MToolBox

A bioinformatics pipeline for human mtDNA analysis from high throughput and automated sequencing data

                                                                                                          

What is MToolBox?   MToolBox software implements an effective computational strategy for human mitochondrial genomes assembling and analysis from mitochondria-targeted and off-targeted sequencing data. A filtering based on quality score (≥25), depth of coverage (≥5) and exclusion of reads mapping on both nuclear and mitochondrial sequences (NumtS) is carried out for variant calling and genomes reconstruction from Whole Exome and Genome Sequencing studies. Functional annotation and prioritization analysis of detected variants are also performed through variants recognition by the two mitochondrial reference sequences commonly used, the revised Cambridge Reference Sequence (rCRS) and the Reconstructed Sapiens Reference Sequence (RSRS), and by a related macro-haplogroup consensus sequence (MHCS) after haplogroup assignment for each analyzed sample. Traditional Sanger sequencing data may be also submitted for this analysis. A summary file and a compressed folder (.tar.gz) containing all the output files generated by the pipeline can be downloaded once the analysis is completed, including a VCF file and FASTA files for each sample analyzed. The summary of the analysis include -        statistics about coverage of reconstructed mitochondrial genomes (for NGS data) -        haplogroup(s) prediction -        functional annotation of prioritized variants.   For details, see MToolBox output files.   How to use MToolBox -        Input format -        Reference Sequences -        Filtering and extra options Output files Command line tool   MToolBox workflow

 

How to use MToolBox

 

MToolBox output files

MToolBox default output files are:

- VCF_file.vcf: contains all the mitochondrial variant positions against the mitochondrial reference sequence identified in the whole set of submitted samples and other meta-information

- mt_classification_best_results.csv: reports for each sample the best haplogroup prediction. If the sorting results in more than one best haplogroup prediction with equal probability, the output will enclose all of them

- prioritized_variants.txt: brief annotation of prioritized variants (variants recognized by the 3 reference sequences, sorted in ascending order of nucleotide variability scores)

- OUT_<sample_name> folders, produced for each sample, containing:

- outmt.sam: reads mapped onto the human mitochondrial DNA

- logmt.txt: GSNAP log file for mitochondrial DNA mapping

- outmt.fastq: fastq file of single reads extracted from outmt.sam file

- outmt1.fastq: fastq file of paired reads extracted from outmt.sam file

- outmt2.fastq: fastq file of paired reads extracted from outmt.sam file

- outhumanS.sam: single reads mapped onto the entire human genome

- loghumanS.txt: GSNAP log file for human genome mapping (single reads)

- outhumanP.sam: paired reads mapped onto the entire human genome

- loghumanP.txt: GSNAP log file for human genome mapping (paired reads)

- OUT.sam: alignments of reads uniquely mapped on mitochondrial genome

- OUT2.sam: alignments of reads uniquely mapped on mitochondrial genome, after processing with IndelRealigner and/or MarkDuplicates. This file will be generated anyway, even if these two processes have been disabled

- mtDNAassembly-table.txt: the main table describing the assembly position by position

- mtDNAassembly-Contigs.fasta: a fasta file including all reconstructed contigs

- mtDNAassembly-coverage.txt: a text file including the coverage per contig and per mitochondrial known annotation

- logassemble.txt: the log file of the assembleMTgenome.py script

- sorted.csv: a table with each haplogroup whose prediction is > 90%

- merged_diff.csv file: reports the SNPs between the query genome and each of the three sequences RSRS, rCRS and hg_MHCS (haplogroup specific Macro-Haplogroup Consensus Sequence)

- <sample_name>.csv: a table where, for all the haplogroups present in the Phylotree Build 15, are reported the same data as in the file sequence_name>.sorted.csv, except for the Missing Sites field

- annotation.csv: a further elaboration of the file merged_diff.csv, providing, for each mt variant allele between the query genome and each of the three sequences RSRS, rCRS and hg_MHCS, several annotations based on different resources: HmtDB, Mitomap, pathogenicity prediction software MutPred, Polyphen-2 and SNPs&GO, 1000 Genomes variants frequency, links to OMIM, dbSNP and Mamit-tRNA. An overall disease score ranging between 0 and 1 is also provided. It was calculated as a weighted average of all the pathogenicity prediction scores considering both the number of times each method gives the right prediction and the number of times each method gives the best probability compared with the other ones on a training dataset of 53 non synonymous variants selected among mitochondrial diseases or cancer associated mutations (Mitomap). The higher the score, the higher the possibility the variant site is deleterious.

Further explanations of files content can be found here.

 

MToolBox command line software

For large datasets, the user could use the command line version of MToolBox. It works on Unix systems and requires Python 2.7 and the preliminary installation of SAMtools, GSNAP and MUSCLE.

 

How to cite

If you use MToolBox, please cite:

Claudia Calabrese*, Domenico Simone*, Maria Angela Diroma, Mariangela Santorsola, Cristiano Gutta', Giuseppe Gasparre, Ernesto Picardi, Graziano Pesole, Marcella Attimonelli.

MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing. Bioinformatics 2014; doi:10.1093/bioinformatics/btu483.