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.

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

“…It is uncertain whether these biases are specific to permafrost samples or whether they will be present in other types of environmental samples. It has already been suggested that these representational biases could be partly related to the GC content of the amplified genomes (Bodelier et al, 2009), which is in line with the fact that Actinobacteria (high G þ C) were systematically underestimated and Firmicutes (low G þ C) were overestimated following MDA in our study. These results clearly call for more caution in interpreting quantitative results from metagenomic studies that use whole-community genome amplification.…”

“…We also performed complementary analyses on the unamplified DNA to confirm and quantify the trends observed in the metagenomic libraries. Recent studies reported that MDA caused relatively little biases for the sequencing of single genomes (Pinard et al, 2006), but indicated that MDA caused biases in community composition when used on mixed communities (Abulencia et al, 2006;Bodelier et al, 2009). qPCR analyses indicated here that the bias in community composition introduced by MDA is quite strong.…”

Hadal biosphere: Insight into the microbial ecosystem in the deepest ocean on Earth

“…For the preparation of sufficient template DNA, amplified environmental DNA was used for the analyses. In fact, the composition of environmental DNA must be biased during the genome amplification; however, the technique has advantages in environmental molecular studies using insufficient amounts of DNA (26,27). The archaeal amoA gene clone analysis suggested the distinctive distribution of four major amoA subgroups (A, Ba, Bb, and D) along the water column (Figs.…”

Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

“…Three methanotrophic subgroups (Table 1) were quantified by pmoAbased quantitative PCR based on assays described elsewhere (22). The type Ia and II assays were carried out as described previously (7). For the type Ib assay, DNA standards were prepared by dilution of a known amount of PCR product amplified from a reference clone by using the 189-Mc468 primer set (22).…”