Primer selection
We designed two new reverse primers, 938iR (5’-GGCAAATGCTTTCGC-3’,
hereafter referred to as Wolf938) and 964iR (5’-ACTTTCGTTCTTGATYRR-3’,
hereafter referred to as Wolf964). The primer design put special
emphasis on the coverage of Haptophyta, which are important contributors
to Arctic eukaryotic microbial communities, and which were known to be
underrepresented in previous meta-barcoding studies (Bradley et al.,
2016). We combined the resulting new primers with the well-established
universal forward primer 528iF (5’-GCGGTAATTCCAGCTCC-3’) (Elwood, Olsen,
& Sogin, 1985). For the design, we used the primer design function
within the ARB software (Ludwig et al., 2004) tested against the SILVA
reference database SSU Ref v.119 (Yilmaz et al., 2014). Primers were
chosen to have the largest possible coverage across all phyla and to
cover an amplicon within 450 bp. We then evaluated these new primers
with the widely used primer pairs designed by Stoeck et al. (Stoeck et
al., 2010) and Bradley et al. (Bradley et al., 2016) by performingin silico PCR using the SILVA TestPrime tool (Klindworth et al.,
2013).