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bwameth.py

bwameth.py is a Python script written by Brent Pedersen that implements bisulfite sequencing (BS-Seq) alignment using the in-silico three-letter genome approach. It converts all cytosines to thymines in both the reference and the reads (C-to-T on the forward strand, G-to-A on the reverse), aligns the converted sequences with bwa-mem (or optionally bwa-mem2), and then recovers the original read sequence from the aligner’s tag output to tabulate methylation. bwameth.py supports single-end and paired-end reads from the directional bisulfite protocol and is published at https://arxiv.org/abs/1401.1129.

When you’d use it

Use bwameth.py when you need a battle-tested, community-supported bisulfite aligner that runs on top of the standard bwa-mem or bwa-mem2 you have already installed, and when you prefer a Python wrapper over a self-contained binary. It also remains the reference for downstream tabulation tools such as MethylDackel and SNP callers such as biscuit that expect the bwameth.py output format. For the actual methylation tabulation and variant calling steps, bwameth.py’s author recommends those dedicated tools rather than the tabulation utilities bundled with the original script.

How it relates to bwa-mem3

bwa-mem3 mem --meth is a single-binary drop-in replacement for the bwameth.py alignment pipeline. It inlines the C-to-T and G-to-A conversion, runs the bwa-mem3 alignment engine (with all of its correctness fixes and SIMD speedups), rewrites the @SQ headers to collapse the per-strand contig pairs back to canonical chromosome names, applies chimera QC, and emits a @PG ID:bwa-mem3-meth header. The output BAM is compatible with the same downstream tabulation tools that consume bwameth.py output. The Methylation Reference section documents the full implementation in detail, including the YS:Z:, YC:Z:, and YD:Z: tags and the --set-as-failed and --do-not-penalize-chimeras flags.

Tip — Interop with the bwameth.py c2t step

If your pipeline already performs its own C-to-T conversion before alignment, see Interop with external bwameth.py c2t for how to pass pre-converted reads to bwa-mem3 mem --meth without double-conversion.

See also: Methylation Reference: Overview · Quick start: methylation alignment · Best Practices — Methylation defaults · Interop with external bwameth.py c2t