> For the complete documentation index, see [llms.txt](https://help.connected.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.connected.illumina.com/illumina-ffpe-dna-prep-with-exome-2.5/additional-information/germline-variants.md). # Germline variants ## Exploring germline variants that might play a role in tumor analysis In somatic T/N analysis, DRAGEN uses a subtraction model for SNVs. VCF will only emit variants where there is no (or very limited) alternate reads in the normal sample. In other words, variants that are found in both the normal and somatic sample will NOT be emitted in the SNV/INDEL VCF (the output \*.hard-filtered.vcf.gz contains only somatic variants). To extract germline variants, it is recommended to run an additional analysis, the[ DRAGEN Germline pipeline](https://help.dragen.illumina.com/product-guides/dragen-v4.3/dragen-recipes/germline-wes), on the normal sample. Note that in this scenario, the germline and somatic variants are output from separate analyses, so the variants may need to be computationally combined. ### Germline variants & Germline tagging in T/N analysis #### Germline variants DRAGEN supports a command-line flag (`--vc-report-germline-variants`, also displayed as "Enable Small Variant Calling on Normal Samples" on the BioInsight platforms) in which the primary output VCF (\*.hard-filtered.vcf.gz) will contain both somatic and germline variants together. However, this setting is **NOT** equivalent to running a dedicated germline caller. The germline calls from T/N somatic mode are a useful approximation for things like LOH analysis and B-allele frequency (used by the Allele-Specific Copy Number (ASCN) module during CNV calling), but they lack the sensitivity tuning that a dedicated germline pipeline provides. #### Germline tagging DRAGEN supports a command-line flag (`--vc-enable-germline-tagging`, also displayed as "Enable Common Germline Variant Tagging" on the BioInsight platforms) that enables post-processing to look up variants in [public databases of human variation](https://help.dragen.illumina.com/dragen-v4.4/product-guide/dragen-v4.4/nirvana) to add INFO/GermlineStatus to the primary output VCF (\*.hard-filtered.vcf.gz). That is, where the VCF in T/N default analysis includes only somatic variants, some of these somatic variants may match a known allele from a database and then be tagged as germline when this flag is enabled. Germline tagging can be seen of as an additional layer of annotation: the matched normal provides one line of evidence (variant not detected in normal → label somatic), while the population database provides another piece of evidence (variant is seen in healthy people → label germline). Note that the germline tagging flag has implications for other settings. First, annotation must be enabled or DRAGEN will error. Second, if calculating TMB is enabled, the additional flag of `--tmb-skip-db-filter` must be set to false or DRAGEN will error. By default in T/N mode, TMB calculation skips the database filter because all variants are somatic in the primary output VCF and consequently included in the TMB calculation. However, if tagging variants as germline, then only those variants labeled as Somatic should be included in the TMB calculation. ### Germline and somatic variants in Tumor-only analysis The matched normal provides a more reliable indicator of somatic variants than comparisons with public database of variants. Database germline tagging for SNV incorrectly tags \~2% of germline variants as somatic and falsely classifies some somatic variants as germline (i.e. false positives and false negatives for somatic SNVs). Additionally, there is limited support for differentiating between germline and somatic CNVs. If a paired normal sample is available, the highest performance for the detection of both somatic and germline variants is achieved by implementing the Tumor/Normal Somatic Variant Calling workflow and additionally the dedicated Germline pipeline on the Normal sample. --- # Agent Instructions This documentation is published with GitBook. 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