Epireads and the epiBED Format
Bisulfite sequencing data may be able to inform how methylation from neighboring positions are linked and how methylation is linked to mutations (provided they can be unambiguously determined). We introduce the epiBED format, which combines the utility of the epiread format with the standard-compliant BED format. The epiBED format is a compact data format used to store CpG (and, in the case of NOMe-seq, GpC) methylation, as well as SNP and indel information from the same read. It can be useful for estimating the epiallele fraction and clonal structure/cell population, which is similar to MethylPurify.
Generating Epireads in the epiBED Format
The original epiread format (proposed by the methpipe team), only includes information related to CpGs. BISCUIT builds off this format to put it in a standard-compliant format, while also containing information related to filtered bases, SNPs, indels, and, in NOMe-seq mode, GpC methylation. To generate an epiBED file, run
# For WGBS data
biscuit epiread -@ NTHREADS [-B snps.bed] /path/to/my_reference.fa my_output.bam | \
sort -k1,1 -k2,2n > my_epireads.epibed
# For NOMe-seq data
biscuit epiread -@ NTHREADS [-B snps.bed] -N /path/to/my_reference.fa my_output.bam | \
sort -k1,1 -k2,2n > my_epireads.epibed
The snps.bed file can be obtained by running biscuit vcf2bed -t snp my_pileup.vcf.gz. If no SNP file is given, the output will not include information related to SNPs. Note, it is suggested that you sort your epiBED file before running bgzip and tabix to compress and index your epiBED file. This is due to how softclipping is handled in the BAM file versus how it is handled in the epiBED file. The difference can cause some reads to not be coordinate sorted, which is required when bgzipping and tabixing.
For more help with epiread, run biscuit epiread in the terminal or check out the epiread help page.
Generating Legacy File Formats
In addition to the epiBED format, biscuit epiread continues to produce the BISCUIT epiread format and the pairwise file format for biscuit asm. While these are available, it is suggested you use the epiBED format instead, as it aligns with the output from biscuit pileup, whereas the epiread and pairwise file formats are not guaranteed to match the results from pileup.
# Original epiread format with WGBS data
biscuit epiread -@ NTHREADS -o my_output.epiread -O [-B snps.bed] /path/to/my_reference.fa my_output.bam
# Original epiread format with NOMe-seq data
biscuit epiread -@ NTHREADS -o my_output.epiread -O -N [-B snps.bed] /path/to/my_reference.fa my_output.bam
# Pairwise format with WGBS data
biscuit epiread -@ NTHREADS -o my_output.epiread -P [-B snps.bed] /path/to/my_reference.fa my_output.bam
# Pairwise format with NOMe-seq data
biscuit epiread -@ NTHREADS -o my_output.epiread -P -N [-B snps.bed] /path/to/my_reference.fa my_output.bam