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Mutserve Tests

Mutserve2 is a variant caller for the mitochondrial genome to detect homoplasmic and heteroplasmic sites in sequence data.

For easier execution and pre- and postprocessing steps, mutserve2 has been integrated into the mtDNA-Server 2

Quick Start

Mutserve requires sorted and indexed CRAM/BAM files as an input.

curl -sL | bash


Available Tools

Currently two tools are available.

  • call: Variant Calling of homoplasmic and heteroplasmic positions.
  • annotate: Annotation of mutserve variants (generated with mutserve call).

Mutserve Variant Calling

curl -sL | bash
./mutserve call --reference rCRS.fasta --output HG00096.vcf.gz --threads 4 *.bam 

Please use this reference file when using BAQ (disabled by default since v2.0.0).

Mutserve Annotation

Mutserve allows to annotate the variant file (.txt) with a predefined annotation file

./mutserve annotate --input variantfile.txt --annotation rCRS_annotation_2020-08-20.txt --output AnnotatedVariants.txt


Parameter Default Value / Comment Command Line Option
Input Files sorted and indexed BAM/CRAM files
Output Name output file; supported: *.txt, *.vcf, *vcf.gz --output
Reference reference file --reference
Threads 1 --threads
Minimum Heteroplasmy Level 0.01 --level
Define specific mtDNA contig in whole-genome file null --contig-name
Output Fasta false --writeFasta
Output Raw File false --writeRaw
MappingQuality 20 --mapQ
BaseQuality 20 --baseQ
AlignmentQuality 30 --alignQ
Enable Base Alignment Quality (BAQ) false --baq
Disale 1000 Genomes Frequence File false --noFreq
Call deletions (beta) false --deletions
Call insertions (beta) false --insertions
Disable ANSI output --no-ansi
Show version --version
Show help --help

Output Formats

Tab delimited File

By default (--output filename does not end with .vcf or .vcf.gz) we export a TAB-delimited file including ID, Position, Reference, Variant & VariantLevel. Please note that the VariantLevel always reports the non-reference variant level. The output file also includes the most and second most base at a specific position (MajorBase + MajorLevel, MinorBase+MinorLevel). The reported variant can be the major or the minor component. The last column includes the type of the variant (1: Homoplasmy, 2: Heteroplasmy or Low-Level Variant, 3: Low-Level Deletion, 4: Deletion, 5: Insertion). See here for an example.


If you want a VCF file as an output, please specify --output filename.vcf.gz. Heteroplasmies are coded as 1/0 genotypes, the heteroplasmy level is included in the FORMAT using the AF attribute (allele frequency) of the first non-reference allele. Please note that indels are currently not included in the VCF. This VCF file can be used as an input for


The focus of mutserve is currenly on SNP calling and not on indels. Please checkout mtDNA-Server 2 to combine SNV with InDel Calling.


Sebastian Schoenherr


Weissensteiner H, Forer L, Fendt L, Kheirkhah A, Salas A, Kronenberg F, Schoenherr S. 2021. Contamination detection in sequencing studies using the mitochondrial phylogeny. Genome Res. 31: 309-316

Weissensteiner H, Forer L, Fuchsberger C, Schöpf B, Kloss-Brandstätter A, Specht G, Kronenberg F, Schönherr S. 2016. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud. Nucleic Acids Res 44: W64–9.