Amplicon of V4 region 18S euk: marine samples
DNA and RNA were extracted from the same filters using the All-Prep DNA/RNA Minikit (Qiagen) as described by Dasilva et al. (2014). Conversion of RNA to cDNA was carried out using the High Capacity Reverse Transcription Kit (Applied Biosystems) following the manufacturerŐs suggestions. We amplified the V4 region of the 18SrRNA gene and cDNA using the forward specific primers from Comeau et al. (2011) coupled with a MiSeqŠ specific linking primer, under PCR conditions described by Comeau et al. 2011. To avoid primer bias, two more reactions with 5 and 10-fold diluted template were also carried out for each sample. Unique pairs of barcodes (tags) were added to the sample amplicons using the TruSeq¨ and Nextera¨ (both Illumina) barcode sets in a nested PCR with the following conditions: initial denaturation at 98ű C for 30s, then 13 cycles of a set of 98ű C for 10s, 55ű C for 30s, 72ű C for 30s, and the reaction finished after a final 72 ű C extension for 4 min and stopped at 4 ű C. The products of the three dilutions were pooled together and purified using the Axygen PCR cleanup kit (Axygen) and then quantified spectrophotometrically with the Nanodrop 1000Ş (ThermoFisher Scientific). Barcoded Amplicons were pooled in equimolar concentration for sequencing on the Illumina Miseq at the Plateforme dŐAnalyses Gnomiques (IBIS, Universit Laval, Qubec, Canada).