Method for retaining even coverage of short insert libraries
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.
1. A method of preparing a short insert library of adaptor-target-adaptor constructs, said method comprising:
(a) preparing a plurality of short target polynucleotide duplex fragments having a distribution of sequences, wherein the short target polynucleotide duplex fragments average less than 150 base pairs in length;
(b) treating the plurality of short target polynucleotide duplex fragments to phosphorylate the 5′ ends of each of the plurality of short 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 short, modified target polynucleotide duplex fragments wherein the plurality of short, modified target polynucleotide duplex fragments is purified, and
wherein all steps in step (b) are performed at a temperature of less than 65° C.; and
(c) ligating adaptor polynucleotides to both ends of each of the plurality of short, modified target polynucleotide duplex fragments to produce a short insert library of adaptor-target-adaptor constructs;
wherein the distribution of sequences of the short target polynucleotide duplex fragments in the short insert library of adaptor-target-adaptor constructs is essentially equal to the distribution of sequences of the short target polynucleotide duplex fragments.
2. The method according to claim 1 , wherein said method treating is carried out at a temperature of less than 55° C.
3. The method according to claim 2 , wherein said method treating is carried out at a temperature of less than 45° C.
4. The method according to claim 3 , wherein said method treating is carried out at a temperature of less than 40° C.
5. The method according to claim 1 , wherein said adaptor polynucleotides comprise an overhanging end complementary to the modified target polynucleotide duplex fragments.
6. The method according to claim 5 , wherein said overhanging end is treated to render the overhanging end resistant to exonucleolysis.
7. The method according to any preceding claim 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.
8. The method according to claim 7 further comprising the steps of:
(e) subjecting the products obtained in step (d) to denaturing conditions, thereby separating the extension products from strands of the adaptor-target-adaptor constructs; and
(f) carrying out a primer extension reaction, wherein a second primer oligonucleotide is annealed to the extension products from step (d), and extended by sequential addition of nucleotides to form additional copies of one strand of each of the adaptor-target-adaptor constructs, wherein the extension products and copies of the adaptor-target-adaptor constructs have common sequences at their 5′ ends and common sequences at their 3′ ends and collectively provide a library of template polynucleotide molecules.
9. The method according to claim 8 , wherein steps (d) to (f) are carried out one or more times to amplify the library of template polynucleotide molecules which have common sequences at their 5′ ends and common sequences at their 3′ ends.
10. The method according to claim 7 , wherein the first primer oligonucleotide used in the primer extension reaction anneals to a mismatched single stranded region on one strand of the adaptor polynucleotides.
11. The method according to claim 8 , wherein the first primer oligonucleotide anneals to adaptor portions of each of the adaptor-target-adaptor constructs and the second oligonucleotide primer anneals to a region of the extended strands produced by extension of the first oligonucleotide primers, this region being complementary to an adaptor portion of the adaptor-target-adaptor constructs.
12. The method according to claim 8 , wherein the first and second primer oligonucleotides are present in the same reaction.
13. The method according to claim 12 , wherein the first and second primer oligonucleotides have different nucleotide sequences.
14. The method according to claim 13 , wherein the first and second primer oligonucleotides are complementary to at least one base of the overhang on the target polynucleotide duplex fragments.
15. The method according to claim 14 , wherein the first and second primer oligonucleotides have been treated to render the primers resistant to exonucleolysis.
16. The method according to claim 1 , wherein the adaptor polynucleotides comprise regions of mismatched single stranded sequence.
17. The method according to claim 16 , wherein the mismatch is in one strand of the adaptor polynucleotides.
18. The method according to claim 16 , wherein the mismatch is in both strands of the adaptor polynucleotides.
19. The method according to claim 16 , wherein the mismatch forms a bubble, wherein the central sequence of the duplex strands is non-complementary.
20. The method according to claim 1 , wherein the target polynucleotide duplex fragments are DNA molecules.
21. The method according to claim 20 , wherein the target polynucleotide duplex fragments are fragments of genomic DNA.
22. The method according to claim 21 , wherein the target polynucleotide duplex fragments are fragments of a whole genome.
23. The method according to claim 20 or 21 or 22 , wherein the target polynucleotide duplex fragments are produced by fragmentation of at least one primary polynucleotide molecule.
24. The method according to claim 23 , wherein fragmentation of the at least one primary polynucleotide molecule is achieved by sonication, nebulization or hydrodynamic shearing.
25. The method according to claim 23 , wherein fragmentation of the at least one primary polynucleotide molecule is carried out by chemical or enzymatic fragmentation.
26. The method according to claim 1 , wherein the target polynucleotide duplexes are derived from cDNA.
27. The method according to claim 1 , wherein the target polynucleotide duplex fragments comprising greater than 50% A/T basepairs are maintained in the library of adaptor-target-adaptor constructs during the steps of the method.