Fastest DNA sequencing technique helps undiagnosed patients find answers in mere hours

A paper describing the researchers' work was published in 'The New England Journal of Medicine'. Euan Ashley, associate dean of the Stanford School of Medicine and the Roger and Joelle Burnell Professor in Genomics and Precision Health, is the senior author of the paper. Postdoctoral scholar John Gorzynski, DVM, PhD, is the lead author.

"A few weeks is what most clinicians call 'rapid' when it comes to sequencing a patient's genome and returning results," said Ashley.

Genome sequencing allowed scientists to see a patient's complete DNA makeup, which contained information about everything from eye color to inherited diseases. Genome sequencing has been vital for diagnosing patients with diseases rooted in their DNA: Once doctors knew the specific genetic mutation, they could tailor treatments accordingly.

Now, a mega-sequencing approach devised by Ashley and his colleagues has redefined "rapid" for genetic diagnostics: Their fastest diagnosis was made in just over seven hours. Fast diagnoses mean patients may spend less time in critical care units, require fewer tests, recover more quickly and spend less on care. Notably, the faster sequencing does not sacrifice accuracy.

Over the span of less than six months, the team enrolled and sequenced the genomes of 12 patients, five of whom received a genetic diagnosis from the sequencing information in about the time it takes to round out a day at the office. (Not all ailments are genetically based, which is likely the reason some of the patients did not receive a diagnosis after their sequencing information was returned, Ashley said.) The team's diagnostic rate, roughly 42 per cent, is about 12 per cent higher than the average rate for diagnosing mystery diseases.

In one of the cases, it took a snappy 5 hours and 2 minutes to sequence a patient's genome, which set the first Guinness World Records title for fastest DNA sequencing technique. The record was certified by the National Institute of Science and Technology's Genome in a Bottle group and was documented by Guinness World Records.

"It was just one of those amazing moments where the right people suddenly came together to achieve something amazing," Ashley said. "It really felt like we were approaching a new frontier."

The time it took to diagnose that case was 7 hours and 18 minutes, which, to Ashley's knowledge, was about twice as fast as the previous record for a genome sequencing-based diagnosis (14 hours) held by the Rady Children's Institute. Fourteen hours is still an impressively quick turnaround, Ashley said. Stanford scientists planed to offer a sub-10-hour turnaround to patients in intensive care units at Stanford Hospital and Lucile Packard Children's Hospital Stanford -- and, over time, to other hospitals too.

To achieve super-fast sequencing speeds, the researchers needed new hardware. So, Ashley contacted colleagues at Oxford Nanopore Technologies who had built a machine composed of 48 sequencing units known as flow cells. The idea was to sequence just one person's genome using all flow cells simultaneously. The mega-machine approach was a success -- almost too much. Genomic data overwhelmed the lab's computational systems.

"We weren't able to process the data fast enough," Ashley said. "We had to completely rethink and revamp our data pipelines and storage systems." Graduate student Sneha Goenka found a way to funnel the data straight to a cloud-based storage system where computational power could be amplified enough to sift through the data in real time. Algorithms then independently scanned the incoming genetic code for errors that might cause disease, and, in the final step, the scientists conducted a comparison of the patient's gene variants against publicly documented variants known to cause disease.

From start to finish, the team sought to hasten every aspect of sequencing a patient's genome. Researchers literally ran samples by foot to the lab, new machines were rigged to support simultaneous genome sequencing, and computing power was escalated to efficiently crunch massive data sets. Now, the team has been optimizing its system to reduce the time even further. "I think we can halve it again," Ashley said. "If we're able to do that, we're talking about being able to get an answer before the end of a hospital ward round. That's a dramatic jump."

Perhaps the most important feature of the diagnostic approach's ability to quickly spot suspicious fragments of DNA is its use of something called long-read sequencing. Traditional genome-sequencing techniques chopped the genome into small bits, spelled out the exact order of the DNA base pairs in each chunk, then pieced the whole thing back together using a standard human genome as a reference. But that approach didn't always capture the entirety of our genome, and the information it provided could sometimes omit variations in genes that pointed to a diagnosis. Long-read sequencing preserved long stretches of DNA composed of tens of thousands of base pairs, providing similar accuracy and more detail for scientists scouring the sequence for errors.

"Mutations that occur over a large chunk of the genome are easier to detect using long-read sequencing. There are variants that would be almost impossible to detect without some kind of long-read approach," said Ashley. It's also much faster: "That was one of the big reasons we went for this approach."

Only recently have companies and researchers honed the accuracy of the long-read approach enough to rely on it for diagnostics. That and a drop from its once-hefty price tag created an opportunity for Ashley's team. To his knowledge, this study was the first to demonstrate the feasibility of this type of long-read sequencing as a staple of diagnostic medicine.

During the study, Ashley's team offered the accelerated genome sequencing technique to undiagnosed patients in Stanford hospitals' intensive care units. They provided established standard of care testing to the study patients along with the experimental rapid gene sequencing, with which they sought answers to two important questions: Are genetics to blame for the patient's ailment? If so, what specific DNA errors are stirring up trouble?

Standard tests screen a patient's blood for markers associated with disease, but they only scan for a handful of well-documented genes. Commercial labs, which often run these tests, were slow to update the molecules for which they screen, meaning it could take a long time before newly discovered disease-causing mutations were integrated into the test. And that could lead to missed diagnoses.

That's why rapid genome sequencing could be such a game-changer for patients ailing from rare genetic disease, Ashley said. Scientists can scan a patient's entire genome for any and all gene variants suggested by the scientific literature, even if that gene is only discovered the day before. Furthermore, if a patient doesn't initially receive a genetic diagnosis, there's still hope that scientists will find a new gene variant linked to the patient's disease down the line.

Interest from other clinicians is already starting to pour in. "I know people at Stanford have heard we can make a genetic diagnosis in a few hours, and they're excited about it," Ashley said. "Genetic tests just aren't thought of as tests that come back quickly. But we're changing that perception. "That's why rapid genome sequencing could be such a game-changer for patients ailing from rare genetic disease, Ashley said.

Scientists can scan a patient's entire genome for any and all gene variants suggested by the scientific literature, even if that gene was discovered the day before. Furthermore, if a patient doesn't initially receive a genetic diagnosis, there's still hope that scientists will find a new gene variant linked to the patient's disease down the line. (ANI)