Method for retaining even coverage of short insert libraries

Patent metadata

Field	Value
Publication number	US-9902994-B2
Application number	US-201414559548-A
Country	US
Kind code	B2
Filing date	Dec 3, 2014
Priority date	Aug 24, 2006
Publication date	Feb 27, 2018
Grant date	Feb 27, 2018

Abstract

The invention relates to a method of preparing a library of template polynucleotides with uniform sequence representation and to use of a library of templates prepared using this method for solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, which contains even representation of all the fragments present in a starting sample of nucleic acid before fragmentation. The invention is especially applicable to the preparation of short insert libraries, where the sample fragments are less than 150 base pairs in length.

First claim

The invention claimed is:

1. A method of solid-phase nucleic acid amplification of template polynucleotide molecules, the method comprising:
(a) preparing a library of template polynucleotide molecules with common sequences as their 5′ ends and common sequences at their 3′ ends by:
(1) providing a plurality of target polynucleotide duplex fragments having a distribution of sequences, and having an average fragment length of less than 150 base pairs;
(2) treating the plurality of target polynucleotide duplex fragments to phosphorylate the 5′ ends of each of the plurality of target polynucleotide duplex fragments and to incorporate a single nucleotide overhang at each of the 3′ ends of each of the plurality of short target polynucleotide duplex fragments, wherein the treating produces a plurality of modified target polynucleotide duplex fragments,
wherein all steps in step (2) are performed at a temperature of less than 65° C.; and

(3) ligating adaptor polynucleotides to both ends of each of the plurality of modified target polynucleotide duplex fragments to produce a library of template polynucleotide molecules;
wherein the distribution of sequences in the library of template polynucleotide molecules is essentially equal to the distribution of sequences of the short target polynucleotide duplex fragments; and

(b) amplifying by solid-phase amplification reaction the template polynucleotide molecules.

2. The method according to claim 1 , wherein said template polynucleotide molecules are amplified in an emulsion.

3. The method according to claim 1 , wherein said template polynucleotide molecules are amplified with a single primer immobilized on a surface.

4. The method according to claim 3 , wherein the surface is a bead or microsphere.

5. The method according to claim 1 , wherein said template polynucleotide molecules are amplified using forward and reverse amplification primers immobilized to a surface.

6. The method according to claim 5 , wherein said template polynucleotide molecules are amplified by solid-phase isothermal amplification.

7. A method of preparing a library of adaptor-target-adaptor constructs that is representative of a pool of target fragments, said method comprising:
(a) providing a plurality of target polynucleotide duplex fragments having a distribution of sequences, wherein the target polynucleotide duplex fragments average less than 150 base pairs in length;
(b) treating the plurality of target polynucleotide duplex fragments to phosphorylate the 5′ ends of each of the plurality of target polynucleotide duplex fragments and to incorporate a single nucleotide overhang at each of the 3′ ends of each of the plurality of target polynucleotide duplex fragments, wherein the treating produces a plurality of modified target polynucleotide duplex fragments,
wherein each step in step b) is performed at a temperature of less than 65° C.; and
(c) ligating adaptor polynucleotides to both ends of each of the plurality of modified target polynucleotide duplex fragments to produce a short insert library of adaptor-target-adaptor constructs;
wherein the target polynucleotide duplex fragments comprising greater than 50% A/T base pairs are maintained in the library of adaptor-target-adaptor constructs during the steps of the method.

8. The method according to claim 7 , wherein said treating is carried out at a temperature of less than 55° C.

9. The method according to claim 7 , wherein said adaptor polynucleotides comprise an overhanging end complementary to the modified target polynucleotide duplex fragments.

10. The method according to claim 9 , wherein said overhanging end is treated to render the overhanging end resistant to exonucleolysis.

11. The method according to claim 7 , further comprising the step of:
(d) carrying out a primer extension reaction, wherein a first primer oligonucleotide is annealed to an adaptor portion of each of the adaptor-target-adaptor constructs and extended by sequential addition of nucleotides to produce extension products complementary to at least one strand of each of the adaptor-target-adaptor constructs, wherein the extension products have common sequences at their 5′ ends.

12. The method according to claim 7 , wherein the target polynucleotide duplex fragments are DNA molecules.

13. The method according to claim 12 , wherein the target polynucleotide duplex fragments are fragments of genomic DNA or fragments of a whole genome.

14. The method according to claim 7 , wherein the target polynucleotide duplex fragments are produced by fragmentation of at least one primary polynucleotide molecule.

15. The method of claim 14 , wherein the fragmentation is performed by sonication, nebulization, hydrodynamic shearing, chemical fragmentation, or enzymatic fragmentation.

16. The method according to claim 7 , wherein the target polynucleotide duplex fragments are derived from cDNA.

17. The method according to claim 7 , further comprising amplifying the adaptor-target-adaptor constructs by a solid-phase amplification reaction.

18. The method according to claim 17 , wherein said adaptor-target-adaptor constructs are amplified in an emulsion.

19. The method of claim 17 , wherein the solid-phase amplification reaction employs a single primer immobilized on a surface or forward and reverse amplification primers immobilized on a surface.

20. The method according to claim 19 , wherein the surface is a bead or microsphere.

21. The method according to claim 19 , wherein said adaptor-target-adaptor constructs are amplified by solid-phase isothermal amplification.

Assignees

• Illumina Cambridge Ltd

Inventors

• Gormley Niall Anthony
• Smith Melanie Anne

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q2533/101

  Primer extension · CPC title