title: Whole-community genome amplification (WCGA) leads to compositional bias in methane-oxidizing communities as assessed by pmoA-based microarray analyses and QPCR.
abstract: Whole-genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single-cell genomes or whole-community metagenomes decreases the minimum amount of DNA needed for subsequent molecular community analyses. The resolution of profiling methods of environmental microbial communities will increase substantially by the use of the whole-community genome amplification (WCGA) procedure, assuming that the original community composition is not affected qualitatively as well as quantitatively. The present study aims to test if WCGA introduces a bias when applied to aerobic proteobacterial methanotrophic communities. For this, first, we subjected samples from freshwater lake sediment to WCGA, and amplified using primers targeting the pmoA gene coding for the α-subunit of the methane monooxygenase enzyme. Second, we analysed community composition using a diagnostic microarray and quantitative PCR (QPCR) assays. These methods clearly demonstrated that the WCGA amplification introduced a bias. Thus, numbers of γ-proteobacterial methanotrophs ('type Ia') increased significantly while the α-proteobacterial methanotrophs ('type II') were not amplified by the WCGA procedure. It is hypothesized that this bias is caused by the differences in GC content, which may compromise the efficiency of the MDA reaction.
authors.affiliation: Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Department of Microbial Wetland Ecology, Rijksstraatweg 6, 3631 AC, Nieuwersluis, the Netherlands. Centre for Infectious Disease Control, National Institute for health and the public environment (RIVM), Bilthoven, the Netherlands. Austrian Research Centers, Department of Bioresources, Seibersdorf, Austria. University of Natural Resources and Applied Life Sciences, Department of Biotechnology, Vienna, Austria.
authors.forename: Paul L E
authors.initials: PL
authors.lastname: Bodelier
authors.forename: Miranda
authors.initials: M
authors.lastname: Kamst
authors.forename: Marion
authors.initials: M
authors.lastname: Meima-Franke
authors.forename: Nancy
authors.initials: N
authors.lastname: Stralis-Pavese
authors.forename: Levente
authors.initials: L
authors.lastname: Bodrossy