NEW YORK (GenomeWeb) – New dyes developed for use in Sanger sequencing work just as well as an established dye, according to an analysis conducted by an Association for Biomolecular Research Facilities research group and presented at the group's annual meeting in San Antonio earlier this week.
Having comparable choices for which dye to use could enable core facilities to save money while providing their customers the same level of service.
Even with the advent of next-generation sequencing technologies, Sanger sequencing has remained a core laboratory workhorse. According to Jessica Podnar, the assistant director of the Center for Biomedical Research Support at the University of Texas-Austin, Sanger sequencing is a moneymaker that helps supplement other services cores may offer. "People still need it and it still makes money," she said.
Though the BigDye Terminator chemistry from Applied Biosystems, now part of Thermo Fisher Scientific, was once the only one available, new dye competitors with different price points have come on the market as its dye patents have expired.
Because there are now new choices, ABRF's DNA Sequencing Research Group (DSRG) sought to examine how the new dyes, EdgeBio's BrilliantDye and MCLab's BrightDye, performed in real-lab scenarios in comparison to the established ABI dye. In addition to a general benchmarking, the DSRG also set out to determine how well the various dye chemistries handled difficult samples and whether the new dyes could be folded into existing lab workflows, results that were presented at the ABRF annual meeting.
"People were waiting for the patent to be up and they weren't sure buying a new dye would work," said Molly Zeller, a senior research specialist at the University of Wisconsin Biotechnology Center's DNA sequencing facility and DSRG member, adding that their findings now show that they do.
For their study, Podnar, Zeller, and their colleagues enlisted three sequencing facilities to investigate the different dyes. For the general benchmark comparison, the labs each used ABI's manufacturer-recommended protocol, while they used a protocol developed by a previous DSRG effort to handle difficult-to-sequence samples. Finally, each lab used its own in-house protocol to gauge how well the dyes worked within existing workflows.
The DNA templates used were a pGEM control template or, for the difficult-to-sequence samples, a GC–rich template or a low-complexity template, and all sequencing was performed on an ABI 3730xl DNA Analyzer.
After receiving their samples, the participating labs first performed a quality control check on the DNA and oligos using their site-specific procedures and standard reagents, including BigDye. This, Podnar said during her talk at ABRF, established a baseline between the sites and showed that their results could be compared.
For their benchmarking effort using pGEM, the three sites followed the manufacturer-recommended protocol, though used a 0.5X dilution — as core labs commonly rely on dilutions as a cost-saving measure. Each site reported similar results across the three dyes.
To test the difficult-to-sequence samples, the researchers turned to a protocol published in the Journal of Biomolecular Techniques in 2009 by a previous ABRF working group that optimized a method to best handle such tricky samples. As Podnar noted, the main differences between that protocol and the manufacturers' recommendations is that it calls for additional dGTP dye terminators, the additive betaine, and an additional 10 PCR cycles.
After first testing the difficult-to-sequence protocol on pGEM — where the results looked similar to those from the recommended protocol — the research sites then ran the high-GC and low-complexity samples. Results from the three sites and three dyes appeared similar, they found, though they noted that the new dyes didn't always perform quite as well.
All the dyes, meanwhile, struggled with the low-complexity samples. "This is difficult to sequence," Podnar said. "This might be as good as you can get, but they look comparable."
Lastly, each of the three sites also tested how well the dyes performed within their own established protocols, by only swapping BigDye out and the new dye chemistries in.
At one site, BrightDye appeared to underperform slightly in their test using pGEM, but the research group found that, broadly, the dyes were comparable.
Overall, this suggested to the researchers that the various dyes are largely interchangeable. "I didn't expect them to all do so well," Podnar said after her talk.
Having different options that work similarly could help cores save money, as the three dyes have different list prices. According to the companies' websites, 100 reactions' worth of Thermo's BigDye costs $1,230 for either version 1.1 or 3.1, while 100 reactions' worth of MCLab's BrightDye is $580 and Edge's BrilliantDye is $970 for either version 1.1 or 3.1.
Podnar noted that while she might not switch which dye her core uses because she has a good relationship with her provider, she said would likely use these findings to help negotiate a better rate. "I wouldn't want to jump ship just because [of] pricing, but I would expect them to treat us now with the idea that we have options," she said.
Wisconsin's Zeller said that she'd consider recommending switching if it saves money, though noted that there have not yet been internal discussions at her institution about this.
"I don't know why there would be a reason to stay with something more expensive, when you can literally just swap it out, save yourself some money, and pass on the savings," she said.