NEW YORK – Fiber-seq, a methylation-based method that adds chromatin accessibility information to any long-read whole-genome sequencing, is growing in popularity and should soon be available in commercial kits.
Developed by Andrew Stergachis' lab at the University of Washington, Fiber-seq uses enzymes to "stencil" DNA with 6mA methylation, an epigenetic mark not normally found in eukaryotic cells. This can then be read out by long-read sequencing from either Pacific Biosciences or Oxford Nanopore Technologies, both of which can detect 6mA.
"If [the DNA is] not stenciled it means something was physically bound and occluding the methyltransferase," Stergachis said. "What could be doing it? There are many possibilities," including various polymerases, transcription factors, and nucleosomes. He added that it can be applied to mitochondrial genomes to look at how it is compacted "and even pull out individual nucleoids forming D loops."
For researchers who were already using long reads, it adds extra layers of information from a small upfront investment. "Anything that gets long reads gets Fiber-seq," said Rachel O'Neill, a researcher at the University of Connecticut with interests in comparative genomics, among other topics. Her lab has been using Fiber-seq since hearing about it in 2022. There's no good reason not to, in her view. "It's just more data on the same sequence," she said.
"I'm very excited about looking at some of these [plant and animal] genomes and see how we are getting species-specific enhancers and different chromatin states."
She's just one of many researchers who are picking up the method, either using a methyltransferase developed by Stergachis or buying them off the shelf from enzyme suppliers such as New England Biolabs. Soon, many more researchers may be able to join in as at least two companies are interested in developing kits.
And with a recent paper published in Nature Genetics by the Stergachis lab showing the utility of Fiber-seq in resolving a Mendelian condition, it could be useful in clinical settings.
Stergachis published on the Fiber-seq method in 2020 in Science and has been working on it ever since. Like short-read sequencing-based methods such as ATAC-seq, CUT&TAG, and CUT&RUN, it provides information on genome accessibility and DNA-protein binding.
The key benefit is that it analyzes a native DNA molecule, not a PCR product, meaning the assay is quantitative. Moreover, as it uses long reads, it can provide long-range information, such as correlating a particular genotype with other measurements, as well as identify regulatory elements in regions that had been inaccessible by short-read methods.
Stergachis' lab, led by postdoc Mitchell Vollger, has also developed Fibertools, a computational suite that they hope will help other labs adopt Fiber-seq.
In parallel, the lab has also developed Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq), a method that marks protein occupancy with a signal that is preserved during PCR and compatible with single-cell sequencing protocols.
"Experimentally, in terms of bench work, [Fiber-seq] is very easy, very straightforward," said Bluma Lesch, a researcher at Yale University who has adopted the method to study stem cells. "Especially compared to things like chromatin immunoprecipitation sequencing, or even CUT&TAG, the standards in the fields for looking at genome-wide chromatin state right now, it's extremely easy to set up and collect the data."
On the computational side, some familiarity with the command line is helpful, she noted. She obtains her key enzyme from the Stergachis lab, though others have turned to off-the-shelf products. Matt Maurano, a researcher at New York University who is using Fiber-seq to study the genetics of psychiatric diseases, said his lab has had success with NEB's EcoG2.
"There's a certain simplicity to it," he said. "The challenge now is how to interpret the data and use it to answer questions that are relevant. There's a lot of potential."
Fiber-seq has enabled his lab to "set up some genome engineering experiments that would be more scalable if we could combine genotyping and phenotyping."
O'Neill uses Fiber-seq to look for species-specific enhancers inside repeated regions as well as to get more data out of long-read sequencing for genome assemblies. "If you make it Fiber-seq, you also get chromatin accessibility and methylation calls from each molecule. It's sort of a three for one," she said.
While many researchers have been happy to adopt Fiber-seq in its current state, it may soon be even easier to implement.
EpiCypher, a firm developing CUT&RUN and CUT&TAG kits, among other products, is "working to fast-track the creation of standardized Fiber-seq enzymes, kits, and end-to end-services," Chief Business Officer Martis Cowles said in an email.
"We have not determined a launch date but are already beginning to engage key opinion leaders for early access to Fiber-seq services," he said. "We anticipate expanding our early access to both products and services in the second half of 2025." He declined to comment on the pricing and IP situations but noted he has been working directly with Stergachis.
PacBio is also interested in commercializing Fiber-seq, according to Chief Operating Officer Mark Van Oene. The firm touted its new 6mA calling ability in its September reveal of the new Sprq chemistry. Given that Fiber-seq got mentioned at PacBio's presentation at last month's JP Morgan Healthcare Conference, the firm sees potential; however, when and how it might approach commercializing the method is unclear.
Following the recent publication of DAF-seq, "we need to revisit which one, if not both, to commercialize first," Van Oene said.