Comparative Analysis of DNA Methylation

To compare and validate TEEM-Seq against more traditional methods of analysis for DNA methylation, we prepared TEEM-Seq libraries from many of the same starling DNA samples that underwent WGEM-Seq and RRBS. For WGEM-Seq, extra material from three of the EM-seq libraries used for bait capture were confirmed clear of adapter dimers on the TapeStation, pooled and sequenced at 2x150 bp reads with 5% PhiX spike-in in 3/4^th of a lane (82.5 Gb) on a HiSeq 4000 at Novogene with the same parameters to provide a whole-genome sequence and partial methylome analysis comparison to our targeted sequencing. For a previously unpublished RRBS study, we used NuGEN (now Tecan) Ovation Methyl-Seq kits to prepare RRBS libraries (with added Promega unmethylated cl857 Sam7 lambda control spike-in of 1 ng per 80–120 ng of sample), which were sequenced in several pools of 16 samples per lane (25 Gb) at 1x100 bp reads with 10% PhiX spike-in on a HiSeq 2500 at the Cornell Institute of Biotechnology Genomics Core, Ithaca, NY. Libraries for RRBS were prepared according to kit protocols. Briefly, samples were digested with MspI at 37°C, ligated with bisulfite-compatible barcoded adaptors at 25°C, end repaired at 60°C, bisulfite converted with the Qiagen EpiTect Fast Bisulfite Conversion Kit, PCR amplified with NuGEN’s standard 12 cycles and 60°C annealing, and purified with Agencourt beads for sequencing. Eight of these samples used previously for RRBS were included in the present study, though one failed enzymatic conversion (mean lambda conversion rate from the original RRBS analysis of these samples was 1.43%). Data from the remaining seven samples (from different sequencing pools) were used as a comparison to the TEEM-Seq and WGEM-Seq derived data generated from the same individual starlings.