title: Illumina:Metagenomics:dairies
design: V3 and V4 regions of 16S rRNA genes were amplified by PCR with specific primers recommended by Illumina (forward=5CGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGC WGCG3; reverse=5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGTATCTAA TCC3) with overhang adapters attached. The amplicons were purified with Agencourt AMPure XP beads (Beckman Coulter, USA) to remove free primers and primers dimers. A second stage PCR was performed to attach dual indices and Illumina sequencing adapters, using the Nextera XT Index kit  (Illumina ). Following, another purification step was performed with Agencourt AMPure XP beads (Beckman Coulter), the libraries were quantified by real-time PCR (Kapa HiFi Hot Start ready Mix, Kapa Biosystems, USA), normalized and the total libraries were pooled, according to instructions provided by Illumina  for the 16S Metagenomic Sequencing Library Preparation guide. The libraries were denatured with sodium hydroxide and the pool samples were loaded into MiSeq flow cell using the MiSeq reagent kit V3 (600 cycles). For internal control of low diversity libraries and to prevent the run to fail, a minimum of 5% of PhiX control kit v3 (Illumina , USA) for each run were used.