14KM1161M
16S rRNA gene hypervariable region V3-V4 amplicon sequencing of canine skin of groin and oral mucosa
To sequence the V3-V4 of the 16S rRNA gene two-step PCR libraries were generated with the primer pair 341F (CCTACGGGNGGCWGCAG) and 806 (GGACTACHVGGGTWTCTAAT) using a high-fidelity polymerase (FastStart High Fidelity, Roche Diagnostics, Rotkreuz, Switzerland) in a total volume of 25 L. The mix contained 1X PCR buffer with 1.8 mM MgCl2, 200 M dNTPs, 0.4 M of each primer and 1.25 U polymerase and 2 l DNA template.  The cycling conditions were as follows: 95C for 3 min, followed by 15 cycles of 95C for 30 s, 50C for 30 s, and 72C for 1 min, followed by 20 cycles of 95C for 30 s, 55C for 30 s, and 72C for 1 min, and a final elongation step at 72C for 8 min. amplicons were purified using a magnetic bead capture kit (AMPure XP, Beckman Coulter, Brea, CA, USA) and quantified and quality-checked using the Agilent DNA 7500 Kit (Agilent Technologies, Waldbronn, Germany). Negative controls of mock swabs were included in all steps. The purified amplicon libraries were mixed with Illumina-generated PhiX control libraries and our own genomic libraries and denatured using fresh NaOH. Subsequently, the Illumina MiSeq platform and a v3 600 cycle kit were used to sequence the PCR libraries.
14KM1161M
SUB4187196
14KM1161M