title: 16s rRNA sequencing of amphibian skin microbiome
design: The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified with single indexed, Illumina compatible primers (341F: CCTACGGGAGGCAGCAG /806R: GGACTACHVGGGTWTCTAAT) following the protocol described by Caporaso et al. (2011). Following PCR, all amplified products were batch normalized using an Invitrogen (Carlsbad, CA, USA) SequalPrep DNA normalization plate and the products recovered from this plate were pooled. The pool was verified and quantified using a combination of the Qubit dsDNA HS (Invitrogen; Carlsbad, CA, USA), Agilent 4200 TapeStation High Sensitivity DNA1000 (Agilent; Santa Clara, CA, USA), and Kapa Illumina Library Quantification qPCR assays (Roche; Indianapolis, IN, USA). The pool was sequenced using an Illumina MiSeq v2 standard flow cell in a 2  250 base pair (bp) paired end format using a v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 341F/806R target specific sequences were added to appropriate wells of the reagent cartridge (Caporaso et al., 2011). Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.