Nucleic acid sample preparation methods
Provided are methods of nucleic acid sample preparation for analysis, including analysis using nanopore sequencing applications. In particular provided are methods that allow for the creation of circular and linear DNA samples that contain asymmetric ends to ligate different and desired adaptors to the end of the sample, methods to create single nucleic acid molecules that automatically form concatemers for higher sequencing throughput; methods to create multiple copies (temperature dependent, multi-primed, open-fold replication) of one original nucleic acid sample, or to create single nucleic acid molecules containing multiple copies of more than one original nucleic acid sample (concatemerized samples) for improved accuracy and throughput are described; and methods for improved modified base calling.
1. 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.
2. The method of claim 1 , wherein the single single-stranded extendable primer region comprises at least one universal base.
3. The method of claim 1 , wherein the single-stranded extendable primer region of the first adaptor comprises a region complementary to a sequence in the sample nucleic acid.
4. The method of claim 1 , further comprising ligating a third adaptor to the end comprising the second adaptor wherein the third adaptor comprises a sequencing primer site.