> 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/tso500/dragen-tso-500-ctdna-guides/dragen-tso-500-ctdna-v2.6/analysis-methods/max-somatic-vaf.md).

# Maximum Somatic VAF

The maximum somatic variant allele frequency (MSAF) is the highest VAF of a confirmed somatic mutation. The MSAF is often used as a surrogate for tumor fraction, especially when the ctDNA tumor fraction is high.

{% hint style="danger" %}
Use caution when using MSAF as a surrogate for tumor fraction due to MSAF limitations:

* Reliable only at higher ctDNA fractions
* Does not account for aneuploidy
* Dependent on detectable somatic mutations
* Can be confounded by germline or clonal hematopoiesis (CH) variants even with filtering strategies in place

The MSAF algorithm in the DRAGEN TSO 500 ctDNA analysis software includes filtering of germline and clonal hematopoiesis variants but does not take into account aneuploidy.
{% endhint %}

## MSAF algorithm

The MSAF is determined using the following steps:

1. Somatic variants determined by the TMB algorithm are used as an input (variants having Status *Somatic* in the `{SampleID}_tmb.trace.tsv` file). Variants that are not in coding regions, MNVs and variants with depth below 500 are filtered out.
2. The remaining variants are ranked by the VAF in descending order
3. The VAF for the highest ranked confident somatic variants is output as MSAF. "Confident somatic variants" are determined analyzing the fragment size of the reads supporting the variants. Circulating tumor DNA (ctDNA) molecules are expected to be shorter, when compared to the normal cell-free DNA (cfDNA) molecules. If the fragment sizes of the reads supporting a variant are significantly shorter than non-supporting reads (p-value < 1x10-5), the variant is considered to be a confident somatic variant.
4. If no such variant exists, the VAF for the highest ranked COSMIC hotspot variant (with COSMIC count > 50) is output as MSAF.
5. If no such variant exists, the VAF for the 4<sup>th</sup> highest ranked variant is output as MSAF.

<figure><img src="/files/syLTO3s9XD88LlVUWuEC" alt=""><figcaption><p>MSAF algorithm in DRAGEN TSO 500 ctDNA Analysis Software</p></figcaption></figure>

## Tumor Fraction Estimation

To estimate the tumor fraction, the MSAF value should be multiplied by two and, if desired, expressed as a percentage. For instance, an MSAF value of 0.02 corresponds to an estimated tumor fraction of 4%.

## MSAF output files

The MSAF algorithm outputs results in several files:

1. Metrics Output File, `{SampleID}_MetricsOutput.tsv` as Max\_Somatic\_AF
2. TMB Max Somatic VAF file, `{Sample_ID}.tmb.msaf.csv` as MaxSomaticAF, using the same file format as the [TMB Trace File](https://help.connected.illumina.com/tso500/dragen-tso-500-ctdna-guides/dragen-tso-500-ctdna-v2.6/analysis-methods/pages/XDUcm9V56hKd9obLJqTa#id-3.-tmb-trace-file).


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