Minipileup is a simple pileup-based variant caller. It takes a reference FASTA and one or multiple alignment BAM as input, and outputs a multi-sample VCF along with allele counts:

samtools faidx ref.fa       # index FASTA; bgzip'd FASTA is not supported
minipileup -yf ref.fa aln1.bam aln2.bam > var.vcf

You can adjust mapping quality, base quality, alignment length and allele count thresholds, or specify regions on the command line.

Minipileup does not use realignment or local reassembly and cannot compete with full-pledge variant callers such as GATK on typical input. Nonetheless, you might find it useful for counting alleles in applications GATK is not designed for. In principle, you can parse the samtools mpileup output to generate a VCF but minipileup is faster and more convenient.

Minipileup is adapted from the htsbox pileup command which was initially implemented in 2012 and has been a tool I frequently use to investigate alignment data.

Minipileup briefly follows three steps:

1. Read filtering. This step controls reads used for pileup. You can filter reads by mapping quality (-q), alignment length (-l and -S) or by region (-b).

2. Allele counting. Initial pileup is generated with the htslib bam_mplp_auto() API. At each position, alleles across all input BAMs are grouped and the number of supporting reads for each allele is calculated. Bases of low quality (-Q) or close to reads ends (-T) may be ignored. Minipileup drops alleles supported by too few reads (-s and -a).

3. Output. In the variant VCF mode (-vc and -f), a site is outputted if a non-reference allele remains. The VCF shows the number of supporting reads of each allele in each BAM. Strand information can be optionally obtained (-C).