Methods for sample preparation
View Patent ↗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 in a single reaction vessel or reaction mixture, the method consisting of the following steps:
(a) generating a plurality of double-stranded nucleic acid molecules from a plurality of single-stranded genomic DNA template polynucleotides by performing a single round of amplification, comprising:
(i) contacting the plurality of single-stranded genomic DNA polynucleotides with a pool of primers, each primer comprising a cleavable 5′ end, and a mixture of dNTPs; and
(ii) extending the primers by a polymerase with 5′ to 3′ exonuclease activity to generate a set of polynucleotide extension products that are hybridized to the single-stranded template polynucleotides to form double-stranded nucleic acid molecules having a cleavable 5′ end and a blunt end;
(b) cleaving the cleavable 5′ ends of the double-stranded nucleic acid molecules having a cleavable 5′ end and a blunt end with a cleaving agent to generate double-stranded nucleic acid molecules having a blunt end, and a 3′ overhang end at the cleavage site; and
(c) ligating to the double-stranded nucleic acid molecules having a blunt end and a 3′ overhang end,
(i) double-stranded nucleic acid adaptors, having a complementary 3′ overhang end, to the 3′ overhang end of the double-stranded nucleic acid molecules having a blunt end and a 3′ overhang end, and
(ii) double-stranded nucleic acid adaptors, having a blunt end to the blunt end of the double-stranded nucleic acid molecules having a blunt end and a 3′ overhang end,
to generate the adaptor-ligated library of double-stranded nucleic acid molecules.
2. The method of claim 1 , wherein the cleavable 5′ end is a nucleotide selected from the group consisting of: rA, rC, rG, rU, and dU.
3. The method of claim 1 , wherein the pool of primers are random primers.
4. The method of claim 1 , wherein the pool of primers are template polynucleotide sequence-specific primers.
5. The method of claim 4 , wherein the template polynucleotide sequence-specific primers further comprise a polynucleotide barcode upstream of the sequence-specific nucleotides.
6. The method of claim 1 , wherein the cleaving agent is RNase H.
7. The method of claim 1 , wherein the polymerase with 5′ to 3′ exonuclease activity is DNA Polymerase I.