Method for retaining even coverage of short insert libraries
First Claim
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, andwherein 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.
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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.
43 Citations
27 Claims
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1. A method of preparing a short insert library of adaptor-target-adaptor constructs, said method comprising:
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(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, andwherein 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification