Variant Allele Frequency (VAF) is a standard metric used when sequencing an individual’s DNA. This simple ratio provides deep insight into the proportion of a specific genetic change present within a sample of cells. VAF determines not just if a change exists, but exactly how much of the sample harbors that change. The precise quantification offered by VAF helps distinguish between different types of genetic phenomena and guides medical decisions in areas from oncology to hereditary disease screening.
Defining the Variant Allele Frequency
Variant Allele Frequency is a quantitative measure that expresses the abundance of a specific genetic alteration within a DNA sample. To understand VAF, it is helpful to first define an allele, which is one of two or more forms of a gene. A “variant” is a specific DNA sequence that differs from the reference genome, representing a change in the genetic code at a particular location.
VAF is calculated as a ratio: the number of sequencing reads that show the variant divided by the total number of reads covering that specific genomic location. For instance, if 50 out of 100 reads show a specific variant, the VAF is 50 percent. In a standard inherited genetic test, a VAF of approximately 50 percent is expected for a heterozygous change, meaning the variant is present on one of the two chromosome copies in every cell.
When dealing with a sample that contains a mix of cells—some with the variant and some without—the VAF will be less than 50 percent. Consider a tumor biopsy where only a fraction of the cells carry a particular cancer-driving mutation; if 10 percent of the DNA analyzed comes from mutated tumor cells, the VAF will register around 10 percent. This percentage is a direct reflection of the physical proportion of altered DNA molecules in the entire sample.
How VAF is Measured in the Laboratory
The determination of Variant Allele Frequency relies primarily on Next-Generation Sequencing (NGS). This technology allows researchers to rapidly read millions of short DNA fragments, called reads, which are then aligned back to a reference human genome map. The lab process begins with meticulous sample preparation, which can involve DNA extraction from a solid tissue biopsy or from a liquid biopsy containing circulating free DNA.
Once the DNA is sequenced, specialized bioinformatics software performs the VAF calculation by counting the occurrences of the specific variant base pair against the total number of times that genomic position was sequenced. A factor known as “sequencing depth” plays a large role in the reliability of the resulting VAF measurement. Sequencing depth refers to the average number of times a specific nucleotide position is read during the experiment.
High sequencing depth is necessary to accurately detect variants that are present at low frequencies, such as those below 5 percent. For example, if a variant exists in only 1 percent of the DNA molecules, the sequencing platform must read that location many hundreds or even thousands of times to ensure the detection is statistically reliable.
The Essential Clinical Meaning of VAF in Cancer
In oncology, VAF is a measure that informs diagnosis, prognosis, and treatment monitoring, dealing with somatic mutations that arise during a person’s lifetime. The frequency provides insight into tumor heterogeneity, which describes the existence of different populations of cancer cells, each with its own set of mutations, within a single tumor mass. A variant with a VAF near 40 percent might represent a mutation present in the vast majority of cancer cells, while a variant with a VAF of 5 percent suggests a subclone—a smaller, distinct population of cells that may have evolved later.
Tracking these VAF differences helps researchers understand the evolutionary history of the tumor and predict which cell populations might drive disease progression or resistance to therapy. For example, a high-frequency driver mutation, such as a KRAS G12C variant, is often targeted by specific drugs, and its VAF helps confirm the suitability of that treatment. If a second, lower-VAF mutation appears after treatment begins, it may signal the emergence of a resistant subclone that will soon dominate the tumor.
One of the most impactful applications of VAF is in the analysis of circulating tumor DNA (ctDNA) found in liquid biopsies. As tumor cells die, they release fragments of their DNA into the bloodstream, and the VAF of cancer-specific mutations in the blood plasma directly correlates with the overall tumor burden in the patient’s body. Following surgery or systemic treatment, oncologists monitor the VAF of previously identified mutations to detect Minimal Residual Disease (MRD).
If the VAF drops significantly or becomes undetectable, it suggests a positive response to treatment. Conversely, a sustained or rising VAF in the ctDNA indicates that the treatment is failing or that the disease is relapsing, often many months before imaging scans can detect new tumor growth. A rising VAF in the bloodstream, even at frequencies below 1 percent, serves as an early warning signal, allowing for timely intervention or a change in therapeutic strategy. This quantitative monitoring of VAF has become a standard approach for assessing treatment effectiveness and managing long-term patient surveillance.
VAF Beyond Cancer: Inherited Disease and Mosaicism
While VAF is heavily used in cancer research, its interpretation in inherited, or germline, genetics provides information about a patient’s genetic status. In cases of inherited disorders, VAF confirms the expected pattern of Mendelian inheritance. A VAF of 50 percent confirms that an individual is heterozygous for a disease-associated variant, meaning they carry one altered copy and one normal copy of the gene. If a patient is homozygous for a recessive variant, meaning both copies of the gene are altered, the VAF will be near 100 percent.
VAF also plays a role in identifying genetic mosaicism, a condition where a variant is present in only a fraction of the body’s non-cancerous cells. Unlike a standard inherited mutation (VAF near 50 percent), a mosaic variant will show a VAF significantly lower than expected, perhaps 10 to 30 percent. This lower VAF is linked to the variable severity and presentation of certain genetic syndromes, as the clinical outcome depends on which tissues contain the mutation and how many cells are affected.