By Allison Proffitt
October 6, 2023 | Beginning today, the research team at Children’s Mercy Kansas City is bringing long read sequencing to clinical care, following three years of research and testing of PacBio HiFi sequencing. The transition is expected to speed the diagnostics odyssey for critically ill patients and their families.
“We are now confident that long read sequencing can see all the [genomic] variation that was previously seen by short read sequencing,” explains Tomi Pastinen, MD, PhD., Director, Genomic Medicine Center, Children’s Mercy. “If you put it in numbers, about 10% of the genome is inaccessible by short read genome sequencing and about 3% of known disease genes are not fully resolved by short read sequencing. Therefore, we’re actually moving forward now and launching the [long read] assay clinically for the first time for rare disease patients in our health system,” he tells Diagnostics World.
Expedited genomic testing is standard for critically ill undiagnosed children and babies at Children’s Mercy. Medical geneticists may order karyotypes, selected gene panels, structural variant detection by clinical microarrays, whole exome sequences, or even clinical whole genome sequencing depending on their clinical suspicion. But still the institutional diagnostic rate hovers about 25%. The new long read assay, using PacBio HiFi sequencing, promises to consolidate the testing. “We're going to tell [the clinicians] that all of this is in the single test now; they don't have to worry about the ancillary orders that that [they] may need with short read sequencing,” Pastinen says.
Road to the Clinic
“The secret in the clinical testing field is that most patients who get a genetic test have had a genetic test before,” explains Pastinen, recounting the test-upon-test approach to diagnostic odysseys. At Children’s Mercy, after all of the available tests, 75% of children remained without an answer. “We knew that there had to be more; there had to be more cases to be solved among that 75%,” Pastinen says. Children’s Mercy launched the Genomic Answers for Kids (GA4K) donor-accelerated research program in July 2019 to address the children left in the 75%.
Right away the group began exploring Pacific Biosciences’ long read sequencing, acquiring a Sequel IIe in March 2020. “In the spring of 2021, we started to collaborate with PacBio directly and developed the first hundred deep genomes from rare disease patients,” Pastinen said. GA4K has since expanded their Sequel IIe fleet, and upgraded to two Revio systems, replacing six IIe’s and increasing capacity by a factor of five, he says.
“In close to 1,500 HiFi genome sequences from patients and families of primarily pediatric rare disease, we've been able to first show that there are a number of regions in the genome that are not measured by the previous clinical sequencing,” Pastinen said. The GA4K team has thus far recover over 5% of additional diagnostic yield, he added, “and that really includes the whole gamut of potential hiding places of clinically-significant variation.”
PacBio’s HiFi sequencing has enabled the team to identify repeat expansion diseases, “diseases where the mutation is actually a short tandem nucleotide repeat that is not measurable by short read genome sequencing and that accounts for a good 3% of additional yield!”, Pastinen explains, as well as methylation changes, imprinting defects, and a variety of structural variations. The team has used HiFi long reads to do de novo personalized assembly of genomes, he added.
For interpretation of the long read sequencing, the GA4K team has adapted some existing, third-party AI systems including Emedgene from Illumina and VIA version 7 from BioNano for single nucleotide variants and simple structural variants. “But that gets you only part of the way,” Pastinen says. “In addition to that, our research group has worked a long time—with PacBio and independently of PacBio—to develop modules to deliver reports on other aspects like repeat expansions, methylation changes in clinically significant sites, and larger scale aneuploidies or translocations. Those are all separate components that are not yet encapsulated into a fully integrated software.”
Expected Benefits for the Clinic
Pastinen says the new assays will offer important benefits in clinical implementation. First, he expects an improvement in turnaround time. During the research phase, he says, the team went slowly. “Now that all these tools have been packaged and clinically validated, we can do the analysis in a matter of days and we can offer a turnaround time of two weeks,” he says. This is a relatively rapid turnaround for a single test that will benefit both families and physicians.
Second, he emphasized that offering the long-read sequencing in the clinic will be a boon to access. Underserved minority populations are even less represented in medical research than they are in general healthcare, he says. “We see that in our dataset, so in order to diversify the data that is being produced, and of course offer this to underserved populations, clinical access is key.”
Finally, Pastinen predicts that the comprehensive test will deliver, “unsuspected gains in clinical accuracy.” “Now that we’re implementing it in successive patients that are admitted to the hospital, we will see the true distribution of structural variants, repeat expansion, methylation disorders, typical genetic disorders, complex variants and so on,” he says. While Pastinen expects the test to be offered initially to critically ill inpatients that receive expedited genomic testing now, he wonders if in the future it may be fit for patients with certain phenotypic manifestations, or even all patients in a certain age group.
In fact, Pastinen plans to take a retrospective view of how the new assay impacts patients, comparing historic and current groups with similar characteristics and tracking how the single long-read sequencing test might impact their journeys through the health system.
Pastinen is confident that the long-read sequencing opportunities are just beginning. “We are very early in the discovery potential of long read sequencing,” he says. “I think that as more clinics adopt long read sequencing and submit discovered variations to ClinVar and so on, we will start to learn about new types of variants in human genes and we’re going to start to find new disease genes. But it really requires the expansion from research long read sequencing to the clinical space where most of the patients are getting sequenced today. I hope that our initiative will catalyze other groups also.”