Methods for Preparing a Next Generation Sequencing (NGS) Library from a Ribonucleic Acid (RNA) Sample and Compositions for Practicing the Same
Methods of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample are provided. Aspects of the methods include combining the RNA sample with a first strand cDNA primer and a template switch oligonucleotide under first strand cDNA synthesis conditions, where one of the first strand cDNA primer and the template switch oligonucleotide includes a first post-tagmentation amplification primer binding domain. The resultant product is subjected to amplification conditions sufficient to produce a double stranded cDNA, which is then tagmented with a transposome that includes a second post-tagmentation amplification primer binding domain. The tagmented sample is then subjected to amplification conditions using first and second post-tagmentation amplification primers that include sequencing platform adapter constructs to produce a NGS library. Aspects of the invention further include compositions produced by the methods and kits that find use in practicing the methods.
1 . A method of preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample, the method comprising:
(a) combining:
a RNA sample;
a first strand complementary deoxyribonucleic acid (cDNA) primer comprising a first pre-tagmentation amplification primer primer binding domain;
a template switch oligonucleotide comprising a 3′ hybridization domain and a 5′ second pre-tagmentation amplification primer binding domain;
a reverse transcriptase; and
dNTPs;
in a reaction mixture under conditions sufficient to produce a double stranded product nucleic acid comprising a template mRNA and the template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein one of the first strand cDNA primer and the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain; and
(b) contacting the product nucleic acid with first and second pre-tagmentation amplification primers complementary to the first and second pre-tagmentation amplification primer binding domains under amplification conditions sufficient to produce a product double stranded cDNA.
2 . The method according to claim 1 , wherein the method further comprises tagmenting the product double stranded cDNA with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon end domain and a second post-tagmentation amplification primer binding domain to produce a tagmented sample.
3 . The method according to claim 2 , wherein the method further comprises amplifying the tagmented sample by contacting the tagmented sample with:
a first primer comprising a first post-tagmentation amplification primer domain, a first NGS indexing domain and a first NGS adapter domain; and
a second primer comprising a second post-tagmentation amplification primer domain, a second NGS indexing domain and a second NGS adapter domain;
under PCR amplification conditions sufficient to produce a NGS library.
4 . The method according to any of claims 1 to 3 , wherein the RNA sample comprises messenger RNA and the method comprises producing the NGS library from mRNA.
5 . The method according to any of claims 1 to 4 , wherein the first strand cDNA primer comprises the first post-tagmentation amplification primer binding domain.
6 . The method according to any of claims 1 to 4 , wherein the template switch oligonucleotide comprises the first post-tagmentation PCR primer binding domain.
7 . The method according to any of the preceding claims , wherein the first and second pre-tagmentation amplification primer binding domains are identical and the first and second pre-tagmentation amplification primers are identical.
8 . The method according to any of the preceding claims , wherein the transposase comprises a Tn5 transposase.
9 . The method according to claim 8 , wherein the transposon end domain comprises a Tn5 transposon end domain.
10 . The method according to any of the preceding claims , wherein the method further comprises pooling the double stranded product cDNA with a second double stranded product cDNA to produce a pooled cDNA sample, and then tagmenting the pooled cDNA sample.
11 . The method according to any of the preceding claims , wherein the RNA sample is one that is produced from a single cell.
12 . The method according to any of the preceding claims , wherein the method further comprises subjecting the NGS library to an NGS protocol.
13 . The method according to any of the preceding claims , wherein the method further comprises quantitating one or more RNA species of the RNA sample.
14 . A composition comprising a template mRNA and a template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein one of the first strand cDNA comprises and template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain.
15 . A composition comprising a double stranded cDNA produced from the composition according to claim 14 .
16 . A composition comprising a tagmented sample produced by tagmenting a double stranded cDNA according to claim 15 with a transposome comprising a transposase and a transposon nucleic acid comprising a transposon end domain and a second post-tagmentation amplification primer binding domain to produce a tagmented sample.
17 . A NGS library produced by amplification of a tagmented sample according to claim 16 .
18 . A kit comprising:
a first strand cDNA primer comprising an 3′ RNA hybridization domain and a 5′ first pre-tagmentation amplification primer binding domain; and
a template switch oligonucleotide comprising a 3′ hybridization domain and a 5′ second pre-tagmentation primer binding domain;
wherein one of the first strand cDNA primer and the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain.