16s rRNA V2 bacterial amplicons from hellbender:skin
We isolated DNA from skin swabs using the PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA) following the protocol described in Appendix A. We amplified the bacterial 16S rRNA V2 region using primer pair 27F/338R (Fierer et al., 2008) with the attachment of connector sequences (Appendix B). We ran each sample in triplicate, and each reaction consisted of 5 킠 of template DNA, 12.5 킠 of MyTaq Master Mix (Bioline, Tauton, MA), 1 킠 of 10 mM forward and reverse primers, and 6.5 킠 of PCR water (MoBio Laboratories Inc., Carlsbad, CA) for a total of 25 킠 per reaction. PCR conditions consisted of 95좧 for 2 minutes, 30 cycles of 94좧 for 45 seconds, 50좧 for 60 seconds, and 72좧 for 90 seconds, followed by 72좧 for 10 minutes. We pooled microbiome amplicon triplicates and cleaned the products using the UltraClean PCR Clean-up kit (MoBio Laboratories Inc., Carlsbad, CA). We performed a second PCR on the PCR products to add-on dual-index barcodes connected to Illumina sequencing adaptors (Hernandez-Gomez et al., 2017) to the ends of amplicons. The PCR consisted of 5 킠 of clean amplicons, 12.5 킠 of MyTaq Master Mix, 1 킠 of forward and reverse barcode primers, and 6.5 킠 of water for a total of 25 킠 reactions. PCR conditions consisted of 95좧 for 2 minutes, 5 cycles of 94좧 for 45 seconds, 65좧 for 60 seconds, and 72좧 for 90 seconds, followed by 72좧 for 10 minutes. We quantified the PCR products using a Qubit Fluorometer (Invitrogen Corp, Carlsbad, CA). We pooled microbiome samples in equimolar amounts and cleaned using the UltraClean PCR Clean-Up kit. The microbiome library was sequenced on a MiSeq machine (Illumina Inc. San Diego, CA) using the Reagent Kit V2 to produce 250 bp paired end reads. Raw reads were paired using PANDASEQ.