BALTIMORE – Nanopore sequencing has reached the realm of rapid genetic disease diagnostics after a Stanford University team recently demonstrated its ability to deliver diagnoses to critically ill patients in record time. But while the approach appears to shatter Illumina’s monopoly in clinical rapid whole-genome sequencing, its accuracy, cost, and scalability still need to be proven for broad real-world applications.
In a paper published in the New England Journal of Medicine last week, the Stanford team and its collaborators described their ultrarapid nanopore whole-genome sequencing approach, which involved streamlined sequencing, cloud-based bioinformatics, and customized variant prioritization.
Led by Euan Ashley, professor of medicine, genetics, and biomedical data science at Stanford, the researchers sequenced the genomes of a dozen critically ill patients at two hospitals in Stanford between December 2020 and May 2021 using Oxford Nanopore Technologies’ highest-throughput sequencer, the PromethIon 48.
Of the dozen study participants, five received a sequencing-based genetic diagnosis, translating to a diagnostic rate of approximately 42 percent. The shortest time between sample collection and reaching a diagnosis was 7 hours 18 minutes, nearly cutting the previous world record — set using Illumina sequencing — in half.
"We looked at literally every part of the pipeline and said, ‘How can we make it faster?’" said Ashley.
Specifically, his team optimized DNA extraction and library preparation for yield, quality, and speed using a modified Puregene genomic DNA extraction protocol from Qiagen and customized library preparation from Oxford Nanopore.
Notably, instead of sequencing a patient’s DNA library on one flow cell, they distributed the sample equally over 48 flow cells, generating as much as 200 Gb of data in as little as 1 hour 50 minutes, according to the authors. They also reduced per-sample sequencing costs by washing and reusing the flow cells after each patient.
To minimize the overhead compute time, the team deployed cloud computing using the Google Cloud Platform to upload, basecall, and align sequencing data in near real time. Post-sequencing, variants were called using the GRCh37 human reference genome, generating a median of almost 4.5 million single-nucleotide variants and small insertions or deletions per patient. Customized variant filtration and prioritization with an ultrarapid scoring system reduced the number of candidate variants for manual review to a median of 29 single-nucleotide and 22 structural variants.
"The Stanford team has said they want to make as much of the pipeline as possible open-source, and we are working with them to enable that," said a spokesperson from Oxford Nanopore, adding that the firm is in the process of re-enabling its Epi2Me cloud-based analysis platform to provide basecalling for rapid turnaround applications.
"Congratulations to this team," said Stephen Kingsmore, president and CEO of Rady Children's Institute for Genomic Medicine in San Diego, who spearheaded rapid whole-genome sequencing, or rWGS, for disease diagnostics. "What a milestone."
Rady Children’s has sequenced the genomes of over 3,000 critically ill children from nearly 80 hospitals across North America and held the previous record for diagnosis time at 13.5 hours, set last year using the SP flow cell on an Illumina NovaSeq.
Kingsmore said the new record is "not at all" surprising to him, noting that feeding a long piece of DNA through a pore and transmitting the sequence signals electrically is intrinsically faster than Illumina sequencing, which uses a sequencing-by-synthesis approach where imaging and DNA synthesis limit speed.
Illumina sequencing "has already been squeezed like a lemon" to improve speed, Kingsmore said. "But Oxford Nanopore is entirely different."
However, Kingsmore argued that even Illumina-based rWGS, which can take up to 18 hours, is still "perfect" for critically ill patients since it delivers a diagnosis the next day. For a rWGS diagnosis workflow to be widely useful in the real world, he said, "it's more about scalability and cost than it is about speed."
Specifically, he pointed out that the Stanford study did not discuss error rate, a longstanding concern with nanopore sequencing. He also noted that the estimated cost mentioned in the paper, $4,971 to $7,318 per patient, is substantially higher than his team’s cost of about $1,500.
In addition, Kingsmore said he is "unsure" whether cloud computing, as devised in Stanford’s workflow, might lead to hiccups with CLIA certification, a necessary step for the technology to be used in clinical diagnostics, since it is something the certifying authorities are "not used to dealing with."
Ashley declined to disclose the sequencing error rate at this time, citing an upcoming technical paper that "has all our benchmarking data" and is pending publication next month. He also said he has "no concerns" with CLIA certification, adding that "the plan is to absolutely go ahead with that."
Yong-Hui Jiang, professor and chief of medical genetics at Yale Medicine, said Stanford's achievement is "quite impressive." While he is also concerned with the error rate associated with nanopore-based rWGS, he said the speed can be beneficial for bedside physicians when deciding how invasively to treat patients whose life is on the line.
In 2019, Yale also started offering rWGS for qualifying critically ill patients using the Illumina platform, Jiang said. But unlike Rady, which sequences roughly 100 patients a month, according to Kingsmore, Yale operates on a much smaller scale — analyzing the genomes of about 30 patients per year. Although the sequencing cost for Yale’s patients is absorbed by the hospital system, Jiang estimated the rWGS cost to be between $5,000 and $10,000 per patient, comparable to Stanford’s range.
Jiang also noted that while the material costs for sequencing can be low, the "soft cost," including data analysis, logistics, and manpower, can be significant. For other hospitals to emulate Stanford’s record-breaking speed would also require wet lab technicians, bioinformaticians, genetic counselors, or clinical geneticists to stand by 24/7 to avoid delays, Jiang said, which is something clinical genetic professionals need to reckon with. "We do not wake up people in the middle of the night to look at the DNA sequence," he said. "That’s not a typical practice right now."
Ashley agreed. "Are genomics labs ready to operate 24/7?" he said. "I think they are not." Staff members in a critical care unit are going to expect to be there during the night as well as during the day, he added, "but if you take a job in a genomics lab, you would be surprised to be asked to come in during the night currently."
Both Jiang and Kingsmore also pointed out that, aside from sequencing and bioinformatics, annotating the pathogenic variants can sometimes be time-consuming, hindering a speedy diagnosis. "We need expert lab directors, genetic counselors to go in, and it can take them 10 hours after the run is done," Kingsmore said. "So, that's a real bottleneck."
Suma Shankar, a physician who serves as director of the precision genomic program at UC Davis Health, said the ultra-fast nanopore WGS approach described by the Stanford team is "awesome" and "can make a difference for metabolic patients," for whom care management is key.
As part of Project Baby Bear, a California state-funded project initiated by Rady Children’s in 2018 to demonstrate the value of rWGS in rapidly diagnosing sick newborns with unexplained conditions, UC Davis does not offer in-house rWGS but ships its samples to Rady.
Shankar said the cost of rWGS for her patients can reach $7,000 to $10,000, and thus, she thinks Stanford’s cost estimate for nanopore sequencing is "actually pretty good."
She pointed out that the Stanford researchers did not sequence parental samples for the pediatric participants, which can be important to determine how a pathogenic variant segregates or whether it is a de novo event, she explained.
Shankar also emphasized that regardless of the sequencing platforms, rWGS "has implications for the entire family and beyond" since the technique deciphers the whole genome. As a result, she said "proper consent and counseling needs to be done" by knowledgeable clinicians or medical genetic professionals, which takes time.
Nonetheless, she said Stanford’s achievement is "very exciting" for the community. "I was a grad student between 2002 to 2005. We were running gels and talking about one day we would be taking a blood draw and running the sequencing and getting an answer within hours," Shankar recalled. "I think the day is here."