2.2 DNA metabarcoding
A two-step PCR approach with two replicates per extract was applied
(Zizka et al., 2019). For the first step, four length-varying primers
for the primer pair fwhF2/EPTDr2n (Leese, Sander et al., 2021; Vamos et
al., 2017) were used, with each of them having a universal tail
attached. Length variation was due to inline shifts (0–3 Ns) between
the universal tail and the primer sequence to maximize diversity between
sequences (Elbrecht & Leese, 2015). For the second step, primers
matching the universal tail with an i5/i7 index and P5/P7 Illumina
adapter attached were used (Buchner et al., 2021a,b). We applied an
improved PCR protocol compared to the previous study (Leese, Sander et
al., 2021), PCR reactions for both PCR steps contained: 1x Multiplex PCR
Master Mix (Qiagen Multiplex PCR Plus Kit), 0.1 μM of each primer, 0.5 x
CoralLoad Dye, 1 μL DNA extract/ unpurified PCR product
(1st step/ 2nd step) filled up with
Rnase-free water to a total volume of 25 μL. Cycling conditions were as
follows: first step PCRs consisted of 5 min initial denaturation at
95 °C, followed by a touchdown of 10 cycles of 30 s at 95 °C, 90 s at
64 °C (- 1 °C each cycle) and 30 s at 72 °C and additional 25 cycles of
30 s at 95 °C, 90 s at 54 °C and 30 s at 72 °C, followed by a final
elongation for 10 min at 68 °C. Second step PCRs consisted of 5 min
initial denaturation at 95 °C followed by 18 cycles of 30 s at 95 °C and
2 min at 72 °C, followed by a final elongation for 10 min at 68 °C.
Eight PCR negative controls were added to check for contamination during
PCR and tag switching. PCR products were quantified and equimolarly
pooled to one library before purification and left-sided size selection
with a ratio of 0.76x (Beckman Coulter). The library was sequenced on a
single flow cell on an Illumina HiSeq X sequencer using the 300 cycle
kit (150 bp paired-end reads) with 5 % Phi-X spike-in added by the
sequencing company Macrogen Europe B.V. Ten of the 102 samples of the
dataset were amplified and sequenced separately in another library, as
they had already been processed in another study (Leese, Sander et al.,
2021). For bioinformatic analysis, sequences from the ten previously
sequenced samples (Short-Read Archive, Project number: PRJNA664693) and
the sequences from the remaining 92 samples (deposited in the European
Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB61051)
were processed together.