Nucleic acid sample preparation methods

A method of preparing asymmetric nucleic acid templates for sequencing, the method comprising:

• (a) contacting a single-stranded sample nucleic acid with a first adaptor molecule comprising a single-stranded extendable primer region and a double-stranded hairpin region;
• (b) ligating the hairpin region of the adaptor to the first end of the sample nucleic acid;
• (c) extending the extendable primer region of the adaptor to copy the sample nucleic acid and form an extended nucleic acid having a single A 3′ overhang of the extended strand, wherein the extended nucleic acid is a first complement to the sample nucleic acid;
• (d) ligating a second adaptor to an end of the extended nucleic acid opposite of the hairpin end, the adaptor comprising a double-stranded duplex region having (i) a first overhang region comprising a single T 5′ overhang, and (ii) a second overhang region comprising a primer site region, thereby forming an asymmetric nucleic acid template, and
• (e) performing an open-fold replication whereby the asymmetric nucleic acid template having the hairpin at one end is opened to form a linear single-stranded molecule, and the linear single-stranded molecule is copied from a primer binding to the primer site region in the second adaptor to form a double-stranded molecule, wherein the double-stranded molecule comprises:
• the sample nucleic acid,
• the first complement to the sample nucleic acid,
• a complement to the first complement to the sample nucleic acid, and
• a second complement to the sample nucleic acid.