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 composition comprising a template mRNA and a template switch oligonucleotide each hybridized to adjacent regions of a first strand cDNA, wherein the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain and does not comprise a NGS adapter domain.
2. A composition comprising a double stranded cDNA produced from the composition according to claim 1 .
3. A composition comprising a tagmented sample produced by tagmenting a double stranded cDNA according to claim 2 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.
4. A NGS library produced by amplification of a tagmented sample according to claim 3 .
5. 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 the template switch oligonucleotide comprises a first post-tagmentation amplification primer binding domain and does not comprise a NGS adapter domain.
6. The kit according to claim 5 , wherein the kit further comprises first and second pre-tagmentation amplification primers.
7. The kit according to claim 6 , wherein the first and second pre-tagmentation amplification primers are identical.
8. The kit according to claim 5 , wherein the kit further comprises a transposome comprising a transposase and a transposon nucleic acid comprising a transposon end domain and a second post-tagmentation amplification primer binding domain.
9. The kit according to claim 8 , wherein the transposase comprises a Tn5 transposase.
10. The kit according to claim 8 , wherein the transposon end domain comprises a Tn5 transposon end domain.
11. The kit according to claim 8 , wherein the kit further comprises:
a first primer comprising a first post-tagmentation amplification primer binding domain, a first NGS indexing domain and a first NGS adapter domain; and
a second primer comprising a second post-tagmentation amplification primer binding domain, a second NGS indexing domain and a second NGS adapter domain.
12. The kit according to claim 5 , wherein the kit further comprises a solid support.
13. The kit according to claim 5 , wherein the kit comprises a lyophilized component.