ERROR-PROOF NUCLEIC ACID LIBRARY CONSTRUCTION METHOD AND KIT
A method and kit for constructing a barcoded single-stranded DNA library are disclosed. The method includes preparing single-stranded DNA molecules each having a dephosphorylated 5′ end, ligating a first adaptor to a 3′ end of each single-stranded DNA molecule, and synthesizing a complementary strand of each single-stranded DNA molecule ligated to the first strand of the first adaptor. The kit includes the first adaptor having a first strand, which includes, from a 5′ end to a 3′ end, a phosphate group, a barcode sequence, and a first primer recognition sequence. The kit also includes a DNA ligase for a ligation between the 5′ end of the first strand of the first adaptor to each single-stranded DNA molecule, and a first primer for the synthesis of the complementary strand. The method allows for analysis of rare mutations and from nucleic acid samples of low quality and quantity.
1 . A kit for constructing a DNA library from a biological sample containing a plurality of nucleic acid sequences, comprising:
a first adaptor, having a first strand comprising, in a direction from a 5′ end thereof to a 3′ end thereof, a phosphate group, a barcode sequence, and a first primer recognition sequence, wherein the barcode sequence is configured to provide barcode information to each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor;
a DNA ligase, configured to allow a ligation between the 5′ end of the first strand of the first adaptor to a 3′ end of each of a plurality of single-stranded DNA molecules, wherein each of the plurality of single-stranded DNA molecules corresponds to one of the plurality of nucleic acid sequences in the biological sample; and
a first primer, comprising a sequence complementary to the first primer recognition sequence of the first adaptor and configured to allow for a single-strand extension reaction to thereby form a double-stranded DNA molecule corresponding to each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor.
2 . The kit of claim 1 , wherein the barcode sequence has a length of about 2-16 nt.
3 . The kit of claim 1 , wherein the first primer has a Tm of about 30-35° C.
4 . The kit of claim 3 , wherein the first primer comprises a sequence as set forth in SEQ ID NO: 914.
5 . The kit of claim 1 , further comprising a solid support, wherein:
the first strand of the first adaptor further comprises an immobilization portion at the 3′ end thereof, configured to allow immobilization of each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor at the 5′ end thereof to the solid support, wherein:
the immobilization portion comprises a first coupling partner, configured to be able to form a stable coupling with a second coupling partner attached to the solid support.
6 . The kit of claim 5 , wherein the stable coupling between the first coupling partner and the second coupling partner is a non-covalent binding.
7 . The kit of claim 6 , wherein the first coupling partner and the second coupling partner are respectively one and another of a coupling pair, selected from one of a biotin-streptavidin pair, a biotin-avidin pair, a biotin-anti-biotin antibody pair, a carbohydrate-lectin pair, or an antigen-antibody pair.
8 . The kit of claim 7 , wherein the first coupling partner comprises a biotin moiety, and the second coupling partner comprises a streptavidin moiety attached to a magnetic bead.
9 . The kit of claim 5 , wherein the immobilization portion further comprises a spacer between the first primer recognition sequence and the first coupling partner.
10 . The kit of claim 9 , wherein the spacer comprises at least one C3 spacer unit.
11 . The kit of claim 1 , wherein the first strand of the first adaptor further comprises an index sequence of about 1-8 nt, wherein:
the index sequence is between the phosphate group and the barcode sequence, or between the barcode sequence and the first primer recognition sequence; and
the index sequence is configured to provide index information for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor.
12 . The kit of claim 1 , wherein the first strand of the first adaptor further comprises a separator sequence of about 2-16 nt, wherein:
the separator sequence is disposed between the phosphate group and the barcode sequence and is configured to serve as a separation marker between the barcode sequence and each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor.
13 . The kit of claim 12 , wherein the separator sequence is further configured to provide index information for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor.
14 . The kit of claim 1 , wherein the first adaptor is single-stranded, and the DNA ligase comprises a single-stranded DNA ligase.
15 . The kit of claim 14 , wherein the single-stranded DNA ligase comprises at least one of CircLigase I or CircLigase II.
16 . The kit of claim 1 , wherein the first adaptor is partially double-stranded.
17 . The kit of claim 16 , wherein:
the first adaptor comprises a single-stranded segment at the 5′ end of the first strand; and
the DNA ligase comprises a single-stranded DNA ligase.
