In silico primer evaluation and taxonomic biases
Results of in silico PCR using the four primer pairs Stoeck (Stoeck et al., 2010), Wolf938 and Wolf964 (this study), and Bradley (Bradley et al., 2016) revealed significant differences in the amplified taxa, indicating potential biases (Supplementary Figure S1). For comparison, we only focused on four major taxonomic groups/phyla that are important in the Arctic realm including Chlorophyta, Haptophyta, Dinoflagellata and Bacillariophyta (Kilias, Wolf, Eva-maria, & Peeken, 2013).
We observed that for Chlorophyta, all four primer pairs targeted between 80% and 90% of all possible chlorophytes included in the reference database. For Haptophyta, only Stoeck covered an average of 1.1%, while the other three primer pairs had over 90% amplification efficiency. For Dinoflagellata, all primer pairs showed a high coverage of over 80%. Lastly, the primer pair Wolf938 only amplified 1% of the bacillariophytes, whereas the Wolf964 covered of about 90%.
Observations in silico were evaluated in situ by PCR-amplification and subsequent sequencing of a mock community comprised of defined concentrations of 18S PCR fragments of 22 eukaryotic microbial species within 18 genera as a template. The relative abundances of these 18 genera in the mock community are shown in Figure 2. The same figure also shows the relative abundances of genera obtained from sequencing with the four different primer pairs. The primer pairs of Stoeck and Wolf938 showed significant overrepresentations of Dinoflagellata, while the Stoeck primer pair showed additionally a near absence of Haptophyta, and amplification with primer pair Wolf938 resulted in an underrepresentation of Bacillariophyta. The primer pairs Wolf964 and Bradley performed similarly and showed a realistic image of the mock community. Two genera (Prasinoderma and Leptocylindrus ) were not detected by any of the primer pairs. The genera Chrysochromulina andSkeletonema did not appear in the assemblages generated by the primer pairs Bradley and Wolf938.