Call methylation¶

bsbit call meth summarizes methylated and unmethylated observations at CG, CHG, and CHH sites. It writes site-level calls in CGmap or extended bedMethyl format.

Inputs¶

Calling requires a prepared BAM and the same reference FASTA used for alignment.

Run methylation calling¶

bsbit call meth \
  -i sample.prep.bam \
  -r GRCh38.fa \
  -o sample.cgmap.gz \
  -f cgmap \
  -t 8

Common options¶

Option	Value	Default	Description
`-i`, `--input`	`PATH`	Required	Coordinate-sorted and indexed BAM
`-r`, `--reference`	`FASTA`	Required	Reference FASTA used to build the alignment index
`-o`, `--output`	`PATH`	Required	Path for methylation calls
`-f`, `--format`	`cgmap` or `bed`	Required	Methylation output format
`-c`, `--compress`	`BOOL`	`true`	Write BGZF-compressed output
`-t`, `--threads`	`N`	`1`	Number of regional calling workers

Advanced parameters¶

Option	Value	Default	Description
`--region`	`CONTIG:START-END`	All contigs	Genomic region to call
`--regions-file`	`BED`	None	BED file containing regions to call; conflicts with `--region`
`--min-bq`	`N`	`20`	Minimum base quality for an observation to be counted
`--min-mapq`	`N`	`20`	Minimum alignment MAPQ for an observation to be counted
`--min-depth`	`N`	`10`	Minimum qualified depth required at a site
`--cg-only`	—	Off	Restrict output to CpG sites
`--ignore-orphan`	—	Off	Skip paired reads without the SAM proper-pair flag

Target regions

A region uses CONTIG:START-END with 1-based inclusive coordinates. Separate multiple regions with commas:

--region chr1:1-100000,chr2:200001-300000

For many regions, use --regions-file, which accepts plain, gzip, or BGZF-compressed BED with 0-based half-open coordinates. It cannot be used together with --region.

Quality filters

Only bases that meet the base-quality threshold and reads whose alignments meet the mapping-quality threshold are included in calling. --ignore-orphan skips paired reads without the SAM proper-pair flag; single-end reads are retained. Sites with fewer than --min-depth qualified observations are omitted. Add --cg-only to omit CHG and CHH sites.

See the CLI reference for accepted ranges and complete parameter details.

Output¶

Calls follow the BAM contig order and are sorted by genomic position. The two strands of a CpG are reported separately. In conventional bisulfite sequencing, an unconverted cytosine is reported as methylated, without distinguishing 5mC from 5hmC.

See the CGmap and extended bedMethyl format descriptions for schemas and coordinate conventions.