Evaluating high-throughput sequencing (HTS) data of microalgae living in melting snow: improvements and limitations

We evaluated the application of high-throughput sequencing (HTS) on the extremophilic habitat of melting snow fields during the Alpine summer season. Closely related members of the Chlamydomonadaceae (Chlorophyceae) are the dominant organisms in these cryophilic communities. The markers 18S rDNA and ITS2 were used to reveal the diversity of the eukaryotic phototrophs. We found that correct OTU assignments strongly depend on the quality of the available reference databases. Moreover, the sole use of one marker can cause misleading results, either because of its low resolution (18S rDNA) or the scarcity of reference sequences (ITS2). We improved the taxonomic assignments of the 18S rDNA and ITS2 HTS data sets by generating additional reference sequences of the locally abundant taxa, and by light microscopic observations of the field samples. Furthermore, we manually inspected the alignments of representative sequences of the most abundant OTUs with their closest reference sequence, as well as performed ITS2 secondary structure analyses. Our evaluation reveals that HTS output needs to be thorougly checked when the studied habitats or groups of organisms are poorly represented in publicly available molecular databases. Here we present an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two-molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended for such studies.