Determining efficiency in ligation using digital PCR
Targeted digital sequencing (TARDIS) is a highly sensitive assay designed to detect circulating tumor DNA (ctDNA) in plasma, addressing the sensitivity limitations of traditional biopsies to enhance residual tumor DNA detection. The ligation efficiency within the TARDIS assay has remained undetermined since publication. This study aimed to determine if digital PCR (dPCR) could be a useful tool to quantify the ligated product. In the TARDIS assay, a blunt-end single-stranded sample fragment is ligated to an adapter, introducing a universal amplification region. dPCR was employed to measure the amount of successfully ligated product, utilizing a modified 5-plex probe quantification assay. Our findings indicate a consistent correlation between varying input amounts and their corresponding output. Additionally, on average, 34% of the DNA input was recovered after ligation, with 12% of the total input being successfully ligated. Overall, dPCR proves to be a practical tool for quantifying ligation efficiency. These results highlight several factors that influence ligation efficiency, such as the effectiveness of the ligase and the magnetic bead purification steps downstream. Future work will focus on testing alternative quantification methods (intercalating-dye), increasing the number of assays, and expanding the number of partitions utilized in the dPCR nanoplate. This additional work aims to enhance the characterization of future ligation optimization steps.
