Methylation Defaults

bwa-mem3 mem --meth ships with scoring and filtering defaults aligned with the bwameth.py reference implementation (in the default collapsed scoring mode). This page is prescriptive: when to keep those defaults and when to override them. For what the flags are and how the scoring modes work, see the reference:

• Scoring modes (collapsed vs genomic) and the full --meth flag set — Methylation Reference → Flags.
• Placement compatibility with bwameth.py (the default collapsed mode is a placement drop-in, not byte-identical — ~1% of records differ in POS/CIGAR/MAPQ, so re-validate if you are pinned to a specific bwameth release) — bwameth.py drop-in mapping.

When to keep the defaults

For standard whole-genome bisulfite sequencing (WGBS) workflows, the defaults (collapsed scoring plus -B 2 -L 10 -U 100 -T 40 -M -C) are appropriate as-is. They were derived from the bwameth.py codebase and are expected by most downstream methylation callers. Unless you have a specific reason to deviate, use:

bwa-mem3 mem --meth -t 16 ref.fa R1.fq.gz R2.fq.gz \
  | samtools sort -@ 4 -o out.bam -
samtools index out.bam

When to override

Low-coverage or targeted bisulfite sequencing. If your library covers a small target region and insert sizes are more variable, consider lowering -T (e.g. -T 20) to recover short or soft-clipped alignments in the target.

Amplicon bisulfite sequencing. Amplicon reads have uniform insert sizes; the default -U 100 is appropriate. However, if your amplicons are short (< 100 bp), consider lowering -L further to reduce clipping at read ends.

Non-standard conversion chemistry. Some library preparations use only one strand conversion (C→T only, not G→A). In such cases, --set-as-failed r suppresses alignments to the reverse-complement strand, which reduces noise from strand-ambiguous alignments:

bwa-mem3 mem --meth --set-as-failed r -t 16 ref.fa R1.fq.gz R2.fq.gz \
  | samtools sort -@ 4 -o out.bam -

Chimera QC is opt-in (off by default, matching Bismark). Leave it off for standard directional EM-seq / WGBS. Turn it on for PBAT / scBS-Seq libraries (where intra-fragment chimerism is common) or when you want bwameth.py-equivalent flagging — pass --chimera-qc (see Flags for the exact heuristic):

bwa-mem3 mem --meth --chimera-qc -t 16 ref.fa R1.fq.gz R2.fq.gz \
  | samtools sort -@ 4 -o out.bam -

Note — Overriding scoring defaults

Scoring flags supplied on the command line override the defaults set by --meth. For example, bwa-mem3 mem --meth -B 4 ... uses -B 4 (not 2). Order does not matter: the defaults are applied after the whole command line is parsed, so -B 4 --meth and --meth -B 4 behave identically.

This applies to -B, -L, -U, and -T. It does not apply to -M and -C: bwa has no option that unsets either one, so --meth sets them unconditionally and they cannot be turned off from the command line. See Flags → -M and split alignments.

Downstream tool compatibility

The --meth output BAM carries the Bismark XR:Z / XG:Z / XM:Z tag set, so it feeds Bismark-aware callers directly — bismark_methylation_extractor, methylKit, methtuple, DMRfinder, epialleleR, MethylDackel, and biscuit per-read tools have all been used successfully. See SAM tags for the tag definitions and which tools read which tag.

See also: Methylation Reference: Overview · SAM tags: XR, XG, XM · Flags: –set-as-failed, –chimera-qc · Quick start: methylation alignment · Output format