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Systematic evaluation of bias in microbial community profiles induced by whole genome amplification

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Original languageEnglish
Pages (from-to)643-657
Number of pages15
JournalEnvironmental Microbiology
Volume16
Issue number3
DOIs
Publication statusPublished - Mar 2014

Abstract

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (

    Research areas

  • MULTIPLE DISPLACEMENT AMPLIFICATION, POLYMERASE-CHAIN-REACTION, ISOTHERMAL DNA AMPLIFICATION, RIBOSOMAL-RNA, SP-NOV., GEN. NOV., HIGH-THROUGHPUT, SINGLE CELLS, SOIL, POPULATIONS

ID: 21492049