The new SPRQ chemistry for the Revio system is making high-quality long-read sequencing more efficient than ever—delivering more data from less input without compromising accuracy or cost efficiency. But what does that actually look like in a real lab?
As the head of University of Bern’s sequencing facility, Dr. Pamela Nicholson and her team are known for taking on some of the trickiest genomic projects, working with challenging samples, and pushing sequencing technology to its limits. When they put SPRQ chemistry to the test, the results were even better than they expected, shifting assumptions about yield, quality, and cost. Here’s how they’re putting SPRQ to work, and what it could mean for the future of sequencing.
Q: Can you tell us a bit about University of Bern and your role at the core service provider?
Dr. Pamela Nicholson: I’m the head of the sequencing facility here at the University of Bern. We serve all the researchers at the university who want to do sequencing from extractions all the way to basic data processing. We also serve one of the biggest hospitals in Switzerland, government institutes, and some industry as well.
Q: What are some of the core areas of expertise and what sets you apart from other providers in the area
We cover everything –– we cover genomics, transcriptomics, epigenomics and we have a particular focus on microbiomics. People come to us because they’ve got difficult projects they want to do, and they know we’re strong on long reads.
Q: What is your consultation process like in helping a customer determine which technology is best for them? What role has HiFi sequencing played in this?
I really focus on finding the technology that will bring them the most for their research questions. If it’s going to provide them with better results to meet their research question, then let’s try and do that.
HiFi sequencing has really made a lot more projects feasible and addresses all the major considerations for a sequencing project. Accuracy and length are not in question with Revio. We struggled with the sequencing yield and throughput of earlier PacBio instruments and Revio really has proved itself to be a game changer for our facility with regards to throughput. In many projects we routinely use all the barcodes that PacBio sells and this just shows the leap forward in sequencing output.
It often comes down to budget for researchers and now, with Revio, many projects are affordable. For example, a PacBio full-length16S rRNA-Seq project is cheaper than an Illumina V3-V4 16S rRNA-Seq at our facility.
Q: What was your first reaction to hearing about the new SPRQ chemistry for Revio?
I really like these kinds of iterations. It’s impressive to follow the relentless innovation, and great to see that this is happening. Of course, with such updates, there are questions like “How can we ensure consistency in our longitudinal projects? What will it mean for people who are between projects? Will there be any changes?”
As a core facility, you always have to ask questions when there’s new chemistry releases. We must test the performance and examine if it really delivers what the company has marketed and presented it to achieve. In the case of Revio and the recent SPRQ chemistry -they really do deliver. SPRQ really is producing quite a lot more data.
Q: Can you tell us about the first few samples you sequenced with SPRQ chemistry?
We started with SPRQ late last December with some really diverse sample types. In particular, we had a project working with naked mole rats, which is genetically a very curious animal, and a project for the Bovine Pangenome Consortium with some really difficult older samples.
With the naked mole rat samples on SPRQ, we got well over 100 Gb. For this project we specifically waited for the updated version of SMRT Link and for the new chemistry because animal and plant projects tend not to run as well as, for example, human blood. These samples were a challenge because they were brain tissue samples that were not in the best condition, having been stored for years and shipped from North America to Europe. We used the Nanobind extraction kit, made the libraries, and typically would have expected between 70–80 Gb, but got 110 Gb with SPRQ. The quality scores were also up with the new chemistry as well, so we were quite happy.
We had a similar experience with the Bovine Pangenome Consortium –– these were blood samples, but really old blood, shipped all over the world for the consortium. Over the last few years, we have processed many of these tricky samples, and some didn’t get quite enough reads. Therefore, with SPRQ, we took the opportunity to pool some of these old libraries together and run them with SPRQ. Previously such libraries returned 60–80 Gb and here with SPRQ we were nearly up to 100 Gb. We are very satisfied with the results and critically, we’ve consistently been seeing results like this since we have switched over to SPRQ chemistry.
Q: What advice would you give to other clients thinking about getting started with long read sequencing?
HiFi sequencing is not as difficult or as expensive as you think. I think a big misconception is that ONT is cheaper, so I see a lot of projects sequencing with them solely because of the cost, but that’s not the case.
PacBio offers fully kitted solutions and protocols so that we don’t have to buy all the important aspects from three different vendors. It becomes expensive when you start counting the other vendors that you have to buy things from for every protocol.
Q: Can you give us insight into where you think long-read sequencing is headed?
I think it’s really the way forward. Before it was only feasible for certain researchers and certain projects, but now it can really cover everything extensively and this is because the protocols and sequencers are constantly evolving and getting better. We do high-quality gold-standard reference genomes with HiFi, Hi-C, and Kinnex full-length RNA sequencing led genome annotations, bulk transcriptomics, as well as long-read single cell RNA-Seq. It is exciting for microbiomics where we clearly see that full-length 16S sequencing with Kinnex is extremely powerful and gives a new dimension to phylogenetic resolution that you will not get from a V3-V4 16S rRNA-Seq project.
| Reads | Yield | Length (Mean) |
|---|---|---|
| 11.2 M | 125.0 Gb | 11.2 kb |
| 11.5 M | 152.3 Gb | 13.3 kb |
| 11.4 M | 164.5 Gb | 14.4 kb |
University of Bern data yield from recent Kinnex single-cell and full-length libraries.
There’s possibly a bit of a latency to see adoption with academic researchers or in the clinic, but when it is taken up, it can really switch a light on in a project. For example, we have had a few long-read RNA sequencing projects that have uncovered critical gene fusions. In these projects, short-read sequencing was not giving enough information and only with PacBio full-length Kinnex kits were we able to detect transcripts spanning two genes and sequence full-length fusion transcripts. The amount of alternative splicing has probably been underestimated and undervalued for years.
People are overwhelmed when they get their Iso-Seq data. They say, “Can this be real?” It is exciting. Long-read RNA sequencing is a new era of transcriptomics.
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