title: MiSeq of Odocoileus virginianus: ear tissue
design: A modified version of the DNA extraction 2XCTAB Protocol for Initial DNA Isolation of Symbiodinium (Santos Lab 2016) was used to extract DNA from tissue samples collected 2007-2013. DNeasy blood and tissue kits (Qiagen, Inc.) were used to extract DNA from tissue samples collected 2014-2017 and from previous samples with low DNA yield. Extractions for 381 individuals were shipped to RTL Genomics (Research and Testing Laboratory, Lubbock TX, United States) for amplicon sequencing of the second exon of MHC-DRB (primers LA31 and LA32; Sigurdardottir et al. 1991; Mikko and Andersson, 1995), which target 307 bp of the peptide binding domain, and 398 bp of the second exon of MHC-DOB (Forward: 5' - AAAGCCCTCCTCTCCAATCC - 3'; Reverse: 5' - CCACCAAGGAGACCCCAAC - 3'). Samples were amplified via a 2-step process. First, the forward primer was constructed by combining the Illumina i5 sequencing primer (5 - TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG - 3) with the MHC-DRB/MHC-DOB exon 2 specific forward primer, and the reverse primer was created using the Illumina i7 sequencing primer (5 - GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG - 3) and the MHC-DRB/MHC-DOB exon 2 specific reverse primer. Amplifications were performed in 25 L reactions using Qiagen HotStar Taq master mix (Qiagen Inc, Valencia, CA), 1 L of each 5 M primer, and 1 L of template on ABI Veriti thermocyclers (Applied Biosystems, Carlsbad, CA). Thermal profiles used for MHC-DRB and MHC-DOB were identical: 1) 95C for 5 minutes; 2) 33 cycles of 95C for 1 minute; 3) 50C for 30 seconds; 4) 72C for 1 minute; 5) final extension of 72C for 10 minutes; 6) 4C hold. Products from this first state of amplification were then added to a second PCR based on qualitatively determined concentrations. Primers for the second PCR used primers that were based on the Illumina Nextera PCR primers (Forward: 5 - AATGATACGGCGACCACCGAGATCTACAC[i5index]TCGTCGGCAGCGTC - 3; Reverse: 5 - CAAGCAGAAGACGGCATACGAGAT[i7index]GTCTCGTGGGCTCGG - 3) using the following thermal profile: 1) 95C for 5 minutes; 2) 10 cycles of 94C for 30 seconds; 3) 54C for 40 seconds; 4) 72C for 1 minute; 5) final extension of 72C for 10 minutes; 6) 4C hold. All amplification products were visualized with eGels (Life Technologies, Grand Island, NY). After pooling the products equimolar, each pool was size-selected in 2 rounds using SPRIselect Reagent (BeckmanCoulter, Indianapolis, IN) in a 0.75 ratio for both rounds. These size-selected pools were then quantified using the Qubit 4 Fluorometer (Life Technologies). The DNA extraction from 1 individual failed to amplify and was therefore removed from the dataset prior to sequencing. Successfully amplified size-selected pools were loaded on an Illumina MiSeq (Illumina, Inc., San Diego, CA) 2x300 flow cell at 10 pM. The amplicons were then sequenced for 300 bp length paired-end reads on the Illumina MiSeq platform for a targeted minimum of 10k reads per individual.