Variant Allele Fraction: What the Percentage on a Report Means

The Basic Calculation

If 1,000 sequencing reads cover a position and 300 contain the variant, the variant allele fraction is 30 percent. It is a direct measurement from the data, distinct from the biological question of what fraction of cells carry the mutation.

For a germline heterozygous variant in a pure normal sample, the expected fraction is about 50 percent; for a homozygous variant, close to 100 percent.

What Lowers a Somatic VAF

In a tumour sample, a clonal heterozygous mutation present in every cancer cell is diluted by normal cells. At 40 percent tumour purity, its fraction is roughly 20 percent. Lower purity lowers it further.

A subclonal mutation, present in only some cancer cells, is lower still. So a low fraction can mean low purity, subclonality, or both, and these have different implications.

What Raises or Distorts a VAF

Copy-number changes shift the fraction. If the mutant allele is amplified, or the wild-type allele is deleted (loss of heterozygosity), the variant can approach or exceed 50 percent even in an impure sample. This is common for tumour-suppressor mutations such as TP53.

PCR amplification bias and sequencing artefacts can also distort low-level fractions, which is one reason very low VAF calls need orthogonal confirmation.

VAF in Blood-Based Testing

In circulating tumour DNA, the fraction reflects how much tumour DNA is shed into the blood, which varies widely. A true driver can appear at well under 1 percent.

Blood tests can also pick up variants from clonal haematopoiesis, age-related expansion of blood-cell clones, typically in genes such as DNMT3A, TET2 and TP53, which can be mistaken for tumour signal.

Interpretation Notes

A low VAF does not automatically mean a variant is unimportant; a subclonal resistance mutation at 2 percent can still explain treatment failure.

VAF should be read together with tumour purity, local copy number and the assay's limit of detection, not in isolation.

Key Takeaways

• ·VAF is the fraction of reads carrying a variant, a measurement not a biological quantity.
• ·Low tumour purity and subclonality both reduce a somatic VAF.
• ·Copy-number gain or loss of heterozygosity can raise it toward or above 50 percent.
• ·In blood, low VAF is expected, and clonal haematopoiesis can mimic tumour variants.