Workflow

Build the reference index, process each sample through alignment and calling, then combine the methylation results into a matrix.

Index bsbit index

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Each sample

Align bsbit align

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Prepare BAM samtools

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Call bsbit call

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Combine bsbit combine

Stages

Stage	Command	Main input	Output
Index	bsbit index	Reference FASTA	Reusable .bsbit alignment index
Align	bsbit align	Index and FASTQ	Input-order BAM
Prepare BAM	samtools	Alignment BAM	Coordinate-sorted, duplicate-handled, indexed BAM
Call methylation or SNVs	bsbit call	Prepared BAM and matching reference	Methylation output and/or VCF
Combine	bsbit combine	Sorted per-sample methylation call files	Methylation level and/or count matrix

Sequencing data support

bsbit supports both directional and non-directional libraries with either single-end or paired-end data. Directional mode is the default; use --non-directional for non-directional libraries.

Preprocessed RRBS and targeted reads are accepted when chemistry and orientation match.

Limitations and roadmap

The following capabilities are not available in the current release and may be added in future:

• PBAT and other library protocols, including assay-specific preprocessing and interpretation
• Broader variant calling, including indels and haplotype-aware analysis
• Standard-stream (-) input and output
• CRAM and additional output formats
• ARM architectures, including Apple Silicon