METHODS FOR PROCESSING NUCLEIC ACID SAMPLES
The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules and cDNA and/or DNA libraries using modified reverse transcriptases.
1 . A method for preparing a complementary deoxyribonucleic acid molecule comprising:
(a) fragmenting a ribonucleic molecule to yield a plurality of RNA fragments;
(b) removing a 3′-phosphate group, a 2′-phosphate group, and cyclic 2′3′ phosphate from one or more of said RNA fragments, thereby generating one or more dephosphorylated fragmented RNAs;
(c) adding a poly-A tail to said one or more dephosphorylated fragmented RNAs;
(d) adding, to said one or more dephosphorylated fragmented RNAs:
i. a primer adapter comprising an oligo-T sequence;
ii. an acceptor adapter; and
iii. a non-naturally occurring R2 enzyme having at least 95% identity to SEQ ID NO: 20 and a processivity of 20 nucleotides or longer wherein said non-naturally occurring R2 enzyme reverse transcribes a sequence from said one or more dephosphorylated fragmented RNAs in a 3′ to 5′ order, wherein said R2 enzyme jumps to a 3′-end of said acceptor adapter upon reaching the 5′ end of said one or more dephosphorylated fragmented RNAs.
2 . The method of claim 1 , wherein said acceptor adapter comprises a nucleotide analogue.
3 . The method of claim 2 , wherein said nucleotide analogue is at the 5′ end of said acceptor adapter.
4 . The method of claim 3 , wherein said nucleotide analogue is at the 5′ end of said primer adapter.
5 . The method of claim 3 , further comprising removing one or more non-annealed primer-adapter of (d)(i) prior to adding said non-naturally occurring R2 enzyme and/or wherein said one or more non-annealed primer-adapter is removed with an immobilized poly A oligo and/or, wherein said acceptor adapter comprises a 3′-dideoxy nucleotide at the acceptor-adapter 3′-end.