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British Study Tests RainDrop Methylation Sequencing Method at Nanogram Sample Sizes

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Researchers from University College London, have shown that a targeted bisulfite sequencing method using RainDance Technologies' microdroplet PCR system can accurately measure DNA methylation levels at sample sizes as low as 100 nanograms.

The team — which refined the technique they call RainDrop BS-seq as a higher throughput method for following up results from epigenome-wide association studies — published the results in a study in the journal Epigenetics this month.

The group's first author Dirk Paul told In Sequence that the study was an effort to put the technique through its paces in terms of both sample volumes and types.

"We wanted to challenge the instrument, to see if we could use nanogram-size samples, and with different sample types like cancer cell lines and whole blood," he said.

"Our [overall goal in developing the method] was to use this for EWAS validation. In these studies, discovery is done on microarrays, and follow up validation is done using, for example, pyrosequencing. But with EWAS getting larger and larger, these [current follow up] methods are not as practical."

Paul and his colleagues tested their RainDrop BS-seq method on a range of starting DNA quantities and qualities using different cell types, comparing the results to those obtained using microarrays. The group also tested whether by adding MDA-based whole-genome amplification, they could push to even smaller DNA inputs.

Using two primer panels — a "sarcoma panel" including 27 loci containing 212 CpG sites represented on the Illumina 450K array, and a "biomarker panel" that included 462 loci associated with ongoing epigenetic biomarker validation work also represented on the 450K array — the researchers performed RainDrop BS-seq, using RainDance's Thunderstorm system and an Illumina MiSeq, on a variety of sample input sizes and types.

According to the authors, the SC panel was applied to whole blood, an osteosarcoma cell line, and a single FFPE sample, while the BM panel was used only on the blood and sarcoma cells.

The team tested 1500, 1000, 250, and 100 ng DNA sample sizes from each sample type. In parallel, the group evaluated a second set of samples at 250, 100, 50, and 10 ng of genomic DNA using whole-genome amplification.

According to the authors, for both the blood and sarcoma samples, methylation profiles generated using a starting DNA amount down to 100 ng correlated very well with Illumina 450K array data, while results using whole-genome amplification were a poorer match.

"We are pretty happy," Paul said. "We managed to get it working with nanogram quantities of DNA and it correlates really well with the 450K array, so [the method looks] perfect for EWAS validation."

"Using a [whole genome amplification] step we did also go all the way to 10 nanograms, which is ridiculously low, and that did work, but the correlation is not as high as we would hope for," he explained. "We also didn't have great success with the FFPE samples [because] after bisulfite conversion DNA is really degraded, so for the PCR steps in these microdroplets the amplification was just not really good."

Based on the results, Paul and his coauthors wrote that RainDrop BS-seq appears "suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics."

The UCL team is currently involved in a large European epigenomics consortium called BluePrint. As part of that effort, Paul said, he and his colleagues are participating in a study pitting different targeted methylation sequencing methods against each other.

"There are up to 20 groups involved around the globe and we all got the same samples and have the same target regions, and the task was for all the groups to test the same samples with our method of choice," he said.

"The study is still underway. We processed all the samples and our data looks really good, but we don't know yet how we compared to other methods," he added.

Additionally, Paul said the UCL group is forging ahead with their own clinical research goals using RainDrop BS-seq.

"We have a lot of EWAS studies underway and we are looking forward to validat[ing] a lot of findings with this method," he said. Specifically, he said, the team has recently made some EWAS discoveries in urological cancer and they hope to follow up on those findings.

“We identified an epigenetic signature for urological cancer for disease detection, [and] we are in the process of recruiting patients [and will use] RainDrop BS-seq to validate the biomarker in [this cohort]," he said.

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