title: 16S of bile: Canis lupus familiaris
design: Extracted DNA was quantified via PicoGreen analysis and used for bacterial 16S rRNA gene amplicon sequencing. Total DNA (12.5 ng) was amplified using universal primers targeting the V4 region of the bacterial 16S rRNA gene[39]. The sequences of the primers were: 515F - 5? TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA 3? and 806R - 5?GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT 3?. Overhang adapters were appended to the 5? end of each primer sequence for compatibility with the Illumina sequencing platform. Master mixes contained 12.5 ng of total DNA, 0.2 M of each primer and 2 KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA). The thermal profile for the amplification of each sample had an initial denaturing step at 95C for 3 minutes, followed by 25 to 32 cycles of denaturing of 95C for 30 seconds, annealing at 55C for 30 seconds for 16S rRNA and a 30 second extension at 72C, a 5 minute extension at 72C and a final hold at 4C. For each sample, a cycle-optimization qPCR was first performed to determine the cycle at which each began logarithmic amplification. Amplicons from each sample were collected at the optimized cycle to prevent over-cycling of highly concentrated samples while allowing ample time for lower input samples to amplify. Each 16S amplicon was purified using the AMPure XP reagent (Beckman Coulter, Indianapolis, IN). In the next step each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dual index barcodes (index 1(i7) and index 2(i5)) (Illumina, San Diego, CA) to the amplicon target. The thermal profile for the amplification of each sample had an initial denaturing step at 95C for 3 minutes, followed by a denaturing cycle of 95C for 30 seconds, annealing at 55C for 30 seconds and a 30 second extension at 72C (8 cycles), a 5 minute extension at 72C and a final hold at 4C. The final libraries were again purified using the AMPure XP reagent (Beckman Coulter), quantified with Quant-iT PicoGreen dsDNA Reagent (Molecular Probes, Thermo Fisher Scientific, Waltham, MA), and normalized prior to equimolar pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge MiSeq instrument (Illumina). Automated cluster generation and pairedend sequencing with dual reads were performed according to the manufacturers instructions.