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J Microbiol Methods. 2011 Jan;84(1):12-8. doi: 10.1016/j.mimet.2010.10.001. Epub 2010 Oct 19.

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples.

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1
Department of Ecology & Genetics, Limnology, Uppsala University, Norbyvägen 18D, SE-75236 Uppsala, Sweden.

Abstract

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.

PMID:
20940022
DOI:
10.1016/j.mimet.2010.10.001
[Indexed for MEDLINE]

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