mtDNA-Server 2

mtDNA-Server 2 provides a free service for the analysis of human mitochondrial DNA data, currently focusing on reliable identification of heteroplasmy (>= 1%) and contamination. We also provide post-processing guidelines that should be applied after each automated analysis.


Run a job using our cloud web service or checkout the source code on GitHub.


First, users upload BAM files and an Input Validation step is executed. For all samples, which are passing Input Validation, a FastQC report is created, summarized with MultiQC. Next, one of the available variant callers (mutserve or Mutect2) can be selected. Detected variants are annotated and a contamination check as well as a haplogroup assignment process are executed (using the previously published haplocheck and haplogrep3 tools from our institute). All created files are finally summarized in an interactive HTML report.

Overall Architecture


The sign-up process on mitoverse is straight-forward. After accessing the landing page, click on "Sign up". Mitoverse allows to register with or without an email. In case no email is specified, mitoverse does not send a job status email.

Sign up

Job Submission

After sign-up is completed, user are able to submit new jobs. Users can submit BAM files and specify numerous parameters:

Input Files

The "Input Files" field enables you to select one or multiple mtDNA aligned reads in BAM Format. These files contain the genetic information necessary for the analysis performed by the mtDNA-Server 2.


In the "Mode" field, you can select the mode for analysis. Options include fusion, mutect2, and mutserve. Choosing the appropriate mode ensures that the analysis is tailored to your specific requirements.

Detection Limit

The "Detection Limit" field allows you to select the detection limit for the analysis. This parameter determines the sensitivity of the analysis to detect mutations or variations in the input data.

Apply Coverage Estimate

With the "Apply Coverage Estimate" field, you can choose whether to apply coverage estimation. This option helps in assessing the depth and uniformity of coverage across the input data, aiding in the accuracy of the analysis results.

Coverage Subsampling

Specify the desired mean coverage by entering the number of coverage you want to use. mtDNA-Server 2 automatically subsamples the uploaded reads to achieve this mean coverage. Enter "0" to deactivate coverage subsampling.

Minimal Base Quality

In the "Minimal Base Quality" field, specify the minimal base quality required for analysis. This parameter ensures that only high-quality bases are considered during the analysis process, improving the reliability of the results.

Minimal Map Quality

The "Minimal Map Quality" field allows you to specify the minimal map quality required for analysis. This parameter helps filter out reads with poor mapping quality, ensuring that only accurately mapped reads contribute to the analysis. |

Submit a job

Job Monitoring

Mitoverse returns constant feedback to the users about the current job status (waiting, running, finished) and also return details about each job step.

Submit a job

Job Results

All results are available in the Results tab. This currently includes a QC-report, the annotated variants, haplogroup and sample information as well as an interactive report (report.html).



After the job has been finished, users can download the interactive report to explore the data in detail which can also be easily shared with collaborators without a login.


By clicking on a sample name, a new tab opens with more details of the sample and all detected variants and heteroplasmies:



The "Variants" section includes files related to variant analysis.

the file variants.annotated.txt contains detailed information about the detected variants, including annotations. It is a tab delimted file and has the following columns:

Column Description
Mutation Defines variant with POS and ALT allele
POS Position of the variant according to the rCRS, see rCRS_annotated
REF Reference allele of the rCRS, see also rCRS_annotated
ALT Alternative allele observed on the current position
Substitution Type of Substitution: Transition (A>G, G>A, T>C, C>T) or transversion (other)
Maplocus 40 different loci, including 2 MT-DLOOP(1,2), 2 MT-RNR1(2), 13 genes MT-ATP6 -> MT-ND6, 22 tRNA and noncoding (empty)
Category 5 different categories: tRNA, rRNA, Coding, Control Region and “-” (noncoding)
Phylotree17_haplogroups How many haplogroups in phylotree 17 have a variant
Phylotree17_clades How many different occurrences (fluctuation rate) in different clades can be observed
HaploGrep2_weight Weight based on the log-transformed value on the Phylotree17_clades. Value between 1 (highest value) and 10 (only occurring in 1 clade)
RSRS_SNP Is this SNP a RSRS defining SNP, see RSRS_vs_rCRS 1=yes, 0=no
KGP3_SNP Was this SNP observed in the 1000 Genomes Project Phase 3 (low-coverage) data? 1=yes, 0=no
AAC Amino Acid Change, denoted with the short Amino Acid Symbol, position on protein and the new amino acid change, e.g. M1L
CodonPosition Position of the codon defining the mRNA sequence, values: 1,2,3 or NA
AminoAcid The amino acid encoded by the reference based codon, e.g. M for Methionine
NewAminoAcid The amino acid encoded by the alternative codon, e.g. L for Leucine
AminoAcid_pos_protein Amino acids position on the produced protein
MutPred_Score Pathogenicity Score based on MutPred – see MutPred – values between 0 (benign) and 1 with values > 0.5 potentially deleterious
mtDNA_Selection_Score Pathogenicity Score as presented in Pereira et al, Pereira et al, log-transformed MutPred score – values between 0 and ~ 3 - values > 0.5 potentially deleterious
CI_MitoTool Conservation Index (CI) as used in MitoTool, see 10.1016/j.mito.2010.09.013 and Ruiz-Pesini et al
OXPHOS_complex Indicates whether a variant is on a gene encoding one of the  OXPHOS complexes I, III, IV or V
NuMTs_dayama SNP observed in the 1000 Genomes Project Phase 1 data occuring as part of a NUMT (Dayama et al 10.1093/nar/gku1038) ? Numbers indicating how many fragments with SNP were found
Helix_count_hom Homoplasmic variant count in HelixMTdb
Helix_count_het Heteroplasmic variant count in HelixMTdb (see previous entry)
Helix_vaf_hom Variants Allele Frequency of homoplasmic variants in HelixMTdb = count / (n=~195,000)
Helix_vaf_het Variants Allele Frequency of heteroplasmic variants in HelixMTdb = count / (n=~195,000)
Helix_haplogroups Haplogroups (major clades) found with the variant in HelixMTdb e.g. H:7 indicates that haplogroup H was found to harbour the variant in 7 samples
rCRS_Surr_seq Surrounding nucleotides based on the POS of the current variant on the reference sequence rCRS e.g. (CCCTC[T/A]AAATC)
LowComplexityRegion Checks if rCRS_Surr_seq includes homopolymeric stretches of length 4 or longer (0 = no, 1=yes)
DuplSeq_rCRS Is this motif (rCRS_Surr_seq) length 11bps found on a different position on the rCRS (0=no, 1=yes)
DuplSeq_rCRS_pos If motif is found as duplicate (see previous field), coordinates of the position on the rCRS are provided here

Auxiliary Files

The "Auxiliary Files" section includes additional files generated during the analysis.


Weissensteiner Hansi, Lukas Forer, Christian Fuchsberger, Bernd Schöpf, Anita Kloss-Brandstätter, Günther Specht, Florian Kronenberg, and Sebastian Schönherr. mtDNA-Server: Next-Generation Sequencing Data Analysis of Human Mitochondrial DNA in the Cloud. Nucleic Acids Research: gkw247. doi:10.1093/nar/gkw247. 2016