REF_CTRL8_16S
Sequencing of the V3V4 region of the 16S rRNA gene (reference nucleotide interval, 341805 nt) was performed using the Illumina MiSeq system at the Unidad de Genmica del Parque Cientfico de Madrid (FPCM). DNA concentration was measured with Picogreen (Thermo Fisher) and 3 ng of each sample were used for the first PCR round using the Q5 hot Start High-Fidelity DNA Polymerase (New England Biolabs) and 100 nM of primers, in a final reaction volume of 25 l. The primers used were CS1-341F (5-ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCA G-3) and CS2-805R (5- TACGGTAGCAGAGACTTGGTCTGA CTACHVGGGTATCTAATCC-3). Cycling conditions were 98C 30 s; followed by 26 cycles of 98C 10 s, 50C 20 s, and 72C 20 s; with a final elongation step at 72C 2 min. A second PCR round was conducted using 1 l of the first PCR reaction, the same enzyme and 400 nM of primers (F: 5-AATGATACGGCGACCACCGAGATCTACACTGACGACATG GTTCTACA-3/R: 5-CAAGCAGAAGACGGCATACGAGAT-[10 nucleotides barcode]-TACGGTAGCAGAGACTTGGTCT-3, Fluidigm, Access Array Barcode Library for Illumina Sequencers) in a 20 l reaction volume. Cycling conditions were the same as in the first round with the exception that only 12 cycles were used. PCR products were checked and quantified with Bioanalyzer (Agilent) and purified with AMPure Beads (Beckman Coulter). The amplicon pool was quantified by qPCR using Kapa-SYBR FAST qPCR kit for LightCycler480 and a reference library from the Unidad de Genmica FPCM. An equimolar pool of amplicons was sequenced in Illumina MiSeq (2  300 paired-end run) following the manufacturers instructions and using the MiSeq Reagent Kit v3 600 Cycles.