eastern newt microbiomes
We isolated DNA from skin swab samples using the DNeasy PowerSoil DNA Isolation Kit (Qiagen N.V., Hilden, Germany) following the modifications to the manufacturers protocol described in Hernndez-Gmez et al., (2017b). To control for contamination, we included unused swabs (i.e., negative extraction controls) and researcher glove swabs in our DNA extractions. We amplified the bacterial 16S rRNA V4 region using primer pair F515/R806 with the attachment of connector sequences that allows for the attachment of barcode/sequencing adaptors (Hernndez-Gmez et al., 2017a). We ran each sample in triplicate, and each reaction consisted of 5.0 L of template DNA, 7.5 L of 2X MyTaq Master Mix (Bioline, Tauton, MA, USA), 1.0 L of 1 nM forward and reverse primers, and 1.5 L of sterile water for a total of 15 L per reaction. PCR conditions consisted of 94 C for 3 min, 30 cycles of 94 C for 45 s, 50 C for 60 s, and 72 C for 90 s, followed by 72 C for 10 min. We pooled amplicon triplicates and cleaned the products using the UltraClean PCR Clean-up kit (Qiagen N.V., Hilden, Germany). We performed a second PCR on microbiota amplicons to ligate dual-index barcodes paired with Illumina sequencing adaptors (Hernndez-Gmez et al., 2017a) to the ends of amplicons. The PCR consisted of 5.0 L of clean amplicons, 7.5 L 2X MyTaq Master Mix, 1.0 L of 1 nM forward and reverse barcode primers, and 1.5 L of water for a total of 15 L reactions. PCR conditions consisted of 94 C for 3 min, 5 cycles of 94 C for 45 s, 65 C for 60 s, and 72 C for 90 s, followed by 72 C for 10 min. We quantified the PCR products using a Qubit Fluorometer (Invitrogen Corp, Carlsbad, CA, USA), pooled samples in equimolar amounts, and cleaned the sample pool using the UltraClean PCR Clean-Up kit. We overnighted the barcoded sample pool on dry ice to the Cornell University Biotechnology Resource Center. The sample pool was sequenced on a MiSeq machine (Illumina, Inc. San Diego, CA) using the reagent kit V2 to produce 250-bp paired end reads.