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How to carry out Variant Allel Frequency analysis, many novices are not very clear about this, in order to help you solve this problem, the following editor will explain for you in detail, people with this need can come to learn, I hope you can gain something.

VAF is the abbreviation of Variant Allel Frequency, which is usually called variant allele frequency. The formula is as follows

From the point of view of the formula, it describes the proportion of the number of reads corresponding to the mutant allel to the total number of read at the locus. In the VCF file, the field AD is usually used to represent the sequencing depth of the allel, the sequencing depth of multiple allel is concatenated with commas, and the DP represents the total sequencing depth of the site, as an example is as follows

AD = 4Jing 8

DP = 12

The AD field has 4 and 8 allel. In diploid organisms, the first number represents the sequencing depth of the ref allel, and the second number represents the sequencing depth of the alt allel, so the VAF=8/12 of the locus in the sample.

For mutation sites, there is another frequency, called MAF, whose full name is Minor Allel Frequency, which describes the frequency of sub-alleles in the population. From here, we can also see the different support of VAF and MAF. These two concepts are for allel frequency. MAF describes the frequency of allel in the population, while VAF describes the frequency of allel in a sample.

What does the size of the value of VAF mean? In diploid organisms, for example, assuming that this locus is heterozygous in all cells, 50% of the chromosomes contain ref allel and the other 50% contain alt allel, then the VAF value of this locus should be 0.5 in the sequencing results. For germline genotype, the VAF value of a reliable mutation site should be around 0.5.

If VAF deviates a lot from 0.5, it means that the corresponding chromosome region is no longer two copies in the cell. For example, the value of VAF is 0.25, which means that the chromosome containing alt allel accounts for 1pm 4, and the chromosome ref allel accounts for 3pm 4. According to the assumption that all cell mutations are exactly the same, only when there are three copies in each cell, two copies are ref allel and one copy is alt allel. There will be a 0.25 situation.

For the detection of reproductive variation, it is considered that the deviation of VAF comes from the change of copy number. For somatic cell detection, it is more believed that the shift of VAF comes from the heterogeneity of tumor cells. When sampling tumor tissue, it will inevitably be mixed with normal somatic cells, and considering the complexity of tumor microenvironment itself, other cell types such as immune cells will also be infiltrated in tumor tissue. Tumor cells themselves can also be divided into different subtypes. Taking into account the above factors, the tumor sample is a mixture of multiple cell types. It is more believed that the deviation of VAF comes from the change of the proportion of different cell types.

In order to further simplify this model, tumor samples are divided into normal somatic cells and tumor cells. Ref allel is equal to normal cells x2 + tumor cells, and alt allel is equal to tumor cells. The value of VAF can reflect the ratio of tumor cells to normal cells and can be used to speculate tumor purity. In tumor genome research, VAF is also regarded as a marker, and the average value of VAF at all loci in the sample is taken as an index to compare the differences in the distribution of VAF in different groups and tumors, and the samples can also be grouped according to the level of VAF for survival analysis.

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