METHODS FOR SAMPLE PREPARATION
The disclosure provides for single amplification and double amplification methods for preparing nucleic acid samples for sequencing.
1 . A method for generating an adaptor-ligated library of double-stranded nucleic acid molecules comprising, in a single reaction vessel or reaction mixture:
(a) generating, from a plurality of single-stranded polynucleotides, a plurality of double-stranded DNA molecules by:
i. contacting said plurality of polynucleotides with a pool of primers comprising a cleavable 5′ end; and
ii. extending said primers to generate first extension products hybridized to said plurality of polynucleotides thereby forming double-stranded duplexes, wherein said first extension products are formed from single template molecules;
(b) cleaving the cleavable 5′ end with a cleaving agent, thereby generating double-stranded DNA molecules, wherein said double-stranded DNA molecules have a blunt end and an overhang end; and
(c) ligating one or more double-stranded adapters, wherein a first set of adapters comprises a 3′ overhang complementary to the overhang end of the double-stranded DNA molecules and a second set of adapters does not comprise the 3′ overhang, to the double-stranded DNA molecules, thereby forming the adapter-ligated library using only a single round of extension; and, optionally,
(d) sequencing the adapter-ligated library,
wherein the plurality of single-stranded polynucleotides is present at a quantity less than about 5 nanograms.
2 . The method of claim 1 , wherein the cleavable 5′ end is selected from the group consisting of: rA, rC, rG, rU, or dU.
3 . The method of claim 1 , wherein the ligating occurs with an efficiency greater than 90%.
4 .- 12 . (canceled)
13 . The method of claim 1 , wherein the pool of primers comprises random primers.
14 . The method of claim 1 , wherein the pool of primers comprises target-specific primers.
15 . The method of claim 14 , wherein the target-specific primers comprise barcode sequence upstream of the target-specific sequence.
16 .- 30 . (canceled)
31 . The method of claim 1 , wherein said cleaving agent is a uracil-DNA glycolyase.
32 . The method of claim 1 , wherein said cleaving agent is an RNaseH.
33 . The method of claim 1 , wherein said extending further comprises incorporating one or more cleavable dNTPs, thereby generating an extended strand comprising cleavable dNTPs.
34 . The method of claim 33 , wherein said extended strand is cleaved and said sequencing does not comprise sequencing said extended strand.
35 . The method of claim 34 , wherein sequencing reads from said sequencing do not comprise errors due to extension or PCR performed prior to said sequencing.