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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Ekologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Ecology (hsv//eng)

Nyckelord

*Biodiversity

*Environmental Microbiology

Archaea/*classification/*genetics

Computational Biology

DNA Primers/*genetics

DNA

Archaeal/chemistry/genetics

High-Throughput Nucleotide Sequencing/methods

Metagenomics/*methods

Molecular Sequence Data

Polymerase Chain Reaction/methods

RNA

Archaeal/genetics

RNA

Ribosomal

16S/*genetics

Sensitivity and Specificity

Freshwater ecology

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)