Methods for preparing a next generation sequencing (NGS) library from a ribonucleic acid (RNA) sample and compositions for practicing the same

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 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 only 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;

(c) 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; and

(d) amplifying the tagmented sample by contacting the tagmented sample with:

a first post-tagmentation primer comprising a first post-tagmentation amplification primer domain complementary to the first post-tagmentation amplification primer binding domain; and

a second primer comprising a second post-tagmentation amplification primer domain complementary to the second post-tagmentation amplification primer binding domain.

2. The method according to claim 1 , wherein

the first post-tagmentation primer further comprises a first NGS indexing domain and a first NGS adapter domain; and

the second post-tagmentation primer further comprises a second NGS indexing domain and a second NGS adapter domain.

3. The method according to claim 1 , wherein the RNA sample comprises messenger RNA and the method comprises producing the NGS library from mRNA.

4. The method according to claim 1 , wherein the first strand cDNA primer comprises the first post-tagmentation amplification primer binding domain.

5. The method according to claim 1 , wherein the template switch oligonucleotide comprises the first post-tagmentation amplification primer binding domain.

6. The method according to claim 1 , wherein the first and second pre-tagmentation amplification primer binding domains are identical and the first and second pre-tagmentation amplification primers are identical.

7. The method according to claim 1 , wherein the transposase comprises a Tn5 transposase.

8. The method according to claim 7 , wherein the transposon end domain comprises a Tn5 transposon end domain.

9. The method according to claim 1 , 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.

10. The method according to claim 1 , wherein the RNA sample is one that is produced from a single cell.

11. The method according to claim 1 , wherein the method further comprises subjecting the NGS library to an NGS protocol.

12. The method according to claim 1 , wherein the method further comprises quantitating one or more RNA species of the RNA sample.

13. The method according to claim 4 , wherein the first strand cDNA primer comprises an oligodT domain 3′ of the first pre-tagmentation amplification primer binding domain.

14. The method according to claim 1 , wherein the first strand cDNA primer further comprises a source barcode domain.

15. The method according to claim 1 , wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs.

16. The method according to claim 15 , wherein each of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation amplification primers comprises one or more nucleotide analogs.

17. The method according to claim 15 , wherein the one or more nucleotide analogs is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, linkage modifications, 5′ and/or 3′ end modifications, one or more fluorescently labeled nucleotides, and combinations thereof.

18. The method according to claim 17 , wherein the one or more nucleotide analogs comprises a 5′ end modification.

19. The method according to claim 1 , wherein at least one of the first strand cDNA primer, template switch oligonucleotide and pre-tagmentation primers comprises a 5′ polymerase blocking modification.

20. The method according to claim 19 , wherein each of the first strand cDNA primer and pre-tagmentation primers comprises a 5′ polymerase blocking modification.

21. The method according to claim 19 , wherein the 5′ polymerase blocking modification is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, and combinations thereof.

22. The method according to claim 1 , wherein the 3′ hybridization domain comprises a homonucleotide stretch.

23. The method according to claim 1 , wherein the 3′ hybridization domain comprises a heteronucleotide stretch.