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University of Groningen Working to Replace Sanger-based Tests with Targeted Sequencing


Researchers in the department of genetics at The Netherlands' University of Groningen are working to develop targeted next-generation sequencing panels to replace the Sanger sequencing that has previously been the group's standard in several disease areas.

The group described the creation and validation of its first panel, for cardiomyopathies, in a study published online ahead of print in Human Genetics this month.

Birgit Sikkema-Raddatz told CSN this week that her team has also now developed a panel for inherited cancers and is interested in working on moving from Sanger sequencing to NGS in other disease areas as well.

"We started with cardiomyopathies because that is one main focus of our research department," Sikkema-Raddatz said, "but we have also now developed an onco-panel and we will also develop a panel for epilepsy."

Up to 60 genes have been implicated in hereditary cardiomyopathies — too great a number to make Sanger sequencing feasible, according to the researchers.

In the study, the team wrote that regular routine diagnostics using Sanger involves stepwise testing, which can take more than a year to complete for only 10 genes. The group's targeted sequencing approach could theoretically cut that down to two weeks, the authors wrote, although currently the hospital is aiming for a reporting time of between four and six weeks.

Sikkema-Raddatz said that while her group is using whole-exome sequencing in its research, issues with incomplete representation and coverage can lead to mutations being missed, so the group decided that for its clinical diagnostic plans a more targeted approach would be necessary.

According to Sikkema-Raddatz, the Groningen University hospital has now been using the team's cardiomyopathy panel clinically for about two months.

The experience has shown that the new approach can increase the number of successful diagnoses fairly significantly. "In our results [in the study] we got an increase from 30 to 60 percent diagnostic efficiency with the switch to next-gen sequencing and that's a real benefit for the patient," she said. "Of course, these are our first results, but now we have more than 200 patients and we are seeing this even more clearly."

The group created the panel, which includes 48 genes associated with hereditary cardiomyopathies, using Agilent's SureSelect target enrichment kit for sequencing on an Ilumina MiSeq.

"We tested several enrichment products but decided to go for the Agilent kit because the coverage was a little better than, for instance, the one from Illumina. But it's a little arbitrary," Sikkema-Raddatz said.

In the study, the team tested the panel on a total of 84 subjects and found that it was fully concordant with Sanger results when a threshold of 30 reads per base pair coverage was set. It was also highly reproducible and specific, the group wrote.

However, for a small percentage of genes — about 50 out of 1,134 in the study — the panel did not yield the 30 reads per base pair the group decided on as a threshold for accuracy. Sikkema-Raddatz said that in light of this, her team is performing Sanger sequencing in parallel for those genes in this group that have well-known clinical relevance to make sure there is complete coverage.

"We have several targets that are not as well covered and usually those targets are the same [each time], so we have a parallel panel for Sanger sequencing that we are doing," she said.

Alternately, the group wrote in the study that variants that are not covered, or are badly covered, could just be excluded from the clinical report. Beyond this, the group does not see the need for additional confirmation by Sanger for discovered variants.

According to Sikkema-Raddatz, the group is planning to develop additional diagnostic panels using the same strategy, and it plans to stick with the MiSeq for its clinical targeted sequencing activities.

"We are using the HiSeq for exome sequencing," she said, "but that is all research-based. With the MiSeq, you have results in two days from sequencing, so we are using that for the panels."

Sikkema-Raddatz said the team's second project, a hereditary cancer panel, includes 73 genes that have been previously associated with tumor syndromes — mainly Lynch syndrome and hereditary breast cancer.

Looking forward, she said the group is also interested in working on moving its epilepsy testing protocol to targeted NGS and in developing a panel for inherited skin diseases.