Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads

J Microbiol Methods. 2011 Nov;87(2):208-12. doi: 10.1016/j.mimet.2011.08.003. Epub 2011 Aug 17.

Abstract

Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10 ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Bacterial / genetics*
  • Environmental Microbiology*
  • Genome, Bacterial*
  • Limit of Detection
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques / methods*
  • Sequence Analysis, DNA
  • Vibrio cholerae O1 / genetics*
  • Vibrio cholerae O1 / isolation & purification

Substances

  • DNA, Bacterial

Associated data

  • GENBANK/CP002555
  • GENBANK/CP002556