Method of library preparation avoiding the formation of adaptor dimers

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.