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.
Claims17
A method for obtaining a sequence, the method comprising:
- ligating adapters to a plurality of nucleic acid fragments in a sample, each nucleic acid fragment having a length between 5 kilobases and 40 kilobases;
- quantifying the amount of nucleic acid fragments present in the sample to which adapters have been ligated on each end, thereby quantifying the amount of amplifiable nucleic acid fragments;
- determining a dilution factor based on the quantified amount of amplifiable nucleic acid fragments;
- partitioning the sample based on the dilution factor such that each partitioned portion comprises, on average, at least two amplifiable nucleic acid fragments that are a subset of unique sequences;
- performing a polymerase chain reaction on the partitioned nucleic acid fragments;
- sequencing the nucleic acid fragments to obtain sequence reads;
- and
- obtaining a sequence from the reads.
A method for obtaining a sequence, the method comprising:
- obtaining nucleic acid;
- fragmenting the nucleic acid, thereby generating nucleic acid fragments that have a length of 5 kilobases to 40 kilobases;
- ligating adapters to a portion of the nucleic acid fragments, wherein the portion of nucleic acid fragments with adapters ligated at each end are amplifiable nucleic acid fragments;
- quantifying an amount of the amplifiable nucleic acid fragments present in the sample;
- determining a dilution factor based on the quantified amount of amplifiable nucleic acid fragments;
- partitioning the nucleic acid fragments based on the dilution factor, wherein each partitioned portion comprises, on average, at least two amplifiable nucleic acid fragments that are a subset of unique sequences;
- amplifying the partitioned nucleic acid fragments by performing a polymerase chain reaction;
- attaching bar codes to the amplified nucleic acid fragments;
- sequencing the nucleic acid fragments to obtain bar coded sequence reads;
- and
- obtaining a sequence from the reads.
Dependencies are inferred from the claim text.