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Nanopore Sequencing Rapidly Advances, Enabling Genetic Assessment of Newborn in Three Hours


BALTIMORE – After striking a record speed earlier this year, nanopore sequencing continues to make headway in the realm of rapid genetic testing, with a new preprint study led by University of Washington researchers demonstrating that the technology can help elucidate a newborn's genetic risk for a specific disorder in as little as three hours after birth.

The study's ultra-rapid turnaround time, which is the fruition of optimized sequencing workflow and targeted data analysis, marks somewhat of a new advancement for nanopore sequencing in terms of speed. However, researchers still face a journey to harness the case-specific breakthrough into broader clinical applications and routine genetic disease diagnostics.

"The initiative of the study is basically [to explore] can we use sequencing to help us with clinical decision-making more rapidly?" said Danny Miller, a pediatrics geneticist at the University of Washington and the lead investigator of the preprint paper, which is currently available on MedRxiv.

For the study, Miller and his team, which included scientists from Oxford Nanopore Technologies as collaborators, deployed rapid nanopore whole-genome sequencing to evaluate a newborn's genetic risk for acrodermatitis enteropathica (AE), an inherited form of zinc deficiency encoded by a single gene, SLC39A4, given the baby's sibling was previously diagnosed with the condition.

While Miller acknowledged that, for this specific case, the diagnostic turnaround time of three hours was not entirely clinically necessary, he considers this study as "a proof of concept to show that we could do it in other cases."

The first technical hurdle that the researchers had to overcome was to draw a smaller amount of blood from the newborn while providing sufficient native DNA to sustain a high-quality nanopore long-read sequencing run.

"We wanted to be thoughtful about the amount of blood we were starting with," said Miller. "In newborns, we often are faced with [the question of], 'Which lab do you want to order today?' because you can only take so much blood."

After that, the team had to come up with ways to expedite the sequencing process. To achieve that, in addition to optimizing the DNA extraction step, the researchers settled on using Oxford Nanopore's rapid library preparation protocol, which can generate sequencing libraries in as little as 10 minutes and does not require a cleanup step.

After preparing the libraries using both the Rapid Sequencing kit and the PCR-cDNA Sequencing kit from Oxford Nanopore, which took 25 minutes, the sample was sequenced with 20 Oxford Nanopore PromethIon flow cells using the R9.4.1 chemistry on a PromethIon 24 platform.

The raw sequencing data was then transferred to a remote basecalling server, where a custom script monitored the incoming sequencing data, which were base called and subsequently aligned to the GRCh38 reference genome in real time.

Previous genetic testing of the newborn's AE-affected sibling had revealed a single pathogenic variant and a candidate promoter variant underlying the disorder. Informed by that prior knowledge, Miller's team performed real-time targeted analysis, which focused on the reads surrounding the target gene and its two control genes, to rapidly assess the newborn's genetic risk for AE.

After close to one and a half hours of sequencing, or within three hours after the baby's birth, the phased sequencing data were clear to show that the newborn did not inherit either the pathogenic variant or the candidate promoter variant discovered in the sibling. As such, the researchers concluded that the newborn was not at high risk for inherited AE.

The researchers continued the sequencing for an additional four hours, generating approximately 45X coverage of the whole genome after a total of five and a half hours of sequencing, or seven hours since the baby was born. 

"I think this is fantastic," said Stephen Kingsmore, CEO and president of Rady Children's Institute for Genomic Medicine in San Diego, who is at the vanguard of rapid whole-genome sequencing for disease diagnostics. "It's now clear that the Oxford Nanopore sequencer is the fastest whole-genome sequencer in the world, that's an important statement to make."

In January, a team of researchers led by Euan Ashley from Stanford University deployed rapid nanopore sequencing to help diagnose patients in the critical care setting. That study delivered the fastest speed of 7 hours and 18 minutes between sample collection and reaching a diagnosis, nearly cutting the previous world record — set by Kingsmore's group using Illumina sequencing — in half.

"When Euan Ashley did it, there were still questions about could anybody else achieve this, but now a second group has done it," Kingsmore said. "It seems clear that this is something that people need to take seriously."

In addition to speed, Kingsmore also thinks the lower upfront investment for nanopore sequencing also makes the technology "very attractive" to many smaller labs. "I think it's going to democratize the ability to do rapid genome sequencing," he said. "Instead of buying a $1 million NovaSeq instrument and then the reagents, [nanopore sequencing] should allow any small groups in hospitals around the world to be able to recapitulate this."

While Kingsmore still considers Rady Children's as "an Illumina shop" — with four NovaSeqs running every day, he said the hospital is starting to investigate the use of nanopore sequencing to better understand where the technology would fit in its diagnostic workflow. 

