Method of preparing libraries of template polynucleotides
The present invention relates to a method for preparing a library of template polynucleotides and use thereof in methods of solid-phase nucleic acid amplification. More specifically, the invention relates to a method for preparing a library of template polynucleotides that have common sequences at their 5′ ends and at their 3′ ends.
1. A method for sequencing a library of nucleic acids, comprising:
(a) obtaining a library of nucleic acids comprising a plurality of double-stranded polynucleotides having identical forked polynucleotide adapters at each end, wherein the forked polynucleotide adapters comprise partially complementary first and second polynucleotide strands that are annealed to each other, and wherein at least one of the first or second polynucleotide strands comprises a nucleotide sequence selected from SEQ ID NO:01, SEQ ID NO:02, or a nucleotide sequence complementary to SEQ ID NO:04; and
(b) sequencing the library of nucleic acids.
2. The method of claim 1 , wherein the sequencing comprises successive addition of nucleotides to a free 3′ hydroxyl group of a polynucleotide, and determining the nature of an added nucleotide after each addition.
3. The method of claim 1 , wherein the double-stranded polynucleotides of the plurality of double-stranded polynucleotides are different from one another.
4. The method of claim 1 , wherein at least one of the first and second polynucleotide strands comprises the nucleotide sequence complementary to SEQ ID NO:04.
5. The method of claim 4 , wherein one of the first and second polynucleotide strands comprises the nucleotide sequence of SEQ ID NO:02.
6. The method of claim 1 , wherein the first polynucleotide strand comprises the nucleotide sequence of SEQ ID NO:01.
7. The method of claim 6 , wherein the second polynucleotide strand comprises a nucleotide sequence of SEQ ID NO:02.
8. The method of claim 1 , further comprising amplifying the library of nucleic acids prior to step (b).
9. The method of claim 8 , wherein the amplifying is performed in solution.
10. The method of claim 8 , wherein the amplifying comprises a solid-phase nucleic acid amplification reaction on a solid support.
11. The method of claim 10 , wherein the solid support comprises glass, polyacrylamide, latex, dextran, polystyrene, polypropylene, gold, or silicon.
12. The method of claim 8 , wherein the amplifying is performed with a single primer.
13. The method of claim 12 , wherein the primer is bound to a surface.
14. The method of claim 1 , wherein a sequence of at least 10 consecutive nucleotides at the 5′ end of the first strand of the adapters and a sequence of at least 10 consecutive nucleotides at the 3′ end of the second strand of the adapters are not complementary to each other, such that a mismatched region of at least 10 consecutive nucleotides on each strand remains in single stranded form when the first and second polynuleotide strands are annealed.
15. The method of claim 14 , wherein a sequence of at least 20 consecutive nucleotides at the 5′ end of the first strand of the adapters and a sequence of at least 20 consecutive nucleotides at the 3′ end of the second strand of the adapters are not complementary to each other, such that a mismatched region of at least 20 consecutive nucleotides on each strand remains in single stranded form when the first and second polynucleotide strands are annealed.
16. The method of claim 1 , wherein the plurality of double-stranded polynucleotides comprises fragments of cDNA.
17. The method of claim 1 , wherein the plurality of double-stranded polynucleotides comprises fragments of genomic DNA.
18. The method of claim 17 , wherein the fragments of genomic DNA are derived from a single subject.
19. The method of claim 17 , wherein the fragments of genomic DNA are derived from a plurality of subjects.
20. The method of claim 17 , wherein the plurality of double-stranded polynucleotides comprises human nucleic acids.