Method for constructing a sequencing library based on a single-stranded DNA molecule and application thereof
A method for constructing a sequencing library based on a single-stranded DNA molecule is provided comprising: (1) forming a poly(C)n tail at a 3′-terminus of the single-stranded DNA molecule, to obtain a single-stranded DNA molecule with the poly(C)n tail with n representing a number of base C, and n being an integer ranging from 5 to 30; (2) obtaining a double-stranded DNA molecule by using an extension primer based on the single-stranded DNA molecule with the poly(C)n tail, with the extension primer comprising a H(G)m unit at a 3′-terminus thereof, H being base A, base T or base C, m being a number of base G, and m being an integer ranging from 5 to 15; and (3) ligating an adapter to one terminus of the double-stranded DNA molecule remote from the H(G)m unit, and amplifying the resulting ligation product to obtain an amplification product forming the sequencing library.
1. A method for constructing a sequencing library based on a single-stranded DNA molecule, comprising:
(1) forming a poly(C) n tail at a 3′-terminus of the single-stranded DNA molecule, to obtain a single-stranded DNA molecule with the poly(C) n tail with n representing a number of base C, and n being an integer ranging from 5 to 30;
(2) obtaining a double-stranded DNA molecule by using an extension primer based on the single-stranded DNA molecule with the poly(C) n tail, with the extension primer comprising a H(G) m unit at a 3′-terminus thereof, H being base A, base T or base C, m being a number of base G, and m being an integer ranging from 5 to 15; and
(3) ligating an adapter to one terminus of the double-stranded DNA molecule remote from the H(G) m unit, and amplifying the resulting ligation product to obtain an amplification product forming the sequencing library.
2. The method of claim 1 , wherein the single-stranded DNA molecule has an amount of at least 25 pg.
3. The method of claim 2 , wherein the single-stranded DNA molecule has an amount of 25 pg to 1000 pg.
4. The method of claim 1 , wherein n is an integer ranging from 15 to 25.
5. The method of claim 4 , wherein n is 20.
6. The method of claim 1 , wherein poly(C)n tail is formed by using a terminal deoxynucleotidyl transferase.
7. The method of claim 1 , wherein in step (2), the double-stranded DNA molecule is obtained by using a KAPA 2G Robust HS.
8. The method of claim 1 , wherein m is 9.
9. The method of claim 1 , wherein the extension primer has a sequence of SEQ ID NO:1.
10. The method of claim 1 , wherein the extension primer comprises a selection marker formed at a 5′-terminus of the extension primer.
11. The method of claim 1 , wherein the selection marker is a biotin.
12. The method of claim 11 , in step (3), further comprising purifying the resulting ligation product by using a bead specific to the biotin before the amplification.
13. The method of claim 1 , wherein step (3) further comprises:
(3-1) annealing single-stranded nucleic acids having nucleotide sequences of SEQ ID NOs: 2-3 respectively to form a semi-adapter;
(3-2) ligating the semi-adapter with one terminus of the double-stranded DNA molecule to obtain a double-stranded DNA molecule with the semi-adapter; and
(3-3) amplifying the double-stranded DNA molecule with the semi-adapter by using nucleotides of SEQ ID NOs: 4-7 as primers.
14. The method of claim 13 , wherein in step (3-2), ligating the semi-adapter with one terminus of the double-stranded DNA molecule is performed by using a Rapid DNA ligase.
15. The method of claim 14 , wherein in step (3), amplifying the resulting ligation product is performed by using a primer comprising an Index sequence.
16. The method of claim 15 , wherein the primer comprising an Index sequence is one selected from a set of indexed-primer consisting of SEQ ID NO: 8-19.
17. A method for determining a sequence data of a target region in a chromatin, comprising:
subjecting a chromatin to random fragmentation to obtain a chromatin sample with a length ranging from 200 bp to 500 bp;
subjecting the chromatin sample to a Chromatin Immunoprecipitation to obtain a double-stranded DNA sample by using an antibody specific to the target region;
subjecting the double-stranded DNA sample to a denaturation treatment to obtain a single-stranded DNA molecule;
constructing a sequencing library based on the single-stranded DNA molecule by a method according to claim 1 ;
sequencing the sequencing library to obtain a sequencing result; and
determining the sequence data of the target region in the chromatin based on the sequencing result.
18. A method for determining a methylation information in a genome, comprising:
subjecting at least a part of the genome to a bisulfate treatment to transform an unmethylated cytosine into a uracil and obtain a transformed genome sample;
subjecting the transformed genome sample to random fragmentation to obtain a double-stranded DNA sample with a length ranging from 200 bp to 500 bp;
subjecting the double-stranded DNA sample to a denaturation treatment to obtain a single-stranded DNA molecule;
constructing a sequencing library based on the single-stranded DNA molecule by a method according to claim 1 ;
sequencing the sequencing library to obtain a sequencing result; and
determining the sequence data of the target region in the chromatin based on the sequencing result.