CountMut

Unified ultra-fast strand-aware mutation counter — C backend, Python wrapper.

CountMut counts base/substitution ratios from BAM files with fast C core and a thin Python wrapper. It fuses the two classic ways of walking a BAM:

• pileup-based (bam_mplp_auto / pysam pileup) — fast, sees indels/ref-skips, general.
• read-walk (countmut's "no pileup") — walk reads directly, only touch the target sites.

Both produce identical output, and the tool can process whole genomes in parallel (threads).

Why it's fast & correct

• The hot loop (BAM read, pileup, per-(site,strand) base counting, mate-overlap dedup, quality/conversion classification) is in C (backend/countmut_core, built on the self-contained htslib subset from lh3/minipileup).
• Strand-aware (countmut biological-strand rule for paired-end reads).
• Paired-end overlap dedup: at an overlapping position a fragment is counted once, choosing the best mate by (mapq, read1, base-qual) — the thing minipileup gets wrong.
• Parallel: divides the genome into bins and processes them across threads (--threads).
• Memory-clean (verified under AddressSanitizer).

Install

pip install -e .
# or, to prebuild the C core:
make backend

Quick start

# strand-aware A->G mutation count (bisulfite / m6A style)
countmut -i in.bam -r ref.fa -o mut.tsv --ref-base A --mut-base G

# per-site base counts (perbase/mpileup style)
countmut -i in.bam -r ref.fa --mode base -o depth.tsv

# alleles -> VCF (minipileup style)
countmut -i in.bam -r ref.fa --mode allele --vcf -o allele.vcf

Modes

Filtering with expressions (-e / -p)

Filtering is done with samtools-style filter expressions — there are no separate --min-mapq/--min-baseq/--trim-* flags; write them as expressions instead.

• -e, --expression <STR> — per-base read filter (samtools SAM fields).
• -p, --pile-expression <STR> — per-site filter (pileup fields).

Grammar is the samtools filter=STRING expression language (C-style precedence, &&/||/!, bit fields, tags, regex). See docs/filter_grammar.md.

# keep high-quality, non-5prime, properly paired reads
countmut -i x.bam -r ref.fa -e "mapq >= 20 && bq >= 20 && dist5 >= 2 && flag & PROPER_PAIR"

# restrict to one RG group
countmut -i x.bam -r ref.fa -e "tag('RG') == 'sampleA'"

# report only A-reference sites with depth >= 5 and > 2 G alleles
countmut -i x.bam -r ref.fa -p "ref == 'A' && depth >= 5 && g > 2"

Read variables: mapq, flag (+ flag.dup, flag.unmap, ...), qname, pos, endpos, pnext, rname, mrname, tlen, qlen, rlen, ncigar, seq, qual, sclen, hclen, bq, dist5/dist3, strand, [NM]/[RG] tags, avg(qual), exists([NM]), sqrt(mapq), ...

Site variables: depth, pos, ref, a c g t n, ins, del, ref_skip, fail.

When -e/-p is given, counting runs on the Python engine (the C core cannot evaluate strings). Without expressions, the fast C backend is used.

Engine selection

--engine {auto|read-walk|pileup} (default auto):

• auto → read-walk for mutation (targeted sites), pileup for base/allele.
• read-walk / pileup → force a strategy.

Options

-i/--input, -r/--reference, -o/--output
--mode {mutation,base,allele}   --engine {auto,read-walk,pileup}
--region, --threads/-t
--ref-base, --mut-base, --pad, --save-rest
--split-strand, --count-indels, --min-depth, --min-allele-support, --vcf
-e/--expression, -p/--pile-expression

Design

countmut/
  cli.py               rich CLI (routes to the backend)
  backend.py           builds/loads the C binary; calls it; Python fallback
  pipeline.py          region binning + parallel dispatch
  model.py             FilterConfig / MutationConfig / StrandConfig / EngineConfig
  engine_readwalk.py   read-walk engine
  engine_pileup.py     pileup engine
  formatter.py         TSV/VCF renderers
  expression.py        samtools-style filter-expression engine
backend/
  countmut_core.c      the computation core (htslib subset)
  countmut_core_main.c CLI wrapper
  Makefile             builds the `countmut_core` binary

Both engines fill the same SiteColumn (per-site, per-strand base counts), so the two "ways" are interchangeable; the C backend implements the pileup engine.

References this tool learns from

• minipileup — pileup walk, filters, allele counting
• perbase / pbr — mate-aware overlap dedup, base counts
• countmut — biological strand, bisulfite NS/Zf/Yf tiers
• mpileup / cpup — base-count output
• samtools --input-fmt-option filter=STRING — the expression grammar

License

MIT