title: MHC IIB PBR Amplicons of hellbender: eastern subspecies
design: We extracted genomic DNA from blood samples using a standard proteinase K/phenolchloroform/isoamyl alcohol procedure (Sambrook & Russell, 2001) and re-suspended in 100 킠 of TLE buffer (10 mM Tris-Cl, 0.1 mM EDTA). To amplify the MHC IIB PBR gene, we performed 25 킠 PCRs consisting ~7.5 킠 of template DNA (20 ng/ 킠), 12.5 킠 of MyTaq MasterMix, 1 킠 of forward and reverse hellbender MHC IIB primers, and 6.5 킠 of water. PCR conditions consisted of 2 minutes at 94좧 for 2 minutes, 35 cycles of 94좧 for 30 seconds, 58.9좧 for 30 seconds, and 72좧 for 30 seconds, followed by 72퍬 for 10 minutes. we cleaned the PCR products using a low-ethanol precipitation protocol (Sambrook and Russell, 2001). We performed a second PCR on 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 MHC barcoded amplicons in equimolar amounts, and purified on a 2% agarose gel. We isolated amplicons from the band corresponding to the expected amplified product size using a custom gel extraction protocol. The MHC samples were sequenced on a MiSeq machine (Illumina Inc. San Diego, CA) using the Reagent Kit V2 to produce 250 bp paired end reads.