Transposon end compositions and methods for modifying nucleic acids
First Claim
1. A method for generating and sequencing a library of DNA fragments having first and second tag sequences, comprising:
- (a) providing a target DNA;
(b) providing a plurality of transposome complexes, wherein(1) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a first polynucleotide comprising a Tn5-type transposon end sequence and a first tag sequence; and
(2) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a second polynucleotide comprising a Tn5-type transposon end sequence and a second tag sequence;
wherein the transposome complexes of (1) and (2) comprise different sequences;
(c) incubating the target DNA and transposome complexes under conditions whereby multiple transposon end sequences and associated tag sequences are inserted into the target DNA and the target DNA is fragmented into a multitude of double-stranded DNA fragments comprising one of the first or the second polynucleotides attached to each 5′
end of the DNA fragments;
(d) non-selectively amplifying the double-stranded DNA fragments; and
(e) sequencing the DNA fragments simultaneously in a single multiplex format.
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Accused Products
Abstract
The present invention provides methods, compositions and kits for using a transposase and a transposon end for generating extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro, then using a DNA polymerase for generating 5′- and 3′-tagged single-stranded DNA fragments without performing a PCR amplification reaction, wherein the first tag on the 5′-ends exhibits the sequence of the transferred transposon end and optionally, an additional arbitrary sequence, and the second tag on the 3′-ends exhibits a different sequence from the sequence exhibited by the first tag. The method is useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including processes for metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and comparative genomic sequencing (CGS), including massively parallel DNA sequencing (so-called “next-generation sequencing.).
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Citations
26 Claims
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1. A method for generating and sequencing a library of DNA fragments having first and second tag sequences, comprising:
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(a) providing a target DNA; (b) providing a plurality of transposome complexes, wherein (1) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a first polynucleotide comprising a Tn5-type transposon end sequence and a first tag sequence; and (2) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a second polynucleotide comprising a Tn5-type transposon end sequence and a second tag sequence; wherein the transposome complexes of (1) and (2) comprise different sequences; (c) incubating the target DNA and transposome complexes under conditions whereby multiple transposon end sequences and associated tag sequences are inserted into the target DNA and the target DNA is fragmented into a multitude of double-stranded DNA fragments comprising one of the first or the second polynucleotides attached to each 5′
end of the DNA fragments;(d) non-selectively amplifying the double-stranded DNA fragments; and (e) sequencing the DNA fragments simultaneously in a single multiplex format. - 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)
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26. A method for generating and sequencing a library of DNA fragments having first and second tag sequences, comprising:
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(a) providing a target DNA; (b) providing a plurality of transposome complexes, wherein (1) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a first polynucleotide comprising a Tn5-type transposon end sequence and a first tag sequence; and (2) at least one transposome complex comprises a hyperactive Tn5 transposase and at least a second polynucleotide comprising a Tn5-type transposon end sequence and a second tag sequence; wherein the transposome complexes of (1) and (2) exhibit different sequences; (c) incubating the target DNA and transposome complexes under conditions whereby multiple transposon ends and associated tag sequences are inserted into the target DNA and the target DNA is fragmented into a multitude of double-stranded DNA fragments that exhibit one of the first or the second tag sequences attached to each 5′
end of the DNA fragments;(d) joining a tag to the 3′
ends of the DNA fragments from step (c) to generate a library of di-tagged DNA fragments, each exhibiting a 5′
first or second tag sequence and a 3′
tag sequence,(e) non-selectively amplifying the library of di-tagged DNA fragments; and (f) sequencing the di-tagged DNA fragments simultaneously in a single multiplex format.
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Specification