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megSAP - DNA analysis (tumor samples, tumor/normal pairs)

Analysis pipeline

Somatic analysis of tumor/normal DNA sample pairs and of tumor-only DNA samples is performed with somatic_dna.php.

Mapping single samples

The somatic pipeline requires requires tumor and normal sample BAM files.
Thus, they need to be mapped first using the analysis.php with the parameter -steps ma -somatic.
For more information, see the documentation at single sample DNA analysis.

Somatic variant calling (tumor/normal)

After mapping, the tumor and normal BAM files are used to perform somatic variant calling withformat.

The main parameters that you have to provide are:

  • t_bam - the tumor DNA sample BAM file
  • n_bam - the normal DNA sample BAM file (only required for tumor/normal analysis)
  • -out_folder - the output folder

Please have a look at the help:

> php megSAP/src/Pipelines/somatic_dna.php --help

Output

The main output files are listed below.

Variant calling step (vc, an):

  • somatic_var.vcf.gz - SNVs and short indels called with Strelka2 (sample pairs) or FreeBayes (tumor sample only)
  • somatic_var_annotated.vcf.gz and somatic.GSvar - annotated variants
  • somatic_var_structural.vcf.gz and somatic_var_structural.tsv - structural variants called with manta
  • somatic_bafs.igv - B-allele frequencies in IGV-compatible tabular format

Copy-number variation calling step (cn):

  • somatic_clincnv.tsv - copy-number variations called with ClinCNV

Microsatellite analysis step (msi):

  • somatic_msi.tsv - microsatellite instabilities called with MANTIS

Additional annotation steps

RNA annotation (an_rna): If you have data from the RNA pipeline, this data can be annotated in the an_rna step. This will annotate read depth, allele frequency and TPM from the RNA sample to each variant contained in the GSvar file. Provide the bam file using the parameter -t_rna_bam and the RNA sample name using the parameter -rna_id. This step also annotates reference gene expression from The Human Protein Atlas. Therefore, you have to specify a reference tissue type. It can be passed via the parameter -rna_ref_tissue.

CGI annotation (ci): This step will send the SNVs and CNVs from somatic.GSvar and somatic_clincnv.tsv to CancerGenomeInterpreter.org. The results will be downloaded into the out folder and annotated to both files. Provide a tumor type using the parameter -cancer_type for more accurate results, acronyms can be found on CancerGenomeInterpreter.org.

Somatic variant calling (tumor only)

Somatic variant calling (tumor only)

Somatic variant calling is also possible without normal sample.
However this leads to a lot of false-positive somatic variant calls because artefacts cannot be removed efficiently.
To use the tumor-only pipeline, simply skip the n_bam parameter of somatic_dna.php.

To remove false-positives, filtering on variant allele frequencies from public databases can be done.
This can be done interactively in GSvar or using VariantFilterAnnotations from ngs-bits.
For 1% AF filter specify this line in the filter definition:

Allele frequency    max_af=1

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