title: Amplicon of canis lupus: gut microbiota
design: Metabarcoding analyses using sequencing of amplicon were investigated throughout the hypervariable genomic region (V3-V4 region 16SrRNA gene amplification), using an NGS approach on Illumina Platform. From 180-220 mg of each fecal sample the total amount of DNA was extracted. Using the Illumina 16srRNA protocol indexed NGS libraries were built. PCR was firstly used to amplify template from a DNA sample using specific denatured primers targeting the 16S V3 and V4 region.  Then, AMPure XP bead removed free primers and primers dimers allowing to complete a second PCR to attach dual index and Illumina sequencing adapters using the Nextera XT Index kit. The final library was diluted 1:50 and sequenced using the Illumina Miseq technology in 2x300bp run using 20% of PhiX library as control