18 . The kit of claim 16 , wherein:
the first adaptor further comprises a second strand, comprising a first portion at a 5′ end thereof and a second portion at a 3′ end thereof, wherein:
the first portion of the second strand has a length of at least 1 nt and forms a double-stranded duplex with the 5′ end of the first strand;
the second portion has a length of at least 1 nt and forms a single-stranded overhang in the first adaptor; and
the DNA ligase comprises a bandage strand-facilitated DNA ligase.
19 . The kit of claim 18 , wherein the first adaptor comprises a set of adaptors, each configured such that a second portion of a second strand thereof comprises a random sequence.
20 . The kit of claim 18 , wherein the first adaptor comprises one or more adaptors, each configured such that a second portion of a second strand thereof comprises a specific sequence.
21 . The kit of claim 18 , wherein the bandage strand-facilitated DNA ligase comprises at least one of T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, or Taq Ligase.
22 . The kit of claim 5 , further comprising a second adaptor, configured to ligate to a free end of the double-stranded DNA molecule corresponding to each of the plurality of single-stranded DNA molecules immobilized to the solid support at an immobilized end thereof, wherein the second adaptor comprises a third strand and a fourth strand, wherein:
the fourth strand comprises:
a second primer recognition sequence, configured to provide a priming site for amplification of the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules; and
a phosphate group at a 5′ end thereof;
and
the third strand comprises a sequence complimentary to a 5′-end sequence of the fourth strand, and is configured to form a duplex with, and thereby to ensure a stability of, the 5′-end sequence of the fourth strand.
23 . The kit of claim 22 , wherein the fourth strand further comprises at least one functional sequence at a 5′ end of the second primer recognition sequence, wherein the at least one functional sequence comprises at least one of a second index sequence, a second barcode sequence, or a sequencing primer sequence.
24 . The kit of claim 22 , wherein the third strand further comprises, at a 5′ end of thereof, at least one of:
a cap structure, comprising a sequence that does not match with a 3′-end sequence of the fourth strand, and configured to avoid concatenation of the second adaptor in a ligation reaction;
an overhang sequence, forming a single-stranded segment for the second adaptor; or
a functional moiety.
25 . The kit of claim 22 , further comprising a pair of primers, configured to amplify the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules therethrough, wherein:
one of the pair of primers comprises a sequence corresponding to at least a portion of a sequence of the first primer in the first strand of the first adaptor; and
another of the pair of primers comprises a sequence corresponding to at least a portion of the second primer recognition sequence in the fourth strand of the second adaptor.
26 . A method for constructing a DNA library from a biological sample containing a plurality of nucleic acid sequences utilizing the kit according to claim 1 , comprising:
preparing a DNA sample from the biological sample, wherein the DNA sample comprises a plurality of single-stranded DNA molecules, each having a dephosphorylated 5′ end;
ligating a first strand of a first adaptor to a 3′ end of each of the plurality of single-stranded DNA molecules, wherein the first strand of the first adaptor comprises a phosphate group, a barcode sequence and a first primer recognition sequence along a direction from a 5′ end thereof to a 3′ end thereof; and
synthesizing a complementary strand for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to obtain a barcoded double-stranded DNA molecule corresponding thereto.
27 . The method according to claim 26 , wherein the plurality of nucleic acid sequences in the biological sample comprise a plurality of DNA sequences, and the preparing a DNA sample from the biological sample comprises:
shearing the plurality of DNA sequences into a plurality of DNA fragments; and
performing dephosphorylation reaction and dissociation reaction to obtain a plurality of single-stranded DNA molecules, each having a dephosphorylated 5′ end.
28 . The method according to claim 27 , wherein each of the plurality of DNA fragments has a size of about 100-300 bp.
29 . The method according to claim 27 , wherein the performing dephosphorylation reaction and dissociation reaction comprises:
at least one cycle of:
performing a dephosphorylation reaction; and
performing a dissociation reaction;
or
at least one cycle of:
performing a dissociation reaction; and
performing a dephosphorylation reaction.
30 . The method according to claim 26 , wherein the first adaptor comprises a single-stranded segment at a 5′ end of the first strand thereof, and the ligating a first strand of a first adaptor to a 3′ end of each of the plurality of single-stranded DNA molecules comprises:
performing a ligation reaction through a single-stranded DNA ligase such that the 3′ end of each of the plurality of single-stranded DNA molecules is ligated to the 5′ end of the first strand of the first adaptor.
