D version of Annovar program [52] with RefSeq genes to determine specific genomic features: promoter regions (2.5 kb upstream and 500 bp downstream from the nearest TSS), genic (further defined as 500 bp downstream from the nearest TSS), exons, introns, and intergenic (defined as greater than 100 kb up/downstream from the nearest TSS). UCSC CpG island definitions were used to define CpG islands. RWPE-1 DNase-seq data from the Encyclopedia of DNA Elements (ENCODE) project [GEO accession: GSM1008595] was correlated to MACS peak sets in both RWPE-1 and 22Rv1 cells. The genomic feature, RWPE-1 UCSC DNAseI peaks, was defined as within 5 kb of a DNase peak. The following options for ChiPpeakAnno HS-173 chemical information pubmed ID:https://www.ncbi.nlm.nih.gov/pubmed/26100631 were used: PeakLocForDistance=”middle,” FeatureLocForDistance=”middle” for DNase I HS and CpG data, and FeatureLocForDistance=”TSS” for RefSeq gene annotations. MACS data was further analyzed using DiffBind R package (v.1.12.0) [53] to determine three consensus peak sets by requiring that peaks from one, two, or all three replicates be present at the same location. The third consensus peak set was subsequently defined as differentially methylated regions (DMRs) between RWPE-1 and 22Rv1 cells (fold change >1, FDR < 0.1).Bioinformatic analysis of hMeSeal-seqboth MACS peaks to be present for a consensus peak to be called. The first consensus peak set was subsequently defined as differentially hydroxymethylated regions (DHMRs) between RWPE-1 and 22Rv1 cells (fold change >1).Bioinformatic analysis of hMeDIP-seqSequencing image analyses and base calling were performed using Off-Line Basecaller software (OLB V1.8). After passing Solexa CHASTITY quality filter, sequenced reads were mapped to the reference human genome (GRCh37, hg19) using Bowtie (V2.1.0) [49]. Significantly enriched regions/peaks of hydroxymethylation were determined using model-based analysis of ChIPseq (MACS, V2) algorithm [50], by comparing immunoprecipitated, enriched samples to input, non-enriched samples (p value < 10-4). Annotation was performed using the UCSC RefSeq database, where peaks were mapped to the nearest gene and specific genomic features were determined: promoter (2500 kb upstream and 500 bp downstream from the TSS); gene body (500 bp downstream of the TSS to the TTS), including introns and exons; and intergenic (remaining regions not defined as "promoter" or "gene body" located more than 100 kb from the nearest TSS). UCSC CpG island definitions were used to define CpG islands. RWPE-1 DNaseseq data from the ENCODE project [GEO accession: GSM1008595] was correlated to MACS peak sets in both RWPE-1 and 22Rv1 cells. The genomic feature, RWPE-1 UCSC DNAseI peaks, was defined as within 5 kb of a DNase peak.Statistical analysis: correlation between genome-wide DNA methylation and hydroxymethylation marksSequenced reads were mapped to the reference human genome (GRCh37, hg19) using Bowtie (v0.12.7) [49]. Repitools package [54] was used as an enrichment diagnostic screen of sequenced samples, enriched and input. Significantly enriched regions/peaks of hydroxymethylation were determined using model-based analysis of ChIP-seq (MACS) algorithm [50], by comparing bound, enriched samples to input, non-enriched samples. Annotation was performed using ChIPpeakAnno R package (v.1.12.0) [51] and a customized version of Annovar program [52] with RefSeq genes to determine specific genomic features: promoter, genic, exons, introns, and intergenic (previously described for MBD-seq). UCSC CpG is.
