Novel primers for 16S rRNA-based archaeal community analyses in environmental samples
2011 (English)In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, no 1, 12-18 p.Article in journal (Refereed) Published
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 165 rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340E-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.
Place, publisher, year, edition, pages
2011. Vol. 84, no 1, 12-18 p.
Archaea, 16S rRNA, PCR, Primer design, Community composition
IdentifiersURN: urn:nbn:se:uu:diva-148929DOI: 10.1016/j.mimet.2010.10.001ISI: 000286848600003OAI: oai:DiVA.org:uu-148929DiVA: diva2:403908