"It's very likely that we will have long-read sequencing before the end of the year," Kingsmore said. "For us, it is probably going to be a backup technology; we will use it when we don't think short-reads sequencing is going to work."

Commenting on the preprint study, Kingsmore said the simplified methods presented by the researchers could potentially speak to greater repeatability and scalability. However, "some of the text and the document suggests that it's still technically pretty tricky to do this," he noted, although he did not specify a particular procedure or protocol.

That said, Kingsmore thinks nanopore sequencing still faces the infrastructural challenge of driving technology adoption. "The thing to remember is that Illumina has been doing this for 15 years, so they have an entire ecosystem. … They offer you kind of a soup-to-nuts, semi-automated solution; it's not plug-and-play, but it's pretty close," he noted. "When it comes to nanopore, you still have to build out all the rest of the pieces."

Additionally, Kingsmore pointed out it is unclear from the preprint paper how much the technology cost per genome to achieve such a rapid speed. According to him, currently, at Rady Children's, the cost for each genome from sample to result at the production level is about $8,000, with a turnaround time of around 30 hours. Meanwhile, the sequencing itself roughly costs $2,000 per genome and has a 15-hour turnaround. "If you're going to do this frequently, you need something that's cost effective," he said.

In terms of the nanopore method's cost, Miller said a ballpark figure for the assay would be tens of thousands of dollars, although it can vary among researchers due to the differing flow cell prices. However, he said the counterpoint is that the cost would still be cheaper than a day in the ICU or many of the medications prescribed to patients.

But beyond cost, Kingsmore listed a few other factors that might constrain the real-world clinical application of ultra-rapid genomic testing. "Once you can get genomes done in a day, you're where you need to be, [but] there are really very few babies where you need a faster result than a day," he argued.

How long it takes a doctor to order the test can be another important factor to consider, he said, adding, "If it takes a week for the doctor to order the test, what's the point of having a one-day turnaround?"

This study "demonstrates the power of the nanopore technology and its capability of producing data fast and allowing the scientific team to monitor the production in real-time, which sets the nanopore technology apart from some of its competitions," said Tomi Pastinen, director of the Genomic Medicine Center at Children's Mercy Kansas City. "To my knowledge, [nanopore sequencing] is the only genome-wide technology that could be applied so rapidly to achieve that kind of high-confidence genotyping."

While Pastinen's group has not yet moved into the newborn sequencing space, his team is known for conducting long-read sequencing on the Pacific Biosciences platform to help solve rare disease cases through the Genomic Answers for Kids (GA4K) project.

Based on the results presented in the paper, Pastinen said it was clear that the early sequencing data would not have allowed researchers to carry out genome-wide interpretation. However, he said for the specific application scenario described in the study, which is effectively confirmatory sequencing for two genetic variants, nanopore sequencing, with its advantage of moving fast, is "completely appropriate."

Even so, Pastinen said not all diseases are likely to benefit from this kind of ultra-rapid sequencing. "For the vast majority of genetic and rare diseases, the ultra-rapid sequencing is not essential," he said, noting that while a sequencing turnaround of a few days can be important for some cases, a large fraction of genetic diseases can wait for a few weeks, since the interventions will typically not make a big difference.

Additionally, given that the study is still a proof of principle, future work is needed in order to expand the methodology for mass adoption and drive it for routine clinical use, Pastinen pointed out.

From a practicality standpoint, Miller, mirroring Kingsmore's and Pastinen's comments, said the honest answer is that there is "a narrow group of cases" that may benefit from the ultra-fast diagnostic turnaround. These include newborns with hyperammonemia or seizures, for instance, where an ultra-rapid genetic diagnosis could provide pivotal information for clinicians to take clinical action.

On the flip side, Miller said cases such as newborns with multiple congenital anomalies may not gain much from a genetic test that has a three-hour turnaround, since the result is less likely to immediately change the treatment course or outcome. But, he said it is still helpful to know the answer within a day or two to have an idea of what's going on.

Still, Miller emphasized that the bigger picture of this study is to help establish a roadmap for other researchers, and by generating and sharing the data, he hopes the study can make an impact in other areas.

"As we learn to do this quickly, we can start to implement this in different places, places that we don't even think about right now," he said, adding that this study and others can hopefully continue to propel nanopore sequencing toward more rapid applications, such as infectious disease testing in the field setting or point-of-care diagnostics.

Moving forward, Miller said the team plans to apply the test on a larger scale for patients in critical care settings, either in NICU, PICU, or even the adult cardiac ICU. In addition, he said the team hopes to continue to improve the test's methods and show its utility in order to drive its clinical adoption and hopefully eventually secure insurance reimbursement.

"A lot of things need to be improved before this becomes standard of care," Miller said. "It's on our list of things that we need to think about."