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Next Article

Selective Whole Genome Amplification for Re-Sequencing Target Microbial Species from Complex Natural Samples

Aaron R. Leichty and Dustin Brisson
Genetics Early online August 5, 2014; https://doi.org/10.1534/genetics.114.165498
Aaron R. Leichty
University of Pennsylvania
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Dustin Brisson
University of Pennsylvania
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  • For correspondence: dbrisson@sas.upenn.edu
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Abstract

Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently-pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The Selective Whole Genome Amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly-processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in greater than 105 fold amplification of the target genomes with less than 6.7 fold amplification of the background. SWGA treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput re-sequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs.

  • culture-free genome sequencing
  • host-parasite genomics
  • microbial genomics
  • phi29
  • population genomics
  • Received April 21, 2014.
  • Accepted July 22, 2014.
  • Copyright © 2014, The Genetics Society of America
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PUBLICATION INFORMATION

Volume 208 Issue 4, April 2018

Genetics: 208 (4)

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  • Methods, Technology, & Resources

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Selective Whole Genome Amplification for Re-Sequencing Target Microbial Species from Complex Natural Samples

Aaron R. Leichty and Dustin Brisson
Genetics Early online August 5, 2014; https://doi.org/10.1534/genetics.114.165498
Aaron R. Leichty
University of Pennsylvania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dustin Brisson
University of Pennsylvania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: dbrisson@sas.upenn.edu
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Citation

Selective Whole Genome Amplification for Re-Sequencing Target Microbial Species from Complex Natural Samples

Aaron R. Leichty and Dustin Brisson
Genetics Early online August 5, 2014; https://doi.org/10.1534/genetics.114.165498
Aaron R. Leichty
University of Pennsylvania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dustin Brisson
University of Pennsylvania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: dbrisson@sas.upenn.edu

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The Genetics Society of America (GSA), founded in 1931, is the professional membership organization for scientific researchers and educators in the field of genetics. Our members work to advance knowledge in the basic mechanisms of inheritance, from the molecular to the population level.

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