title: Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.
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 (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars.
authors.affiliation: Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Susana O L
authors.initials: SO
authors.lastname: Direito
authors.forename: Egija
authors.initials: E
authors.lastname: Zaura
authors.forename: Miranda
authors.initials: M
authors.lastname: Little
authors.forename: Pascale
authors.initials: P
authors.lastname: Ehrenfreund
authors.forename: Wilfred F M
authors.initials: WF
authors.lastname: Röling
mesh_terms: D001482:Base Composition; D015982:Bias; D044822:Biodiversity; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000737:chemistry; D016680:Genome, Bacterial / Q000235:genetics*; D008970:Molecular Weight; D021141:Nucleic Acid Amplification Techniques / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000592:standards; D017422:Sequence Analysis, DNA / Q000592:standards
chemical_list: D004269:DNA, Bacterial