Methods for obtaining a sequence
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsDmitry Pushkarev, Stephen R. Quake, Ayelet Voskoboynik, Michael Kertesz
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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.
Claims143
A method for relating sequence components in a target polynucleotide, the method comprising:
- (a) providing at least one sample comprising one or more target polynucleotides each adjacent to a known nucleotide sequence at at least one end, wherein each target polynucleotide comprises one or more sequence segments with a sequence segment length, and wherein the known nucleotide sequence is capable of serving as an amplification target;
- (b) partitioning the sample into a finite plurality of partitions such that at least a fraction of the sequence segments in at least one partition is estimated to be present as a single copy in the at least one partition with a likelihood that is less than 99.999%;
- (c) subjecting the partition to polynucleotide fragmentation thereby generating a set of fragments;
- (d) obtaining partial or complete sequence information from at least a subset of the fragments;
- and
- (e) relating sequence information from a plurality of fragments into a subset of the one or more target polynucleotides.
A method for relating sequence components in a target polynucleotide, the method comprising:
- (a) providing at least one sample comprising one or more target polynucleotides each adjacent to a known nucleotide sequence at at least one end, wherein each target polynucleotide comprises one or more sequence segments with a sequence segment length of greater than 30 bp, and wherein the known nucleotide sequence is capable of serving as an amplification target;
- (b) partitioning the sample into a finite plurality of partitions such that at least a fraction of the sequence segments in at least one partition is estimated to be present as a single copy in the at least one partition with a likelihood that is less than 100%;
- (c) subjecting the partition to polynucleotide fragmentation thereby generating a set of fragments;
- (d) obtaining partial or complete sequence information from at least a subset of the fragments;
- and
- (e) relating sequence information from a plurality of fragments into a subset of the one or more target polynucleotides.
A method for relating sequence components in a target polynucleotide, the method comprising:
- (a) providing at least one sample comprising one or more target polynucleotides wherein each target polynucleotide comprises one or more sequence segments with a sequence length;
- (b) partitioning the sample into a finite plurality of partitions such that at least a fraction of the sequence segments in at least one partition is estimated to be present as a single copy in the at least one partition with a likelihood that is less than 99.999%;
- (c) subjecting the partition to polynucleotide fragmentation thereby generating a set of fragments;
- (d) obtaining partial or complete sequence information from the fragments;
- and
- (e) relating sequence information from a plurality of fragments into the target polynucleotides.
A method for relating sequence components in a target polynucleotide, the method comprising:
- (a) providing at least one sample comprising one or more target polynucleotides each adjacent to a known nucleotide sequence at at least one end, wherein each target polynucleotide comprises one or more sequence segments with a sequence segment length;
- (b) partitioning the sample into a finite plurality of partitions and subjecting at least one partition to exponential nucleic acid amplification thereby amplifying the one or more target polynucleotides into amplicons such that at least a fraction of the sequence segments originating from the at least one or more target polynucleotides or the amplicons that are exponentially amplified in the at least one partition is estimated to be present as a single copy prior to exponential nucleic acid amplification among the one or more target polynucleotides that get exponentially amplified in the at least one partition with a likelihood that is less than 99.999%;
- (c) subjecting the partition to polynucleotide fragmentation thereby generating a set of fragments;
- (d) obtaining partial or complete sequence information from at least a subset of the fragments;
- and
- (e) relating sequence information from a plurality of fragments into a subset of the one or more target polynucleotides.
A method for relating sequence components in a target polynucleotide, the method comprising:
- (a) providing at least one sample comprising one or more target polynucleotides each adjacent to a known nucleotide sequence at at least one end, wherein each target polynucleotide comprises one or more sequence segments with a sequence segment length of greater than 30 bp;
- (b) partitioning the sample into a finite plurality of partitions and subjecting at least one partition to exponential nucleic acid amplification thereby amplifying the one or more target polynucleotides into amplicons such that at least a fraction of the sequence segments originating from the at least one or more target polynucleotides or the amplicons that are exponentially amplified in the at least one partition is estimated to be present as a single copy prior to exponential nucleic acid amplification among the one or more target polynucleotides that get exponentially amplified in the at least one partition with a likelihood that is less than 100%;
- (c) subjecting the partition to polynucleotide fragmentation thereby generating a set of fragments;
- (d) obtaining partial or complete sequence information from at least a subset of the fragments;
- and
- (e) relating sequence information from a plurality of fragments into a subset of the one or more target polynucleotides.
A kit for relating sequence components in a target polynucleotide, comprising
- (a) a device comprising a substrate with one or more reaction volumes comprising one or more restriction endonucleases capable of polynucleotide fragmentation;
- (b) a device comprising a substrate with one or more reaction volumes comprising one or more oligonucleotides with a known sequence;
- and
- (c) a device comprising a substrate with one or more reaction volumes comprising one or more polymerases capable of polynucleotide synthesis.
A method of forming a linear nucleic acid molecule comprising a target polynucleotide with an adaptor oligonucleotide at each end, the method comprising the steps of:
- (a) providing a target polynucleotide and an intermediate polynucleotide, wherein the intermediate polynucleotide comprises at least two adaptor oligonucleotides;
- (b) forming a circular polynucleotide construct by combining the target polynucleotide with the intermediate polynucleotide at each end;
- (c) cleaving the circular polynucleotide construct within the intermediate polynucleotide to form a linearized molecule.
A composition capable of creating a linear nucleic acid molecule comprising a target polynucleotide with an adaptor oligonucleotide at each end for the identification and isolation of a target polynucleotide, the composition comprising:
- (a) the target polynucleotide;
- (b) an intermediate polynucleotide comprising at least two adaptor oligonucleotides;
- wherein the intermediate polynucleotide further comprises at least one or more elements that allow for circularization, linearization, identification, amplification or isolation.
Dependencies are inferred from the claim text.