Methods of preparing nucleic acids for sequencing
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
1. A method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising:
(a) contacting a target nucleic acid molecule comprising the known target nucleotide sequence with an initial target-specific primer under hybridization conditions;
(b) performing a template-dependent extension reaction that is primed by a hybridized initial target-specific primer and that uses the target nucleic acid molecule as a template;
(c) contacting an extension product of step (b) with a plurality of tailed random primers under hybridization conditions;
(d) performing a template-dependent extension reaction that is primed by a hybridized tailed random primer and that uses the extension product of step (b) as a template;
(e) amplifying a portion of an extension product of step (d) with a first tail primer and a first target-specific primer; and
(f) sequencing an amplification product from step (e) using a first or second sequencing primer;
wherein each of the plurality of tailed random primers comprises (i) a common sequence and (ii) a 3′-end comprising about 3 to about 15 random nucleotides;
wherein the initial target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid;
wherein the first target-specific primer comprises a 3′ portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence and is nested with respect to the initial target-specific primer; and
wherein the first tail primer comprises a nucleic acid sequence identical to a portion of the common sequence.
2. The method of claim 1 , wherein each of the plurality of tailed random primers comprises a barcode or index sequence between the common sequence and the 3′-end.
3. The method of claim 1 , wherein the first target-specific primer is nested with respect to the initial target-specific primer by at least 3 nucleotides.
4. The method of claim 1 , wherein each of the plurality of tailed random primers consists of naturally occurring nucleotides.