Optimized digital polymerase chain reaction enables detection of telomerase reverse transcriptase C228T mutation for prognostic assessment in hepatocellular carcinoma

Figure 4 Illustration of sequencing results and follow-up data in four samples, which were positive for telomerase reverse transcriptase C228T by digital polymerase chain reaction assay, but were all negative by Sanger sequencing. Each column represents a sample of one patient with hepatocellular carcinoma after surgical resection. At the top of the figure, Sanger sequencing results (all were non mutated); In the middle of the figure, the two-dimensional digital polymerase chain reaction plot (the color of line: Green, wild-type; dark blue, mutant type; light blue, droplets with mutant and wild-type allele; dark grey, negative droplets) and the concentration (cp/μL) of the telomerase reverse transcriptase C228T mutant allele targeted by the 6-carboxyfluorescein dye labeled probes. 6-carboxyfluorescein concentration higher than LoD was positive. Clinical follow-up data collected from each patient are shown at the bottom of the figure. The orange and grey bars represent the time from surgery to tumor recurrence and death, respectively. PCR: Polymerase chain reaction.