Methods for improving genome assemblies
AssigneeLos Alamos National Security, LLC
InventorsXiaojing Zhang, Karen Walston Davenport, Lance Duane Green, Shunsheng Han
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Abstract
Advances in sequencing technologies have dramatically reduced costs in producing high quality draft genomes. There are still many contigs and possible misassembled regions in those draft genomes. Described herein are methods for improving the quality of sequencing techniques, and particularly methods for overcoming the loading bias inherent in, for instance, the PacBio sequencing process. Compared to Sanger sequencing technology, the herein described method is not only cost-effective but also can close gaps greater than 2.5 Kb in a single round of reactions. It can also sequence through high GC regions and difficult secondary structures such as hairpin loops.
Claims20
A method of sequencing a pool of at least two amplicons having different lengths, the method comprising:
- mixing an amount of a first amplicon with an amount of a second amplicon, wherein the amounts of the first and second amplicons are selected so there is a molar excess of the longer of the two amplicons in the resultant pooled amplicons;
- and
- subjecting the pooled amplicons to a nucleic acid sequencing reaction.
An improved method for single-molecule real-time (SMRT) sequencing a pool of amplicons having different lengths, wherein the improvement comprises adjusting the amount of at least two of the amplicons included in the pool using the following formula:
- Volume=[PCR size (Kb)]2×[10 ng/PCR concentration (ng/μl)].
A method for gap-filling sequencing of at least one amplicon, comprising:
- subjecting the amplicon to serial sequencing to produce a series of subreads of the same amplicon template;
- selecting a subset of the subreads based on the accuracy of the sequence of a portion of the amplicon;
- and
- using the sequences of the subset of subreads to assemble a consensus sequence for the amplicon.
Dependencies are inferred from the claim text.