NEW YORK (GenomeWeb) – As diagnostic exome sequencing becomes more common for rare disorders, groups are starting to look at ways to boost the success rate of such tests, which has been hovering at around 25 percent. Many have turned to so-called 'medical exomes,' where coverage of disease-related genes is boosted.
Baylor College of Medicine is the latest group to offer such a test. It launched its test, which includes enhanced coverage of over 3,600 genes, in September 2014, Donna Muzny, instructor in the department of molecular and human genetics, said in a presentation at the Advances of Genome Biology and Technology meeting in Marco Island, Fla. In addition, she said, the diagnostic laboratory now also offers a "lightning exome," which performs exome sequencing on the HiSeq 2500 and includes optimized library prep and capture protocols to reduce total turnaround time to less than three days from 20 days.
Aside from offering a medical exome, Muzny said that the Baylor team is trying to bridge the gap between the clinic and research by offering the 75 percent of patients that do not receive a diagnosis from their initial exome test the option to consent to whole-genome sequencing in a research setting. As part of that process, she said that Baylor has validated the HiSeq X Ten system to integrate into its whole-genome sequencing research pipeline for unsolved clinical cases who consent to research.
Baylor launched its exome sequencing test in 2011 and since then has received around 6,000 samples and finalized 5,000. It receives approximately 250 samples per month. The vast majority of patients present with some sort of neurologic disorder, and 85 percent are pediatric cases, Muzny said.
In November, Baylor published findings in the Journal of the American Medical Association from 2,000 consecutive cases referred for clinical exome sequencing between June 2012 and August 2014, reporting a diagnostic rate of around 25 percent.
While exome sequencing can be a comprehensive and effective approach for diagnosing rare, unknown disorders, researchers have long known that the approach is not perfect, in part, because certain regions are not captured well, or at all, from standard exome capture kits.
Emory Genetics Laboratory Executive Director Madhuri Hegde, for instance, has found that off-the-shelf kits cover up to 92 percent of the exome, but poorly cover or miss completely between 11 percent and 20 percent of known clinically relevant exons.
EGL and Personalis were among the first to begin offering exome testing with enhanced coverage in disease genes. EGL's EmExome boosts coverage in around 4,600 genes, while Personalis' ACE Clinical Exome adds coverage to more than 8,000 genes.
Muzny said that Baylor's goal was to use techniques that the lab uses for its 21 clinical gene panels, which cover completely all the genes in the panel, to enhance its exome test. The lab's final version of the medical exome includes enhanced gene coverage of over 3,600 genes chosen from its 21 clinical gene panels, OMIM genes, selected cancer genes, and genes from its solved cases. The spike-in encompasses approximately 2.5mb of sequence.
When it compared the exome plus spike-in to its standard exome test, it found that using the medical exome, more than 3,200 genes were covered completely at 20x or greater, compared to 2,500 genes with its standard exome.
Since implementing the new version in its diagnostic laboratory, it has sequenced 655 cases and diagnosed 153. Of those, seven cases, or 4.6 percent, "would have been missed if the spike-in was not used," Muzny said. Those included cases of both autosomal dominant and autosomal recessive inheritance, as well as both known and novel variants.
Aside from continuing to refine its diagnostic exome test, Muzny said that being able to bridge the gap between research and clinical in a HIPAA-compliant manner would not only help solve some of the 75 percent of unsolved cases, but identify additional disease genes to improve the test even more.
"What we really want to do is access unsolved cases from the clinical side and bring them to the research side to solve more of the cases and facilitate discovery," she said.
Baylor, in collaboration with Johns Hopkins University, is one of the National Human Genome Research Institute's Centers for Mendelian Genomics. One of its main goals is to obtain samples for Mendelian disorders and determine the genetic basis of such disorders, Muzny said.
This study offers a good opportunity through which to consent patients to the research project for whom diagnostic exome testing was unsuccessful, she said.
Baylor has such a pipeline in place, where if exome testing does not yield a diagnosis, patients can consent to further sequencing and analysis in a research setting. Under the pipeline, patients decide at the time of initial clinical testing whether to give permission to be recontacted for research.
Thus far, Muzny said, approximately 121 patients with a nondiagnosis have consented for research and 69 trios have been sequenced.
The biggest barrier to consenting patients for research has been going through the process of recontacting the patients and families and consenting both the individual patients as well as, typically, additional family members, Muzny said.
Nonetheless, the Baylor team has already identified three novel genes and has identified additional cases in its database with those mutations, which were not known to be disease causing when they were tested clinically, Muzny said.
To facilitate research-based sequencing, Muzny said that the lab has validated Illumina's HiSeq X Ten instrument, including its TruSeq PCR-free library prep kit and TruSeq Nano library prep kit.
In a research setting, Baylor has now sequenced 31 samples from 10 families on the HiSeq X Ten, and identified a few novel genes. Comparing the two library prep kits to each other using 30x whole-genome sequencing on the X Ten to its clinical exome, Muzny said that the TruSeq PCR-free library prep yields better coverage than the TruSeq Nano kit of 1,821 disease genes. In addition, whole-genome sequencing on the HiSeq X Ten performs about as well as Baylor's standard exome test did before the spike-in.