negative extraction
16S rRNA gene amplification and library preparation was performed as recommended by the Illuminar 16S metagenomic protocol. In short, modified primers combining the illumina overhang adapter sequence and the 16S V3-V4 region target were used (341F (5'- CCTACGGGNGGCWGCAG) & 806R (5'- GGACTACHVGGGTWTCTAAT)). In a second PCR, unique dual-index barcodes were added and the amplicon were purified and sequenced on the  Illumina Nextseq 2000