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End-to-end examples (MToolBox)

Prerequisites

Installation, reference bundles, and all dependencies must be completed beforehand.

➡️ Installation

Architecture

MToolBox supports x86_64 only. ARM-based systems, including Apple Silicon (M1/M2/M3), are not supported.

1. Before running mtDNA calling you must have a bam file coming from wes/wgs

Does it matter if I ran WES/WGS with GATK 3.5 or GATK 4.6?

No. CBIcall will detect and use the bam files produced by either version.
Just make sure that bam files are available — FASTQ input is not supported.

CBIcall expects BAM file infrom a previous run:

CNAG999_exome
└── CNAG99901P_ex  <--- ID taken from here
    └── cbicall_bash_w?s_single_gatk-* <- The script expects that you have a BAM file inside this directory

Note on nomenclature

Please see this page.

2. Create a parameters file

Create a YAML file, e.g. mit_single.yaml:

mode:            single
pipeline:        mit
workflow_engine: bash
gatk_version:    gatk-3.5
sample:          CNAG999_exome/CNAG99901P_ex

3. Run CBIcall

bin/cbicall -p mit_single.yaml -t 4
  • -p selects the YAML parameters file
  • -t sets the number of threads

4. Inspect outputs

After completion, you will find:

CNAG999_exome/CNAG99901P_ex/cbicall_bash_wes_mit_gatk-3.5_*/
  01_mtoolbox/
  02_browser/

5. Visualize variants in the browser

Please see:

02_browser/README.txt

The results are reported both as a HTML table and as downloadable files.

See snapshot

browser

Downloadable files:

  • mtDNA JSON A JSON file with the results from mit_prioritized_variants.txt.
  • Report: A tsv file including all the annotations for each variant. Name of the file mit_prioritized_variants.txt.
  • Haplog: A tsv file including the predicted haplogroup for each sample. Name of the file mt_classification_best_results.csv.
  • VCF: A text file consisting of all the variants in the VCF format. Name of the file VCF_file.vcf.

HTML table:

In this tab SG-ADVISER mtDNA displays a browsable table consisting of the most relavant fields relative to the variant annotation:

  • Sample: The full name of each sample.
  • Locus: The location on the mitochondrial chromosome.
  • Variant_Allele: The position in the mitochondrial chromosome + the alternative allele format.
  • Ref: The reference allele (mitochondrial reference genome: RSRS).
  • Alt: The alternative allele(s).
  • Aa_change: The amino acid change if the variant falls in a coding region.
  • GT: Genotype. 0:Ref, ≥1:Alt(s).
  • Depth: The number of times this position is covered by reads.
  • Heterop_Frac: The heteroplasmic fraction. Note that the confidence interval can be retrieved from the downloadable VCF file.
  • Other: For other fields please consult MToolBox's manual.

For advanced parameters, multi-sample analyses, mtDNA workflows and troubleshooting, see the Usage and FAQ sections.

1. Before running mtDNA calling you must have bam files coming from wes/wgs

Does it matter if I ran WES/WGS with GATK 3.5 or GATK 4.6?

No. CBIcall will detect and use the bam files produced by either version.
Just make sure that bam files are available — FASTQ input is not supported.

CBIcall expects BAM file from previous runs:

CNAG999_exome
└── CNAG99901P_ex  <--- ID taken from here
    └── cbicall_bash_w?s_single_gatk-* <- The script expects that you have a BAM file inside this directory

Note on nomenclature

Please see this page.

2. Create a parameters file

Create a YAML file, e.g. mit_cohort.yaml:

mode:            cohort
pipeline:        mit
workflow_engine: bash
gatk_version:    gatk-3.5
sample:          CNAG999_exome

3. Run CBIcall

bin/cbicall -p mit_cohort.yaml -t 4
  • -p selects the YAML parameters file
  • -t sets the number of threads

4. Inspect outputs

After completion, you will find:

CNAG999_exome/cbicall_bash_mit_cohort_rsrs_gatk-3.5*
  01_mtoolbox/
  02_browser/

5. Visualize variants in the browser

Please see:

02_browser/README.txt

The results are reported both as a HTML table and as downloadable files.

See snapshot

browser

Downloadable files:

  • mtDNA JSON A JSON file with the results from mit_prioritized_variants.txt.
  • Report: A tsv file including all the annotations for each variant. Name of the file mit_prioritized_variants.txt.
  • Haplog: A tsv file including the predicted haplogroup for each sample. Name of the file mt_classification_best_results.csv.
  • VCF: A text file consisting of all the variants in the VCF format. Name of the file VCF_file.vcf.

HTML table:

In this tab SG-ADVISER mtDNA displays a browsable table consisting of the most relavant fields relative to the variant annotation:

  • Sample: The full name of each sample.
  • Locus: The location on the mitochondrial chromosome.
  • Variant_Allele: The position in the mitochondrial chromosome + the alternative allele format.
  • Ref: The reference allele (mitochondrial reference genome: RSRS).
  • Alt: The alternative allele(s).
  • Aa_change: The amino acid change if the variant falls in a coding region.
  • GT: Genotype. 0:Ref, ≥1:Alt(s).
  • Depth: The number of times this position is covered by reads.
  • Heterop_Frac: The heteroplasmic fraction. Note that the confidence interval can be retrieved from the downloadable VCF file.
  • Other: For other fields please consult MToolBox's manual.