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.