Reducing GC bias in DNA sequencing using nucleotide analogs
First Claim
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1. A method of analyzing genomic DNA in a sample, the method comprising:
- (a) dividing the sample into a plurality of separate aliquots;
(b) amplifying fragments of the genomic DNA in the separate aliquots to produce a plurality of amplicons, wherein the amplifying is conducted with a population of dNTPs containing dNTP analogs, whereby a number of nucleotides in the amplicons are nucleotide analogs;
(c) removing nucleotide analogs incorporated into the amplicons to form gapped DNA;
(d) treating the gapped DNA such that gaps on opposite strands converge, thereby further fragmenting the genomic DNA;
(e) tagging DNA fragments in different aliquots with different oligonucleotide tags or tag combinations to form tagged fragments;
(f) combining the aliquots to produce a mixture of tagged fragments;
(g) obtaining sequence reads from tagged fragments in the mixture, wherein using the nucleotide analogs to create the gapped DNA has the effect of reducing GC bias in the sequence reads; and
(h) phasing heterozygous loci in the genomic DNA by a process that includes identifying sequence reads for DNA fragments having the same oligonucleotide tag.
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Abstract
The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.
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Citations
24 Claims
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1. A method of analyzing genomic DNA in a sample, the method comprising:
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(a) dividing the sample into a plurality of separate aliquots; (b) amplifying fragments of the genomic DNA in the separate aliquots to produce a plurality of amplicons, wherein the amplifying is conducted with a population of dNTPs containing dNTP analogs, whereby a number of nucleotides in the amplicons are nucleotide analogs; (c) removing nucleotide analogs incorporated into the amplicons to form gapped DNA; (d) treating the gapped DNA such that gaps on opposite strands converge, thereby further fragmenting the genomic DNA; (e) tagging DNA fragments in different aliquots with different oligonucleotide tags or tag combinations to form tagged fragments; (f) combining the aliquots to produce a mixture of tagged fragments; (g) obtaining sequence reads from tagged fragments in the mixture, wherein using the nucleotide analogs to create the gapped DNA has the effect of reducing GC bias in the sequence reads; and (h) phasing heterozygous loci in the genomic DNA by a process that includes identifying sequence reads for DNA fragments having the same oligonucleotide tag. - View Dependent Claims (2, 3, 4, 21)
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5. A method of preparing genomic DNA in a sample for sequence determination, comprising:
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(a) dividing the sample into a plurality of separate aliquots; (b) amplifying DNA in the separate aliquots in the presence of a mixture of nucleotides and nucleotide analogs having a ratio of dUTP to dTTP and a ratio of 5-methyl dCTP to dCTP, thereby producing a plurality of amplicons in which 0.05% to 4% of the cytosines and 0.05% to 4% of the thymines are replaced by a nucleotide analog; (c) contacting the amplicons with a reagent such that deoxyuracils incorporated into the amplicons are removed, leaving gaps; (d) contacting the amplicons with a reagent such that 5-methyl cytosines incorporated into amplicons are removed, leaving gaps; and (e) contacting the amplicons with a reagent such that gaps on opposite strands of the DNA converge, thereby creating DNA fragments. - View Dependent Claims (6, 7, 8, 9, 10, 22, 23, 24)
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11. A method for reducing GC bias when sequencing a sample of genomic DNA, wherein the sequencing comprises:
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(i) dividing the sample into a plurality of separate aliquots; (ii) amplifying fragments of the genomic DNA in the separate aliquots to produce amplicons, (iii) combining the amplicons from a plurality of the aliquots into a mixture; (iv) obtaining sequence reads from amplicons in the mixture; and (v) assembling sequence information for the genomic DNA by a process that includes identifying sequence reads for fragments originating in the same aliquot; wherein the method of reducing GC bias comprises; (a) conducting the amplifying in step (ii) in the presence of a combination of dNTPs containing nucleotide analogs, such that some of the nucleotides incorporated into the amplicons are nucleotide analogs; (b) removing nucleotide analogs incorporated into the amplicons to form gapped DNA; and (c) treating the gapped DNA such that gaps on opposite strands converge, thereby producing cleaved fragments for sequencing; wherein using the nucleotide analogs to produce the cleaved fragments has the effect of reducing GC bias in the sequencing. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification