Methods of enriching a target sequence from a sequencing library using hairpin adaptors
Disclosed herein is a method for enriching a sequencing library comprising double-stranded nucleic acid fragments comprising preparing a library of double-stranded fragments having one or more adaptors at ends of the double-stranded fragment; denaturing the double-stranded fragments to form single-stranded fragments; and hybridizing an extension primer that binds to a target sequence of at least one insert in the library of double-stranded fragments and that does not bind to non-target sequences. In an embodiment, the adaptor is a hairpin adaptor, and extension from the extension primer using a polymerase with 5′ to 3′ exonuclease activity removes all or part of a sequence of the hairpin adaptor that is at least partially complementary to the amplification primer sequence. Each fragment may comprise an insert comprising double-stranded nucleic acid and a hairpin adaptor at the 5′ end of one or both strands of the double-stranded fragments. Hairpin adaptors may comprise an amplification primer sequence and a sequence at least partially complementary to the amplification primer sequence.
1 . An adaptor for use in preparing a nucleic acid sequencing library comprising:
a. a first strand having a 5′ end and a 3′ end; and
b. a second strand having a 5′ end and a 3′ end;
wherein a portion of the 3′ end of the first strand and a portion of the 5′ end of the second strand are complementary and form a first double-stranded region,
wherein a portion of the 5′ end of the first strand and a portion of the 3′ end of the second strand are non-complementary; and
wherein the portion of the 5′ end of the first strand comprises a second double-stranded region and a linker portion, wherein the second double-stranded region comprises an amplification primer sequence hybridized to a sequence complementary to the amplification primer sequence, wherein the linker portion is between the amplification primer sequence and the sequence complementary to the amplification primer sequence and wherein the linker portion is not degradable by an exonuclease.
2 . The adaptor of claim 1 , wherein the second double-stranded region and the linker portion comprise a hairpin structure.
3 . The adaptor of claim 1 , wherein the linker portion comprises a non-nucleic acid portion.
4 . The adaptor of claim 1 , wherein the first double-stranded region is at least 5 consecutive nucleotides.
5 . The adaptor of claim 1 , wherein the second double-stranded region is at least 5 consecutive nucleotides.
6 . The adaptor of claim 1 , wherein the 5′ end of the second strand is phosphorylated.
7 . The adaptor of claim 1 , wherein all cytosine bases in the first strand and in the second strand are methylated.
8 . The adaptor of claim 1 , wherein the adaptor further comprises a capture moiety.
9 . A method of preparing a nucleic acid sequencing library comprising:
a. producing a plurality of double-stranded nucleic acid fragments; and
b. attaching the adaptor of any one of claim 1-3 or 4-8 to at least one end of the plurality of double-stranded nucleic acid fragments.
10 . The method of claim 9 , wherein the adaptor is attached via tagmentation.
11 . The method of claim 9 , wherein the adaptor is attached via ligation.
12 . A kit for preparing a nucleic acid sequencing library comprising:
a. the adaptor of any one of claim 1-3 or 4-8 ;
b. at least one primer capable of hybridizing to a portion of the adaptor;
c. at least one enzyme; and
d. dNTPs.
13 . The kit of claim 12 , wherein the at least one enzyme has exonuclease activity.
14 . The kit of claim 12 , wherein the at least one enzyme is a polymerase.
15 . The kit of claim 12 , wherein at least one component is in a lyophilized or dried form.