As new research questions the reliability of exome sequencing, Jason Y. Park, MD, PhD, director of the Advanced Diagnostics Laboratory at Children’s Medical Center Dallas, Texas, is advising laboratorians to educate themselves about the quality issues that surround this testing. Park, who also is an assistant professor of pathology at the University of Texas Southwestern Medical Center and the Eugene McDermott Center of Human Growth and Development, joined with other researchers to see how well four exome methods performed in identifying disease-causing genetic variants. One of his collaborators, Eric Londin, PhD, assistant professor at Thomas Jefferson University’s Sidney Kimmel Medical College, recently presented the group’s findings at the European Society of Human Genetics in Milan, Italy.

Clinical exome sequencing has grown in the last 8 years from a research tool into a clinical diagnostic test, Park explains. Initially, exome testing was used for the diagnosis of diseases with unknown genetic causes, but now it’s “being used as a substitute for testing panels of clinically significant genes,” he says.

Park’s research focused on how well four exome sequencing kits performed in detecting 56 genes identified in 2013 as clinically relevant by the American College of Medical Genetics and Genomics​ (ACMG), explains Park. ACMG recommended that incidental findings on these genes should be reported to patients.

The researchers determined that a high proportion of clinically relevant regions in this subset of genes were not reported on across 44 exome datasets from the four different testing kits and two testing platforms. “At least one of the 56 genes in each exome method was missing sequencing data that would be necessary to report on 40 percent of disease-causing genetic variants. The worst-performing method (from a direct-to-consumer sample) missed sequencing data required to report on 90 percent of such variants in four of the 56 genes,” says Park.

Overall, the quality of the datasets was comparable to those reported from clinical exome laboratories, with 90% of coding nucleotides covered at 10x or better. But in looking at the specific subset of 56 genes, “we identified that many nucleotide locations with known clinical significance had no sequencing data. We had expected that there would be coding areas of genes that would not have sequence data, but we had not anticipated the magnitude of the problem,” he explains.

In retrospect, the lack of coverage in coding sequence makes sense, Park says. “Considering that there are approximately 30 million coding nucleotides in an exome, 90% coverage will leave 3 million without sequencing data. Some of the best clinical exome labs achieve 98% coverage of coding nucleotides, which still leaves 600,000 coding nucleotides without sequencing data.”

This is concerning for a high false-negative rate, he notes. What it means is “if you perform clinical exome testing on a patient, a positive result may be informative, but a negative result may be meaningless.”

Taking into account these issues of gene coverage and the potential false-negative rate, laboratorians should learn the critical quality parameters for exome sequencing and seek out the best-performing exome laboratories, he advises.

“Some clinical exome labs have publicly accessible databases that show the genes with poor coverage. These types of databases can be used by laboratorians to inform their clinicians on which genes may not have adequate sequencing results,” Park says.

Clinical labs that perform exome testing should develop and adopt methods that have better coverage of the clinically relevant portions of the exome, he notes.