in solutio enrichment (UCEs) of sifaka ear tissue samples
One quarter of ear biopsy punches had phenol-chloroform DNA extraction performed, with a goal of using 500 ng of DNA for downstream analyses. Raw DNA was sheared with a Covaris M220 to an average length of 300 bp and cleaned via SPRI purification. Dual indexed iTru style Illumina adapters were obtained from the UGA sequencing core, and we used a KAPA biosystems library preparation kit using 1/4 reactions. Amplification of libraries used the i5 and i7 primers using the following cycle: 98 C for 45 seconds for an initial denature step, followed by ten cycles of: 98 C for 15 seconds, 60 C for 30 seconds, 72 C for 60 seconds, with a final extension of 72 C for 5 minutes. PCR products were SPRI cleaned again and eluted in ddH20. A Qubit 2.0 florometer was used to quantify each sample. A MYbaits kit was used to enrich UCEs for the 5k tetrapod probe set, following manufacturer's protocols following version 3.01 of the manual. All enrichments included multiplexed pools of 3-4 individuals (including 125 or 166 ng of DNA for each sample for a total of 500 ng per pool). Twelve cycles of PCR were performed following enrichment with the following program: five-minute denaturation at 95C and thirty second anneal at 60C followed by a five-minute final extension at 72C modified from the above cycling conditions. qPCR was performed on an ABI QuantStudio 3 with a KAPA Biosystems Illumina library quantification kit. Enrichments were pooled in equimolar ratios and sent out for sequencing at HudsonAlpha on a NextSeq 550 with PE 150 bp runs.