title: Selective whole genome amplification for resequencing target microbial species from complex natural samples.
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 >10(5)-fold amplification of the target genomes with <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 resequencing 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.
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018.
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Leichty
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 dbrisson@sas.upenn.edu.
authors.forename: Dustin
authors.initials: D
authors.lastname: Brisson
mesh_terms: D000818:Animals; D025065:Borrelia burgdorferi / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015723:Gene Library; D016680:Genome, Bacterial; D021141:Nucleic Acid Amplification Techniques; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial
keywords: culture-free genome sequencing; host-parasite genomics; microbial genomics; phi29; population genomics
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