Bisulfite sequencing of equine bone marrow genomic DNA
Genomic DNA was isolated from equine CVID patients (n=2) and healthy control horse (n=1) core bone marrow samples with Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) and unmethylated lambda DNA was obtained (Promega, Madison, WI). Reduced representation bisulfite sequence (RRBS) libraries were prepared by the Cornell Epigenetics Core Facility, Weill Cornell Medical College, New York, NY per Illumina protocol. Libraries were sequenced on the Hi-Seq 2000 at Cornell Institute of Biotechnology, Ithaca, NY. After removal of adapter and primer sequences used in RRBS library construction, sequence reads went through an adaptive quality trimming of low quality trailing bases from the 3’ end. Such adaptive quality trimming (also adaptor trimming) was performed with cutadapt (http://code.google.com/p/cutadapt/).  The filtered reads were mapped to the equine reference genome EquCab2.0 with Bismark v0.6.0.  Bisulfite mapping where reads were converted into a C-to-T and a G-to-A version and then aligned to equivalently converted versions of the reference genome, and the methylation calling where the methylation state of positions involving cytosines was inferred by comparing the read sequence with the corresponding genomic sequence.  Sequence reads that produce a unique best alignment from the four alignment processes against the bisulfite genomes were then compared to the normal genomic sequence and the methylation state of all cytosine positions in the read is inferred using Bismark. The CpGs with read depth  >= 5 were kept as informative CpGs.  To score CpG island (CGI) methylation, we required that the methylation level was determined for ≥10% of their total CpGs and  a CGI must have  >= 5 informative  5 CpGs.  Then  CGIs with an average methylation level ≥75% and ≤25% were called methylated and unmethylated, respectively. The horse CGI list was created by Wu et al. (2010, PMID 20212320) using the model-based method.