TRANSPOSOME ENABLED DNA/RNA-SEQUENCING (TED RNA-SEQ)
Provided herein are methods, compositions, and kits for generating a nucleic acid library. In various embodiments provided herein, transposomes comprising transposases are used in forming dual-end tagged nucleic acid molecules for downstream amplification and nucleic acid molecule processing steps.
1 . A composition comprising a transposome having a polypeptide transposase and a nucleic acid comprising a mosaic end and a first tag, a reverse transcriptase, an oligonucleotide comprising a mosaic end and a second tag.
2 . The composition of claim 1 , wherein the first tag and the second tag share a common nucleic acid sequence.
3 . The composition of claim 1 , wherein the first tag and the second tag comprise distinct nucleic acid sequences.
4 . The composition of claim 1 , comprising a transposome having a polypeptide transposase and a nucleic acid comprising a mosaic end and a second tag.
5 . The composition of claim 4 , further comprising a transposome having a polypeptide transposase and a nucleic acid comprising a mosaic end and a first tag.
6 . The composition of claim 1 , wherein the nucleic acid comprising a mosaic end and a first tag further comprises a tag that identifies a reaction batch.
7 . The composition of claim 5 , wherein the tag that identifies a reaction batch comprises a UMI.
8 . The composition of claim 1 , wherein the oligonucleotide comprises a poly-T segment.
9 . The composition of claim 1 , wherein the oligonucleotide comprises a gene-specific segment.
10 . The composition of claim 1 , comprising a ribonucleic acid sample.
11 . The composition of claim 1 , comprising a deoxyribonucleic acid sample.
12 . The composition of claim 1 , comprising a ribonucleic acid/deoxyribonucleic acid (RNA/DNA) hybrid molecule.
13 . The composition of claim 1 , comprising a template switching oligonucleotide (TSO).
14 . The composition of claim 13 , wherein the TSO comprises a first tag and a mosaic end.
15 . The composition of claim 1 , comprising a protease.
16 . The composition of claim 15 , wherein the protease is active at a temperature no more than 65° C. and inactive at a temperature greater than 65° C.
17 . The composition of claim 1 , comprising a polymerase.
18 . The composition of claim 17 , wherein the polymerase is a DNA polymerase.
19 . The composition of claim 17 , wherein the polymerase is a thermostable polymerase.
20 . The composition of claim 1 , comprising a ligase.
21 . The composition of claim 1 , comprising an enzyme for an amplification reaction of a nucleic acid molecule.
22 . The composition of claim 1 , comprising an oligonucleotide comprising a complementary segment to a portion of the first tag or the second tag.
23 . The composition of claim 1 , wherein an oligonucleotide comprises a sequence adapter.
24 . The composition of claim 1 , wherein the transposase is a Tn transposase, an MuA transposase, or a Vibhar transposase.
25 . The composition of claim 24 , wherein the Tn transposase is selected from Tn3, Tn5, Tn7, and Tn10.
26 . The composition of claim 1 , comprising a dual-end tagged RNA/DNA hybrid molecule comprising 5′ overhang tags at 5′ ends, wherein the individual 5′ overhang tag comprises a first tag or a second tag.
27 . The composition of claim 13 , comprising a blunt ended DNA/RNA hybrid molecule comprising complementary segments to the 5′ overhang tags from 3′ recessed ends of the dual-end tagged RNA/DNA hybrid molecule.
28 . The composition of claim 27 , comprising the blunt ended DNA/RNA hybrid molecules comprising sequence adapters at ends of the blunt-ended DNA/RNA hybrid molecules.
29 . The composition of claim 27 , comprising a sequence library comprising a plurality of blunt ended DNA molecules comprising sequence adapters at ends of the blunt-ended DNA molecules.