Call SNVs¶

bsbit call snp identifies genetic variants from bisulfite sequencing data. Currently, it reports only diploid single-nucleotide variants in VCF format.

Inputs¶

Calling requires a prepared BAM and the same reference FASTA used for alignment. One BAM represents one biological sample.

Run SNV calling¶

bsbit call snp \
  -i sample.prep.bam \
  -r GRCh38.fa \
  -o sample.vcf.gz \
  -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 the VCF output
`--sample-name`	`NAME`	BAM `SM` or filename stem	VCF sample name
`-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
`--min-alt-count`	`N`	`2`	Minimum informative ALT count for candidate selection and `PASS`
`--min-alt-fraction`	`P`	`0.1`	Minimum fraction of qualified depth supporting the strongest ALT
`--min-aq`	`N`	`30`	Minimum ALT-presence quality required for `PASS`
`--min-gq`	`N`	`0`	Minimum genotype quality required for `PASS`; `0` disables `LowGQ`
`--heterozygosity`	`P`	`0.001`	Prior probability that a site differs from the reference
`--underconversion-rate`	`P`	`0.0025`	Probability that an unmethylated cytosine remains unconverted
`--overconversion-rate`	`P`	`0`	Probability that a methylated cytosine is converted
`--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. --min-depth sets the qualified depth required for candidate evaluation, while --min-gq adds LowGQ when genotype confidence is below its threshold. Filtered records remain in the VCF.

Allele thresholds

The strongest non-reference allele must meet both --min-alt-count and --min-alt-fraction before a site is evaluated as an SNV candidate. After genotyping, each selected ALT must also have at least --min-alt-count bisulfite-informative observations; otherwise the call is marked LowAD.

Allele quality (AQ) is the Phred-scaled confidence that a selected ALT is present. A selected ALT below --min-aq adds LowAQ. Increasing these thresholds requires stronger ALT evidence but may reduce sensitivity.

Conversion rates and prior

--underconversion-rate is the probability that an unmethylated cytosine fails to convert and is observed as C instead of T. --overconversion-rate is the probability that a methylated cytosine converts unexpectedly and is observed as T instead of C. The caller uses both rates to distinguish conversion errors from SNV evidence.

--heterozygosity sets the prior probability that a site differs from the reference. Use validated assay-specific estimates when the default rates are not appropriate.

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

Run joint calling¶

bsbit call joint writes methylation calls and SNVs from one pass:

bsbit call joint \
  -i sample.prep.bam \
  -r GRCh38.fa \
  -m sample.cgmap.gz \
  -f cgmap \
  -v sample.vcf.gz \
  -t 8

Shared region, quality, compression, and thread settings apply to both outputs. Add --cg-only to limit only the methylation output to CpG sites. The methylation and VCF paths must be different. See the bsbit call joint CLI reference for joint-specific options.

Output¶

The VCF records GT, GQ, AQ, allele depths, and bisulfite-strand evidence. AQ measures confidence that an ALT is present; GQ measures confidence in the complete diploid genotype. See VCF variant calls for the full fields and filter definitions.

Compressed VCF is tabix-compatible. Create an index when downstream tools require random access:

tabix -p vcf sample.vcf.gz

Once indexed, use standard VCF tools such as bcftools to filter, query, normalize, or combine calls. The prepared genotype data can then be integrated with phenotype or methylation measurements for downstream analyses such as genome-wide association studies (GWAS), allele-specific methylation analysis, and methylation quantitative trait locus (mQTL) mapping.