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

Paula Juliana Pérez Chaparro, John Anthony Mcculloch, Louise Teixeira Cerdeira, Arwa Al-Dilaimi, Lena Lillian Canto De Sá, Rodrigo De Oliveira, Andreas Tauch, Vasco Ariston De Carvalho Azevedo, Maria Paula Cruz Schneider, Artur Luiz Da Costa da Silva

Research output: Contribution to journalArticleResearchpeer-review

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)208-212
Number of pages5
JournalJournal of Microbiological Methods
Volume87
Issue number2
DOIs
Publication statusPublished - 1 Nov 2011
Externally publishedYes

Keywords

  • Multiple displacement amplification
  • Short reads
  • Vibrio cholerae
  • Whole genome sequencing

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