31 . The method according to claim 26 , wherein the first adaptor further comprises a second strand, comprising a first portion at a 5′ end thereof and a second portion at a 3′ end thereof, wherein the first portion of the second strand has a length of at least 1 nt and forms a double-stranded duplex with the 5′ end of the first strand, and the second portion has a length of at least 1 nt and forms a single-stranded overhang in the first adaptor, and the ligating the 5′ end of a first strand of a first adaptor to a 3′ end of each of the plurality of single-stranded DNA molecules comprises:
performing a ligation reaction through a bandage strand-facilitated DNA ligase such that the 3′ end of each of the plurality of single-stranded DNA molecules is ligated with the 5′ end of the first strand of the first adaptor.
32 . The method according to claim 31 , wherein the first adaptor comprises a set of adaptors, each configured such that a second portion of a second strand thereof comprises a random sequence.
33 . The method according to claim 31 , wherein the first adaptor comprises one or more adaptors, each configured such that a second portion of a second strand thereof comprises a specific sequence.
34 . The method according to claim 26 , wherein the synthesizing a complementary strand for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to obtain a barcoded double-stranded DNA molecule corresponding thereto comprises:
annealing a first primer with each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor, wherein the first primer comprises a sequence complementary to the first primer recognition sequence in the first strand of the first adaptor; and
performing a single-strand extension reaction to form a double-stranded DNA molecule for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor.
35 . The method according to claim 34 , wherein the annealing a first primer with each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor comprises:
slowly altering a temperature of a reaction to a working temperature for the single-stranded extension reaction.
36 . The method according to claim 35 , wherein the first primer has a Tm of about 30-35° C., and the slowly altering a temperature of a reaction to a working temperature for the single-stranded extension reaction comprises:
increasing the temperature of the reaction from an original temperature of no more than ˜20° C. to the working temperature for the single-stranded extension reaction at a rate of no more than ˜3° C. per minute.
37 . The method according to claim 26 , wherein the first strand of the first adaptor further comprises an immobilization portion at the 3′ end thereof, configured to be able to form a stable coupling to a solid support, and the method further comprises, between the ligating a first strand of a first adaptor to a 3′ end of each of the plurality of single-stranded DNA molecules and the synthesizing a complementary strand for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to obtain a barcoded double-stranded DNA molecule corresponding thereto:
immobilizing each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to the solid support via the stable coupling between the immobilization portion and the solid support.
38 . The method according to claim 37 , wherein the immobilization portion comprises a first coupling partner, configured to be able to stably bind to a second coupling partner attached to the solid support, wherein:
the first coupling partner comprises a biotin moiety;
the second coupling partner comprises at least one of a streptavidin moiety, an avidin moiety, or an anti-biotin antibody; and
the solid support comprises at least one of a magnetic bead, a filter, a resin bead, a nanosphere, a plastic surface, a microtiter plate, a glass surface, a slide, a membrane, or a matrix.
39 . The method according to claim 37 , further comprising, after the synthesizing a complementary strand for each of the plurality of single-stranded DNA molecules ligated to the first strand of the first adaptor to obtain a barcoded double-stranded DNA molecule corresponding thereto:
ligating a second adaptor to a free end of the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules immobilized to the solid support at an immobilized end thereof, wherein the second adaptor comprises a third strand and a fourth strand, wherein:
the fourth strand comprises:
a second primer recognition sequence, configured to provide a priming site for amplification of the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules; and
a phosphate group at a 5′ end thereof;
and
the third strand comprises a sequence complimentary to a 5′-end sequence of the fourth strand, and is configured to form a duplex with, and thereby to ensure a stability of, the 5′-end sequence of the fourth strand.
40 . The method according to claim 39 , further comprising:
performing a PCR reaction to thereby amplify the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules.
41 . The method according to claim 40 , further comprising, between the ligating a second adaptor to a free end of the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules immobilized to the solid support at an immobilized end thereof and the performing a PCR reaction to thereby amplify the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules:
eluting the double-stranded DNA molecule corresponding to the each of the plurality of single-stranded DNA molecules from the solid support.