2.2. Metabarcoding
Metabarcoding was performed at the National Center of Sequencing
(Genoscope, Paris, France) following protocols published by Brandt et
al. (2019). PCR was performed on two barcoding gene regions (Table 2)
targeting prokaryotes (V4 16S rDNA region) or eukaryotes (V1-V2 18S rDNA
region). Each 25 µl PCR contained Phusion® High-Fidelity PCR 2X Master
Mix (GC Buffer for 18S and HF Buffer for 16SV4; New England Biolabs,
Ipswich, MA US), 0.5 µM of each primer, and approximately 10-20 ng of
DNA template and were filled to volume with molecular water. PCR cycling
conditions were 98°C for the 30 s, followed by a 30 (for 18S) or 29 (for
16SV4) cycles of 98°C for 10 s, annealing at 50°C for 45 s, 72°C for 60
s, and final elongation at 72°C for 10 min. Each library was prepared in
triplicate PCR with a Kapa Hifi HotStart NGS library Amplification kit
(Kapa Biosystems, Wilmington, MA, USA), and triplicates were pooled for
sequencing with the HiSeq technology with the 2x250 bp reading mode.