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megSAP - DNA analysis (tumor only)

Analysis pipeline

Somatic analysis of tumor-only DNA samples is performed with somatic_dna.php.

Mapping tumor sample

The somatic pipeline requires the tumor sample BAM file.
Thus, the tumor sample needs 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

After mapping, the tumor BAM file is used to perform somatic variant calling.

The main parameters that you have to provide are:

  • t_bam - the tumor DNA sample BAM file
  • -out_folder - the output folder

Please have a look at the help:

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

Somatic variant calling without normal sample leads to a lot of false-positive somatic variant calls because artefacts cannot be removed efficiently.

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

Tools used in this analysis pipeline

The tools used in this analysis can be found here: Somatic tools.

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.

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