Short-read genome sequencing, while cost- and time-effective, often misses pertinent genetic information for rare disease diagnosis. This method sequences DNA fragments between 50 and 300 base pairs at a time and struggles with sequencing complex genomic regions and identifying large structural variations. It also requires an amplification process that can introduce errors or sequence bias.
Credit: PacBio and iHope
Long-read sequencing methods can read segments of DNA that are thousands to hundreds of thousands of base pairs, allowing researchers to uncover more complicated regions of the genome. This method can detect the large inversions, deletions or translocations that short-read sequencing misses.
“With long-read sequencing, we can now access parts of the genome that were previously invisible — complex variants and structural changes that we know play a crucial role in rare diseases,” Jesper Eisfeldt, a researcher at the Department of Molecular Medicine and Surgery at the Karolinska Institutet and the lead author of a paper in Nature Genetics exploring how long-read sequencing is changing genetic testing, said in a statement.
iHope partners with PacBio to scale long-read sequencing in low-income countries
iHope, a global rare disease genomics program of Genetic Alliance, has partnered with PacBio to bring long-read whole-genome sequencing (LR-WGS) to its patients. The program supports more than 1,000 patients annually across 25 clinical sites in 14 countries, with a focus on low-income areas.
“The huge advantage of having PacBio come on board is we’re trying to do somewhere between 1,000 and 2,000 patients a year across 25 clinics in these low- and middle-income countries, and having PacBio come on board as a partner is going to allow us to boost that by another 500 to 1,000 patients a year,” said Ryan Taft, chief science officer of Genetic Alliance.
The core of PacBio’s long-read sequencing technology is single-molecule real-time sequencing, explained Stacy Musone, who leads the market development group at PacBio. In this process, a single molecule enters a well and is read by an enzyme. The molecule is circularized in the library prep process to enhance base-calling accuracy by reading the molecule multiple times; this is called a circular consensus read.
Collecting epigenetic data in a single assay
This method also collects epigenetic data by measuring the interpulse duration, the time the polymerase pauses between incorporating nucleotides, and kinetic signals during the integration of the nucleotide, said Musone. “With our sequencing chemistry, you actually are able to understand what marks are present on an individual nucleotide,” she added.
With PacBio’s LR-WGS method, the DNA does not need to be chemically treated before sequencing, meaning epi signatures can be read without a separate methylation genotyping array. “It’s a really important differentiator,” said Musone, “especially when thinking about clinical adoption of long-read sequencing, one of the areas of really high value to clinical testing laboratories is the idea of consolidating multiple workflows to a single assay.”
Many rare disorders have genome-wide methylation changes, which can be diagnostic, said Taft, making epigenetic data sometimes essential for rare disease diagnosis.
“We’re talking about 300 million people across the globe with a rare disease. Right now, these technologies, genomic technologies, are probably getting to maybe the top 1% or top half a percent in the world. If we’re gonna put a dent in this, we all have to really pull together, and that’s sequencing technology companies, it’s tech companies, it’s infrastructure companies that are gonna help us really push the limits of what’s possible for precision medicine,” Taft said.
