Whole-community genome amplification (WCGA) leads to compositional bias in methane-oxidizing communities as assessed bypmoA-based microarray analyses and QPCR
| Autor: | Miranda Kamst, Levente Bodrossy, Marion Meima-Franke, Nancy Stralis-Pavese, Paul L. E. Bodelier |
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| Rok vydání: | 2009 |
| Předmět: |
0303 health sciences
Microarray biology 030306 microbiology Methane monooxygenase Multiple displacement amplification Computational biology Agricultural and Biological Sciences (miscellaneous) Genome Molecular biology 03 medical and health sciences Real-time polymerase chain reaction Microbial ecology biology.protein Gene Ecology Evolution Behavior and Systematics GC-content 030304 developmental biology |
| Zdroj: | Environmental Microbiology Reports. 1:434-441 |
| ISSN: | 1758-2229 |
| DOI: | 10.1111/j.1758-2229.2009.00066.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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