Freshwater Targeted Locus (Loci)

We evaluated a commercially available protocol for simultaneous extraction of DNA, RNA and miRNA, which we adapted for freshwater microbial community samples that were collected on filters. Community composition, assessed by sequencing the V4 region of the 16S rRNA gene was compared to other commonly used, but independent DNA and RNA extraction protocols. We observed that choice of extraction protocol influenced perceived bacterial community composition, with strong method-dependent biases observed for specific phyla, such as the Verrucomicrobia. Microscopic analysis detected similar levels of Verrucomicrobia as the combined DNA/RNA extraction protocol we optimized, which detected this phylum at significantly higher levels than the other protocols we used. A smaller but significant shift in community composition was caused by the use of RNA protection agents, as well as by independent sequencing runs. Despite methodological biases, the origin of the water sample was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.