title: MiSeq sequencing of Bacteria 16S rRNA gene: Sample L09_TF
design: Libraries for sequencing were prepared according to Illumina’s “16S Metagenomic Sequencing Library Preparation” guide (Part # 15044223 Rev. B), with the exception of using Qiagen HotStar MasterMix for the first PCR (“amplicon PCR”) and halving reagents volumes for the second PCR (“index PCR”).  The template specific primers were  (without the overhang adapter sequence): 1) Archaea 16S rRNA gene: Arch516F (5’-TGYCAGCCGCCGCGGTAAHACCVGC-3’) and A806R (5’-GGACTACVSGGGTATCTAAT-3’), 2) bacterial 16S rRNA gene: F343 (5’-TACGGRAGGCAGCAG-3’) and R803 (5’-CTACCAGGGTATCTAATCC-3’), and 3) fungal ITS: ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and 5.8A2R (5’-CTGCGTTCTTCATCGAT-3’).  The first PCR (“amplicon PCR”) was carried out for 30 (bacteria 16S) or 35 (archaea 16S and fungi ITS) cycles with annealing temperatures of 55°C (archaea 16S and bacteria 16S) or of 45°C (fungi ITS). Diluted pooled samples were then loaded on an Illumina MiSeq and sequenced using a 300-cycles MiSeq Reagent Kit v2 (Archaea 16S) or a 600-cycles MiSeq Reagent Kit v3 (bacteria 16S and fungi ITS). 