AChE-seq data analysis

Libraries were sequenced using Illumina HiSeq 2500 100-bp paired-end sequencing in our institutional Epigenomics Shared Facility. The sequencing reads were aligned to the hg19 reference genome using BWA (version 0.7.10). Alignments were generated in the SAM format and then transformed and sorted into BAM files using SAMtools (version 1.2). To remove duplicates and to down-sample all datasets to an equivalent number of reads (127 million), the sorted sequences in BAM format were processed using Picard-tools (version 1.119). Down-sampled unique reads were transformed into BED format using BEDtools2 (version 2.24.0). To identify domains of enrichment from the AChE-seq data, we created genomic windows of 1–1,000 kb using the hg19 reference genome and measured the number of normalized reads for each AChE-seq sample set, including input, using BEDtools2 (version 2.24.0). The enrichment for each AChE-seq sample set (S–G2 and G1) was calculated by the number of reads in each genomic window normalized by the number of input reads in that window. The distribution of the enrichment ratio for each window was visualized using density plots generated using R. For permutation testing, shuffled sequences (100 iterations) were created with BEDtools2 (version 2.24.0). The window size with a bimodal pattern of enrichment and the cutoff defining the enriched windows were determined with the pastecs library in R. These enriched windows were intersected with CytoBand annotations from the UCSC Genome browser using BEDtools2 (version 2.24.0). Custom code and parameters used in this project are available at GitHub (https://github.com/hnst/Sato-et-al).