Guidelines For Phasing Variants Using Bio-Rad Droplet Digital System

Droplet Digital PCR (ddPCR) enable absolute quantification of nucleic acid sequences by dividing reaction mixtures into nanoliter-sized droplets. Well-known applications for Droplet Digital PCR include sensitive rare mutation detection and accurate measurements of copy number variation (CNV). Here, we show that Droplet Digital PCR can also be used to determine which alleles are location on the same chromosome, a process known as phasing.

Drop-Phase involves performing a duplex ddPCR reaction that targets the two variants to be phased. DNA molecules that are long enough to preserve the physical link between the two targeted variants will result in more double-positive droplets than would be expected from random colocalization of unlinked variants. This overabundance of double-positive droplets is measured by QuantaSoft Software and used to calculate the concentration of linked variants in the reaction.

More Information on Digital PCR Systems and Consumables Can Be Found Here

Unlike traditional phasing methods, which are labor-intensive, low-throughput, and limited in distance, Drop-Phase is easy to perform, high throughput, and works over large genomic distances.