DNA extraction, amplification and high-throughput sequencing
The DNA extraction was performed in a dedicated controlled DNA
laboratory (SPYGEN, www.spygen.com)equipped with separate cleanrooms with positive air pressure, UV
treatment and frequent air renewal. Decontamination procedures were
conducted before and after all manipulation. Each filtration capsule was
agitated for 15min on S50 Shaker (Cat Ingenieurbüro™) at 800 rpm. The
buffer was retrieved using a 3 mL BD Disposable Syringe with Luer-Lok™
tips, emptied into a 50mL tube containing 33 mL of ethanol and 1.5 mL of
3M sodium acetate and, finally, stored for at least one night at -20°C.
The DNA extraction and amplification were performed following the
protocol of (28) Pont et al 2018 including 12 separate PCR
amplifications per sample. A teleost-specific 12S mitochondrial rDNA
primer (teleo, forward primer-ACACCGCCCGTCACTCT, reverse primer
-CTTCCGGTACACTTACCATG, (26) Valentini et al. 2016) was used for the
amplification of metabarcoding sequences. Eight negative extraction
controls and two negative PCR controls (ultrapure water) were amplified
(with 12 replicates as well) and sequenced in parallel to the samples to
monitor possible contaminations. The teleo primers were 5’-labeled with
an eight-nucleotide tag unique to each PCR replicate with at least three
differences between any pair of tags, allowing the assignment of each
sequence to the corresponding sample during sequence analysis. The tags
for the forward and reverse primers were identical for each PCR
replicate.
After amplification, samples were titrated using capillary
electrophoresis (QIAxcel; Qiagen GmbH, Hilden, Germany) and purified
using a MinElute PCR purification kit (Qiagen GmbH, Hilden, Germany).
Before sequencing, purified DNA was titrated using capillary
electrophoresis. The purified PCR products were pooled in equal volumes,
to achieve a theoretical sequencing depth of 1,000,000 reads per sample.
Library preparation and sequencing were performed at Fasteris (Geneva,
Switzerland). A total of five libraries were prepared using MetaFast
protocol (Fasteris,https://www.fasteris.com/dna/?q=content/metafast-protocol-amplicon-metagenomic-analysis).
A paired-end sequencing (2x125 bp) was carried out using an Illumina
HiSeq 2500 sequencer on three HiSeq Rapid Flow Cell v2 using the HiSeq
Rapid SBS Kit v2 (Illumina, San Diego, CA, USA) following the
manufacturer’s instructions.