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.