Why Cell Sorting Increases VAFs compared to what it would be in a Bulk Tissue Sample

Increase Sensitivity

- The increased purity of cell populations obtained through FACS can lead to higher quality DNA for sequencing. This results in better coverage and depth in genomic analyses, allowing for the detection of variants at lower frequencies that might not be detectable in bulk tissue samples due to the mixture of cell types and the corresponding genomic material.

Targeted Analysis of Specific Cell Types

- Certain variants might be specific to particular cell types within a tissue. FACS allows for the isolation of these specific cell types for targeted analysis, which can reveal higher frequencies of specific variants that are relevant to the biology of the cell type of interest but would be diluted in the context of the whole tissue.

Purity of Cell Populations

- FACS can isolate cells with high purity based on specific markers or characteristics. In a bulk tissue sample, the presence of various cell types can dilute the signal of the cells carrying the variant of interest, leading to a lower apparent variant allele frequency. By sorting and isolating cells of interest (e.g., tumor cells from a mixed tumor sample), the proportion of cells carrying specific mutations is increased, thus raising the observed variant allele frequency.

Reduction in Background Noise

- Sorting cells can significantly reduce the background noise from non-target cells, dead cells, or debris. This enhanced purity allows for more sensitive detection of low-frequency alleles and mutations that might be missed or underrepresented in the noise of bulk tissue samples.

Clonal Expansion

- Tissues can contain subpopulations of cells with different genetic profiles. Some mutations might confer a selective advantage to cells, leading to clonal expansion. In a bulk analysis, the contribution of these clones to the overall mutation burden might be underestimated if they are in a minority. Sorting cells can help to isolate these clones and accurately measure their mutation burden.