Illumina MiSeq paired end sequencing; Illumina MiSeq sequencing
All nucleic acids (NA) were extracted from samples using the NucliSens reagents (bioMérieux), except for the TSS1, which were already in the form of nucleic acids. Nucleic acids were extracted from BMS samples from TSS2 and BLS samples following the ISO 15216-1 method (ISO 15216-1, 2017). For this, 2g aliquots of DT were incubated in 2ml of proteinase K solution (100µg/ml) for 1 h at 37°C and then for 15 min. at 60°C. The sample was then centrifuged at 3000g for 5 min at room temperature, the total volume of supernatant was recorded and 0.5ml used for nucleic acid extraction (NucliSens, bioMérieux). For the BLS samples, the remaining original RNA generated during the BLS study, stored below -70°C, was used for the present study when possible. When the remaining RNA volume was not sufficient, the ISO 15216-1 method (2017) was used for extraction from remaining proteinase K supernatant, or from extra-DT samples, also stored at -70°C or below, in this order of priority, depending on availability of each material, for each sample.  The RT-PCR method for the metabarcoding analysis consisted of 3 stages: reverse transcription (RT), first round PCR (referred below as PCR) and second round, semi-nested PCR (referred to as N-PCR).  For reverse transcription, a mix of random hexamers (Invitrogen, France) and nonamers (Sigma, France), both at 2.5 µM of final concentration were used as primers with the SuperScript II (Invitrogen, France) reverse transcriptase. The reaction was carried out in a total volume of 20µL, containing 7 µL of nucleic acids extract, according to the manufacturer's instructions, using a PTC-100 (MJ Research Ink), SimpliAmp (Applied Biosystems), Mastercycler Nexus X2 (Eppendorf) at Ifremer, Cefas and DTU. For samples for which several viruses and genes were targeted, duplicate reverse transcription reactions were necessary to generate enough complementary DNA (cDNA) for subsequent analysis. The efficiency of RT was controlled by quantitative PCR (qPCR) on the cDNA, using the same primers, probes and controls as described in ISO 15216-1 (2017), using the Taq polymerase instead of the RT-DNA polymerase enzyme mix and deleting the RT step from the thermal profile. The amplifications were performed using Stratagene Mx3000Pro, Mx3005Pro and Agilent AriaMx real-time thermocyclers.  Following reverse transcription, 10 µL of cDNA was used for each PCR. Then, to enhance specificity and sensitivity of the method, the N-PCR used 5 µL of the first round PCR product. For each amplification reaction mix, the Platinum Taq kit (Invitrogen, France) was used following the manufacturer's instruction, with a final volume of 50 µL, MgCl2 (final concentration 1.5mM), and forward and reverse primers at a final concentration of 0.2pM. Both amplification reactions (PCR and N-PCR) followed the same program, including 40 amplification cycles with an annealing temperature at 50°C. Conventional amplifications were run in the thermocyclers as specified above.  Tagged PCR amplicons were indexed by an additional PCR, purified, quantified and mixed into a pool with equimolar quantities of each amplicon by the subcontractant selected to perfom the sequencing (Institut du Cerveau et la Moëlle, Paris, France). Sequencing was performed on the MiSeq platform (Illumina) to generate paired-end reads of 300nt (2x300).