METHOD OF LIBRARY PREPARATION AVOIDING THE FORMATION OF ADAPTOR DIMERS

• US 20100167954A1
• Filed: 07/30/2007
• Published: 07/01/2010
• Est. Priority Date: 07/31/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Smith, Melanie Anne, Earnshaw, David James, Bignell, Helen Rachel, Gormley, Niall Anthony
• Granted Patent: US 9,328,378 B2
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

The invention relates to a method of preparing a library of template polynucleotides which reduces and/or prevents the formation of adaptor-dimers. The invention also relates to the use of a library of templates prepared using the method of the invention 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 is substantially free of adaptor-dimers.

Claims

1. A method for generating a library of template polynucleotide molecules from one or more primary polynucleotide molecules, said method comprising:

(a) fragmenting said one or more primary polynucleotide molecules to produce target polynucleotide duplexes;

(b) ligating an adaptor polynucleotide construct to both ends of the target polynucleotide duplexes to generate combined ligated adaptor-target-adaptor sequences;

(c) preparing an amplification reaction comprising said combined ligated adaptor-target-adaptor sequences and at least two different primer oligonucleotides, wherein each of said at least two different primer oligonucleotides is complementary to at least a part of the adaptor polynucleotide construct sequence of the combined ligated adaptor-target-adaptor sequences, and complementary to at least one base of either the target polynucleotide duplexes, or an overhang attached to the target duplex, and wherein the two primer oligonucleotides have a modification that makes the primers resistant to exonucleolysis;

(d) performing an amplification reaction wherein said at least two different primer oligonucleotides anneal to complementary parts of the adaptor-target-adaptor sequences and are extended by sequential addition of nucleotides to generate a plurality of amplification products complementary to at least one strand of the combined ligated adaptor-target-adaptor sequences, wherein each of said plurality of amplification products has a first common sequence at its 5′

end and a second common sequence at its 3′

end, and wherein said plurality of amplification products comprises a library of template polynucleotide molecules.

2. A method for generating a library of template polynucleotide molecules from one or more primary polynucleotide molecules, said method comprising:

(a) fragmenting said one or more primary polynucleotide molecules to produce target polynucleotide duplexes;

(b) ligating an adaptor polynucleotide construct to both ends of the target polynucleotide duplexes to generate combined ligated adaptor-target-adaptor sequences, wherein said adaptor polynucleotide construct comprises a nucleic acid strand comprising at least one overhanging base that is modified to render the nucleic acid strand resistant to exonucleolysis;

(c) preparing an amplification reaction comprising said combined ligated adaptor-target-adaptor sequences and at least two different primer oligonucleotides, wherein each of said at least two different primer oligonucleotides is complementary to at least a part of the adaptor polynucleotide construct sequence of the combined ligated adaptor-target-adaptor sequences;

(d) performing an amplification reaction, wherein said at least two different primer oligonucleotides anneal to complementary parts of the combined ligated adaptor-target-adaptor sequences and are extended by sequential addition of nucleotides to generate a plurality of amplification products complementary to at least one strand of the combined ligated adaptor-target-adaptor sequences, wherein each of said plurality of amplification products has a first common sequence at its 5′

end and a second common sequence at its 3′

end, and wherein said plurality of amplification products comprises a library of template polynucleotide molecules.

26. The method of claim 2, wherein at least one of said primer oligonucleotides is longer than the ligated adaptor sequences in the 5′

-direction.

3. A method for generating a library of template polynucleotide molecules from one or more primary polynucleotide molecules, said method comprising:

(a) fragmenting said one or more primary polynucleotide molecules to produce target polynucleotide duplexes;

(b) ligating an adaptor polynucleotide construct to both ends of the target polynucleotide duplexes to generate combined ligated adaptor-target-adaptor sequences, wherein said adaptor polynucleotide comprises a nucleic acid strand comprising at least one overhanging base that is modified to render the nucleic acid strand resistant to exonucleolysis;

(c) preparing an amplification reaction comprising said combined ligated adaptor-target-adaptor sequences and at least two different primer oligonucleotides, wherein each of said at least two different primer oligonucleotides is complementary to at least a part of the adaptor polynucleotide construct sequence of the combined ligated adaptor-target-adaptor sequences, and complementary to at least one base of either the target polynucleotide duplexes, or an overhang attached to the target duplex;

(d) performing an amplification reaction, wherein said at least two different primer oligonucleotides anneal to complementary parts of the adaptor-target-adaptor sequences and are extended by sequential addition of nucleotides to generate a plurality of amplification products complementary to at least one strand of the combined ligated adaptor-target-adaptor sequences, wherein each of said plurality of amplification products has a first common sequence at its 5′

end and a second common sequence at its 3′

end and wherein said plurality of amplification products comprises a library of template polynucleotide molecules.

4. The method of claim 3, wherein fragmentation of the primary polynucleotide molecules is by mechanical fragmentation.

6. The method of claim 3, wherein fragmentation of the primary polynucleotide molecules is by chemical or enzymatic fragmentation.

9. The method of claim 3, wherein the one or more primary polynucleotide molecules are human genomic DNA molecules.

10. The method of claim 3, wherein the adaptor polynucleotide construct has a single nucleotide overhang at either the 5′

or 3′

terminus of each duplex strand and the target polynucleotide duplexes have complementary single nucleotide overhangs at the 5′

or 3′

terminus of each duplex strand.

13. The method of claim 10, wherein the two primer oligonucleotides have a 3′

nucleotide that hybridises to the single nucleotide overhang attached to the target DNA.

15. The method of claim 13, wherein the two primer oligonucleotides have a modification that makes the primers resistant to exonucleolysis.

16. The method of claim 15, wherein said exonuclease resistant modification is a phosphorothioate moiety, a methyl phosphonate moiety, or a 2′

-O-methyl moiety.

20. The method of claim 15, wherein the primer oligonucleotides are treated with an exonuclease prior to amplification.

21. The method of claim 3, wherein the sequential addition of nucleotides is carried out by a high fidelity DNA polymerase possessing exonuclease activity.

24. The method of claim 21, wherein said polymerase is Phusion™

DNA polymerase.

25. The method of claim 3, wherein at least one of said primer oligonucleotides is longer than the ligated adaptor sequences in both the 3′

and 5′

directions.

27. The method of claim 3 further comprising the steps of

(e) preparing clusters from the library of template polynucleotide molecules;

(f) sequencing the clusters by sequencing by synthesis.

28. A library of template polynucleotide molecules prepared by the method of claim 3.

29. An array comprising the library of template polynucleotide molecules of claim 28.

5. (canceled)

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11. (canceled)

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17. (canceled)

18. (canceled)

19. (canceled)

22. (canceled)

23. (canceled)

30. (canceled)

31. A method of reducing the population of adaptor dimers in a library of adaptor modified polynucleotide fragments, the method comprising amplification using primers complementary to at least one base of a polynucleotide fragment inserted between the adaptors, thereby reducing amplification of adaptor dimers.

32. The method of claim 31 wherein the adaptor is exonuclease resistant.

33. The method of claim 31 wherein the amplification primers are exonuclease resistant.