Oligonucleotide replacement for di-tagged and directional libraries
View Patent ↗Transposomes and oligonucleotide replacement methods to make DNA libraries that have distinct 5′ and 3′ tags, and to make directional libraries that are enriched for a desired strand.
1. A method for adding a tag to the double-stranded product of a tagmentation reaction, comprising the steps of:
(a) providing a double-stranded target nucleic acid and a transposome having a transposase with two transposon end sequences: a “transferred strand” and a “non-transferred strand”;
(b) allowing the transposome to fragment the target nucleic acid, whereby the transferred strand is covalently transferred to a first strand of a fragment, and the non-transferred strand remains hybridized to the transferred strand;
(c) removing the non-transferred strand from the transferred strand;
(d) providing a replacement oligonucleotide that comprises a tag sequence, to hybridize to the transferred strand; and
(e) ligating the replacement oligonucleotide to the second strand of the fragment;
thereby generating a tagmentation product having a transferred strand and a replacement oligonucleotide.
2. The method of claim 1 , wherein one strand of the target nucleic acid is chemically modified.
3. The method of claim 2 , wherein the chemical modification is conversion of cytosines to uracils.
4. The method of claim 2 , further comprising the step of
(f) enriching for a desired strand.
5. The method of claim 4 , wherein step (f) comprises treating the fragment with an enzyme that preferentially cleaves a nondesired strand of the fragment.
6. The method of claim 4 , wherein step (f) is performed using a template-dependent polymerase that is sensitive to chemical modifications.
7. The method of claim 1 , wherein the transposome comprises a Tn5 transposase.
8. The method of claim 1 , wherein the transposome comprises a Mu transposase.
9. The method of claim 1 , wherein the transferred strand comprises the ME sequence
SEQ ID NO: 1)
MRWTGTGHWKAVGARACAV
or
(SEQ ID NO: 2)
NSHBGHSHDDRNGAKACAN,
but excluding
(SEQ ID NO: 3)
AGATGTGTATAAGAGACAG.
10. The method of claim 9 , wherein the transferred strand further comprises a tag sequence.
11. The method of claim 1 , wherein the non-transferred strand comprises
(SEQ ID NO: 20)
BTGTYTCBTN 1-10
or
(SEQ ID NO: 21)
NTGTMTCNTN 0-10 .
12. The method of claim 1 , wherein the non-transferred strand is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
13. The method of claim 1 , wherein step (e) further comprises an extension step.