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 \
  -f cgmap \
  -t 8

Common options¶

Option	Argument	Default	Description
`-i`, `--input`	BAM path	Required	Coordinate-sorted and indexed BAM
`-r`, `--reference`	FASTA path	Required	Reference FASTA used to build the alignment index
`-o`, `--output`	Output path	Required	New path for methylation calls
`-f`, `--format`	`cgmap` or `bed`	Required	Methylation output format
`-c`, `--compress`	`true` or `false`	`false`	Write BGZF-compressed output when `true`
`-t`, `--threads`	Positive integer	`1`	Number of regional calling workers

Advanced options¶

Option	Argument	Default	Description
`--region`	`CONTIG:START-END`	All contigs	One 1-based inclusive genomic region
`--regions-bed`	BED path	—	BED3+ file for multiple regions
`--min-bq`	Integer from `0` to `93`	`20`	Minimum base quality for an observation to be counted
`--min-mapq`	Integer from `0` to `254`	`20`	Minimum alignment MAPQ for an observation to be counted
`--min-depth`	Positive integer	`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

Target regions restrict methylation calling to genomic intervals of interest. This is useful for capture panels, selected genes or loci.

Each --region selects one interval using 1-based inclusive coordinates; repeat the option to select additional intervals:

--region chr1:1-100000

For many intervals, use --regions-bed, which accepts plain, gzip, or BGZF-compressed BED3+ with 0-based half-open coordinates. Intervals supplied by both options are merged before calling, and overlaps do not duplicate observations or output calls.

Quality filters

bsbit filters evidence before counting methylated and unmethylated observations:

Alignment: Unmapped, secondary, supplementary, QC-failed, and duplicate-marked records never contribute. Alignments with MAPQ 255 or MAPQ lower than --min-mapq are also excluded. With --ignore-orphan, paired records lacking the SAM proper-pair flag are skipped.
Base: A base contributes only when its Phred quality is at least --min-bq. Where mates overlap, the fragment contributes at most one observation at each genomic position.
Site: A site is written only when at least --min-depth qualified observations remain. --cg-only additionally restricts output to CpG sites, omitting CHG and CHH sites regardless of their depth.

See the CLI reference for accepted ranges and complete option 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.