Panel of Normals (PON)

BALSAMIC provides a functionality to generate a Panel of Normals (PON) for more accurate copy-number filtering of false positives and that can be used as an input for the CNVkit variant caller.

For a more detailed PON use case, please refer to the following documentation:

• CNVkit

• Illumina DRAGEN

• GATK

PON Generation

When creating a new PON reference file, the next steps have to be followed:

1. Identify the samples to be included in the PON and add their fastq files to the fastq directory

Note

One needs to fetch normal samples coming from the same origin, tissue or blood

2. Generate the <CASE_ID>_PON.json configuration file:

balsamic config pon --case-id <CASE_ID> --balsamic-cache </path/reference_cache/> --analysis-dir </path/analysis/> --fastq-path </path/fastq/> --panel-bed </path/panel.bed>

3. Run the BALSAMIC PON workflow:

balsamic run analysis -s </path/analysis/<CASE_ID>/<CASE_ID>_PON.json -r

4. Check for the PON reference finish and output files:

/path/analysis/analysis_PON_finish
/path/analysis/cnv/<panel_name>_CNVkit_PON_reference_<version>.cnn

Using the PON during analysis

BALSAMIC can use a PON reference file if its provided while running CNVkit analysis:

balsamic config case --case-id <CASE_ID> --pon-cnn /path/analysis/cnv/<panel_name>_CNVkit_PON_reference_<version>.cnn --balsamic-cache </path/reference_cache/> --analysis-dir </path/analysis/> --panel-bed </path/panel.bed> --tumor-path </path/tumor.fastq>

Note

In the absence of a PON reference file, CNVkit is capable of generating a flat reference (tumor-only) or normal reference (tumor-normal) file on its own to correct for GC content and regional coverage