Method of library preparation avoiding the formation of adaptor dimers

• US 9,328,378 B2
• Filed: 07/30/2007
• Issued: 05/03/2016
• Est. Priority Date: 07/31/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Earnshaw, David James, Bignell, Helen Rachel, Smith, Melanie Anne
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US12/309,772
• Publication Number:
  US 20100167954A1
• Time in Patent Office: 3,200 Days
• Field of Search: None
• US Class Current: 1/1
• 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) providing blunt end target polynucleotide duplexes; and

performing a tailing reaction to attach a single nucleotide overhang to the 3′

ends of the blunt end 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 the single nucleotide overhang attached to the target polynucleotide duplex, not extending beyond the single nucleotide overhang, and wherein a first primer of the two primer oligonucleotides is between 21-100 nucleotides in length and comprises a 5′

sequence having the sequence of nucleotides 1-21 of SEQ ID NO;

6; and

(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. The method of claim 1, wherein a second primer of the two primer oligonucleotides is between 20-100 nucleotides in length and comprises a 5′

sequence having the sequence of nucleotides 1-20 SEQ ID NO;

5.

3. The method of claim 1, wherein the 5′

sequence of the first primer comprises the sequence 5′

-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3′

(SEQ ID NO;

6).

4. The method of claim 2, wherein the 5′

sequence of the second primer comprises the sequence 5′

-AATGATACGGCGACCACCGAGATCTACACTCTTTCC CTACACGACGCTCTTCCGATCT-3′

(SEQ ID NO;

5).

5. The method of claim 1, wherein the product of step (d) is subjected to solid phase amplification.

6. The method of claim 1, wherein providing blunt end target polynucleotide duplexes comprises fragmentation of the primary polynucleotide molecules by mechanical fragmentation.

7. The method of claim 1, wherein providing blunt end target polynucleotide duplexes comprises fragmentation of the primary polynucleotide molecules by chemical or enzymatic fragmentation.

8. The method of claim 1, wherein the one or more primary polynucleotide molecules are human genomic DNA molecules.

9. The method of claim 1, further comprising the steps of (e) preparing clusters from the library of template polynucleotide molecules;

and (f) sequencing the clusters by sequencing by synthesis.

10. The method of claim 1, wherein the first primer of the two primer oligonucleotides is between 40-100 nucleotides in length.

11. The method of claim 1, wherein the first primer of the two primer oligonucleotides is between 21-40 nucleotides in length.

12. The method of claim 2, wherein the second primer of the two primer oligonucleotides is between 40-100 nucleotides in length.

13. The method of claim 2 wherein the second primer of the two primer oligonucleotides is between 20-40 nucleotides in length.