Emory Genetics Laboratory, which has been offering next-generation sequencing-based panels since 2010 on Life Technologies' SOLiD, has now converted all of its panel tests to the Illumina MiSeq system, while running an exome test on the HiSeq 2000, executive director Madhuri Hegde told Clinical Sequencing News at last week's American College of Molecular Genetics and Genomics meeting in Phoenix., Ariz.
The laboratory has run around 200 exome tests since August 2012, and is aiming to launch new targeted panels, including expanding its existing tests for neuromuscular disorders and congenital disorders of glycosylation, as well as delve into new areas such as cancer.
Hegde said the laboratory would eventually move into the areas of carrier screening and non-invasive prenatal testing. She said the lab is looking to develop an in-house noninvasive prenatal test as well as to offer a test from one of the four US-based companies — Sequenom, Verinata, Ariosa, and Natera.
Additionally, she said the laboratory is collaborating with Harvard University and the Children's Hospital of Philadelphia to develop a "medical exome" that includes full gene coverage for around 4,500 medically important genes.
From SOLiD to PGM to MiSeq
In 2010, Emory launched panels for X-linked intellectual disability, congenital muscular dystrophy, and congenital disorders of glycosylation on Life Technologies' SOLiD platform out of its CLIA- and CAP-certified laboratory (IS 5/25/2010).
About a year ago, the lab was looking to convert its tests to the Ion Torrent PGM because of its faster turnaround time and lower cost compared to the SOLiD (CSN 3/21/2012). But since then, the team has instead switched its panels to the Illumina MiSeq.
Now, Emory has two MiSeqs and one HiSeq 2000, which Hegde said she hopes to upgrade to a 2500.
Exomes vs. Panels
In August, Emory began taking orders for a clinical exome test. The test also includes the entire mitochondrial genome and Sanger validation.
Emory has so far run around 200 exomes, with orders for the test increasing weekly, Hegde said. The diagnostic rate is around 25 percent, she said, but the lab has very strict criteria for deciding that a variant is disease-causing. For instance, said Hegde, if the disease is expected to be recessive and only one pathogenic mutation is identified, the laboratory will not consider that a diagnosis unless a second mutation is identified.
Emory charges $9,000 for the test, which includes exome sequencing, mitochondrial genome sequencing, confirmatory testing, and additional testing on the parents. Turnaround time is about 16 weeks.
Hegde said that insurance companies have been reimbursing between 50 percent and 70 percent of the test's cost. "We've gotten paid on almost every case," she said.
One of the lab's main focuses in developing the test was to be able to offer automated confirmatory testing, she said. The laboratory has designed infrastructure that enables automated primer design of around 18,000 transcripts in its internal mutation database, which speeds up the Sanger confirmation process.
It also has IRB approval to send any negative exome results into research for discovery. In this case, the patient goes through the consenting process again.
Additionally, the lab spent a large portion of last year working on its variant-calling algorithms and gene curation. Two systems it has developed for this purpose, EmBase and EmVClass, can cross-communicate so that as new information is learned about variants and as variants are re-classified, they are automatically updated and alerts are issued. EmVClass currently includes the 700 genes for which Emory has been offering Sanger sequencing tests for the last seven years. Additionally, the database is being populated with genes that are included in its next-gen sequencing panels or that are discovered to be disease-causing from exome sequencing results.
Despite now offering an exome test, Hegde said that there are still many cases where panels make more sense, and as such, the laboratory is continuing to develop new panels and expand existing ones.
One main advantage of panels, said Hegde, is that they offer more complete coverage of the genes of interest than exome sequencing, with a near complete coverage of the entire mutational spectrum, including point mutations, small indels, and large single-exon or multi-exon indels. Additionally, many of Emory's panel tests offer more than just sequencing, but also methylation testing, cytogenetic arrays, or Sanger sequencing for problematic areas.
Clinicians "want to know when to order a panel and when to order an exome," Hegde said. "There's a lot of confusion about what is the right approach and what to do when."
Hegde recommends panels for clinically well-defined cases and exomes when there is a complex or overlapping phenotype or if a lot of testing has already been done and still has not yielded a diagnosis.
Panel testing has proven to be very successful in terms of yielding a diagnosis when the phenotype matches, she said. For example, Emory previously offered single-gene testing for the various subtypes of neuromuscular disorders. Switching to a comprehensive panel that allowed the lab to assay all the subtypes in one test increased the diagnostic yield to 50 percent from 10 percent.
Emory also includes arrays to measure duplications and deletions in many of its panel tests and will soon incorporate dup/del arrays in all of its tests, Hegde said. This is important because targeted sequencing does not pick up these types of variants, and for a disorder like Duchenne muscular dystrophy, duplications and deletions comprise 65 percent of the mutation spectrum, she said.
Medical Exome
Because the current exome kits have holes in their coverage, Hegde said that her lab has been collaborating with Brigit Funke's lab at Harvard and Avni Santani's lab at CHOP to create what they've dubbed a "medical exome."
Agilent's SureSelect kit and NimbleGen's exome capture kit miss 7.4 percent and 10 percent of all the exons, respectively, Hegde said. For each of Emory's targeted panels, if a researcher were to instead use exome sequencing to mine those same genes, "you will miss variants of significance," she said.
Already in tests with the latest version of Agilent's SureSelect kit, Hegde said she has been able to reduce the number of exons not covered to 1.6 percent from 7.4 percent.
The team is also testing NimbleGen exome kits and is considering using an alternate selection method like RainDance. "The goal is to get near 100 percent of all known medically relevant genes," which number around 4,500, she said.
The curated list of genes will be finalized within the next month, she said. The medical exome "will focus on genes that are medically actionable, but that doesn't mean we're taking away coverage of other genes," she said.
Future Tests
Moving forward, Hegde said that Emory is planning to "get into cancer big time." The lab is currently working with physicians at Emory to re-design RainDance's cancer hotspot panel, which targets 54 cancer genes. Additionally, it is working on focused somatic cancer panels.
It is also working on panels for irritable bowel disease, hearing loss, and periodic fever, among others. And, it is expanding some of its current panels and adding deletion and duplication arrays to all of its panels.
The lab also plans to get into reproductive medicine; andis developing a carrier screening test that will initially be array-based but eventually move over to sequencing, Hedge said. Emory is also pursuing the non-invasive prenatal testing market, working on developing an in-house assay as well as considering offering one of the currently available tests to patients.