments are end-repaired and adenine tailed at the 3′-end prior to ligation in step (b).
9. The method of claim 1 , wherein the DNA polymerase used in step (c) is Bacillus stearothermophilus DNA polymerase (Bst Pol) or phi29 DNA polymerase.
10. The method of claim 1 , wherein the dsDNA adapters comprise a unique molecule tag.
11. The method of claim 10 , wherein the dsDNA adapters further comprise a universal primer.
12. The method of claim 1 , wherein a terminal deoxynucleotidyl transferase catalyzes the addition of the 3′ poly-adenine tail in step (d).
13. The method of claim 1 , wherein the set of ssDNA oligonucleotides comprise amplification primers.
14. The method of claim 13 , where the set of ssDNA oligonucleotides further comprise an indexing sequence.
15. The method of claim 1 , wherein the set of ssDNA oligonucleotides comprise a first ssDNA oligonucleotide and a second ssDNA oligonucleotide, wherein the first and second ssDNA oligonucleotides are complementary to a region on the dsDNA adapters.
16. The method of claim 15 , wherein the second ssDNA oligonucleotides further comprise a poly-T region.
17. The method of claim 13 , wherein the method further comprises PCR amplification of the dsDNA fragment-adapter templates to create a sequencing library.