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Droplet Digital Methylation Assay Shows Improvements in LOD, Precision Over Previous PCR Method

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NEW YORK (GenomeWeb) – A newly published study has demonstrated that a method for detecting the presence of methylated alleles using Bio-Rad's Droplet Digital PCR platform is superior to a previous methylation assay based on standard PCR.

In the study, published this week in the journal Epigenetics, researchers led by investigators from the Fred Hutchinson Cancer Research Center showed that their approach, MethyLight ddPCR was able to achieve a significantly lower limit of detection, and also offered other improvements in precision over a previous method called MethyLight PCR.

Hutch researcher and senior author William Grady told GenomeWeb this week that he and his team had three main goals. "First, we wanted to get a sense of what the limits of detection are just in the technical sense to [see] what's the fewest number of molecules of methylated DNA we can detect," he noted.

Second, he explained, the team wanted to determine the ddPCR method's ability to detect methylated alleles in a background of unmethylated DNA, something they expected would be easier because of the ability to partition a reaction into multiple droplets.

Finally, they hoped to show that ddPCR would outperform standard MethyLight PCR in terms of the precision of detection, or the ability to confidently detect low levels of methylated targets in a clinical sample.

According the Grady, MethyLight PCR has been around for a number of years as a method for assaying methylated DNA. Initially, when digital PCR platforms based on array technologies, such as Fludigm's, became available, researchers adopted these as a way to lower the detection limit of standard MethyLight PCR.

Grady's team, which is interested in developing assays for the early detection of colon cancer via specific DNA methylation biomarkers and also through detection of a process called the field effect, required the ability to detect even more infrequently methylated alleles than allowed by array-based digital PCR strategies, so they turned to even newer droplet-based systems.

Initially, the team considered droplet-based digital PCR platforms from both Bio-Rad and RainDance Technologies . At the time, the group was worried that difficulties in creating consistent droplets with the RainDance system could be an issue, Grady said, so they decided to use Bio-Rad.

Overall, Grady and his colleagues were able to show in their study that the resulting ddPCR version of MethyLight offers improvements in sensitivity with a much lower limit of detection, improved absolute quantification of methylated alleles, as well as increased precision in detecting infrequently methylated targets.

To demonstrate these advantages, the group performed a series of experiments using MethyLight ddPCR and the original MethyLight PCR in parallel focusing on two gene targets — NTRK3 and EVL.

The investigators first established the limit of quantification and limit of detection for the ddPCR method compared to standard MethyLight PCR, demonstrating that the droplet approach had a 25-fold lower LOQ and a 20-fold lower LOD.

Next, the group applied their assay and the standard PCR version to a set of two types clinical samples: primary colon cancer tissues with a known high proportion of methylated EVL DNA, and normal colon mucosa biopsies known to have very low levels of EVL methylation.

In four CRC samples, MethyLight ddPCR reduced the coefficients of variation down to at least 65 percent of the CV seen with the conventional PCR approach.

And in the nine normal colon mucosa samples, while conventional MethyLight PCR could not detect the low levels of EVL methylation, ddPCR was able to "selectively detect and absolutely quantify methylated EVL in all nine cases," the authors wrote.

Moving forward, Grady said he and his colleagues have two focuses. First, the group is interested in harnessing the superior LOD and precision of ddPCR to measure methylation differences that could be associated with field cancerization, also known as the field effect.

"If you have a field of 1,000 cells, only one or two maybe have acquired a molecular change like DNA methylation, so we want to be able to detect that. The problem is that with the standard PCR it didn't allow us to get down to that level of detection, but with ddPCR, we can now do that more precisely," Grady said.

Now the researchers are using their ddPCR method to compare high- and low-risk subjects and see whether they can identify field effect changes that correlate with or predict increased risk of developing CRC.

Also, the team is now working to develop methylation biomarker assays for early detection of CRC from stool samples, Grady said.

He and his colleagues have data from previous work using the standard MethyLight PCR in which they have been able to detect about 60 percent of people with colon polyps, and they are now hoping to improve on that with ddPCR.

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