Methods for obtaining a sequence
First Claim
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1. A method for analyzing a plurality of target polynucleotides, the method comprising:
- (a) providing a sample comprising a plurality of target polynucleotide fragments, wherein each target polynucleotide fragment has a length of 5 kb to 40 kb;
(b) performing a size selection on the plurality of target polynucleotide fragments to select 5-7 kb fragments;
(c) ligating amplification adaptors at each end of at least a portion of the 5-7 kb fragments to produce a library of amplifiable fragments, wherein the adaptors serve as primer binding sites;
(d) quantitating via qPCR to establish a number of amplifiable fragments in the library of amplifiable fragments;
(e) determining a dilution factor based on the number of amplifiable fragments in the library of amplifiable fragments;
(f) diluting and partitioning the library into a plurality of partitions based on the dilution factor such that each partition comprises, on average, a unique subset of 500 or more of the amplifiable fragments;
(g) amplifying via polymerase chain reaction the partitioned amplifiable fragments;
(h) subjecting the partitions to polynucleotide fragmentation thereby generating a set of smaller fragments; and
(i) obtaining partial or complete sequence information from at least a subset of the smaller fragments.
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Abstract
The invention generally relates to methods for obtaining a sequence, such as a consensus sequence or a haplotype sequence. In certain embodiments, methods of the invention involve determining an amount of amplifiable nucleic acid present in a sample, partitioning the nucleic acid based upon results of the determining step such that each partitioned portion includes, on average, a subset of unique sequences, sequencing the nucleic acid to obtain sequence reads, and assembling a consensus sequence from the reads.
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63 Claims
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1. A method for analyzing a plurality of target polynucleotides, the method comprising:
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(a) providing a sample comprising a plurality of target polynucleotide fragments, wherein each target polynucleotide fragment has a length of 5 kb to 40 kb; (b) performing a size selection on the plurality of target polynucleotide fragments to select 5-7 kb fragments; (c) ligating amplification adaptors at each end of at least a portion of the 5-7 kb fragments to produce a library of amplifiable fragments, wherein the adaptors serve as primer binding sites; (d) quantitating via qPCR to establish a number of amplifiable fragments in the library of amplifiable fragments; (e) determining a dilution factor based on the number of amplifiable fragments in the library of amplifiable fragments; (f) diluting and partitioning the library into a plurality of partitions based on the dilution factor such that each partition comprises, on average, a unique subset of 500 or more of the amplifiable fragments; (g) amplifying via polymerase chain reaction the partitioned amplifiable fragments; (h) subjecting the partitions to polynucleotide fragmentation thereby generating a set of smaller fragments; and (i) obtaining partial or complete sequence information from at least a subset of the smaller fragments. - View Dependent Claims (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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
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2. A method for analyzing a plurality of target polynucleotides, the method comprising:
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(a) providing at least one sample comprising a plurality of target polynucleotide fragments, wherein each target polynucleotide fragment has of a length of 5 kb to 40 kb; (b) performing a size selection on the plurality of target polynucleotide fragments to select 5-7 kb fragments; (c) ligating amplification adaptors to each end of at least a portion of the 5-7 kb fragments to produce a library of amplifiable fragments, wherein the adaptor serves as a primer binding site; (d) quantitating via qPCR to establish a number of amplifiable fragments in the library of amplifiable fragments; (e) determining a dilution factor based on the number of amplifiable fragments in the library of amplifiable fragments; (f) diluting and partitioning the library based on the dilution factor into a plurality of partitions such that each partition comprises, on average, a unique subset of 500 or more of the amplifiable fragments; (g) amplifying via a polymerase chain reaction the partitioned amplifiable fragments; (h) subjecting the partitions to polynucleotide fragmentation thereby generating a set of smaller fragments; (i) coupling a partition specific barcode to at least a subset of the smaller fragments; and (j) obtaining partial or complete sequence information from at least a subset of the smaller fragments coupled with the partition specific barcodes. - View Dependent Claims (63)
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Specification