Chris Boles is Chief Scientific Officer of Sage Science, where he’s been helping the R&D team develop the new SageHLS (that’s short for HMW Library System), a platform designed to rapidly purify high molecular weight DNA directly from samples. We caught up with him to learn more about it.
Q: What’s so important about having high molecular weight DNA?
A: Working with extremely long DNA has become a lost art in the life science community. Back in the early days of the Human Genome Project, every lab had to think about this as they worked with recombinant BACs, fosmids, Southern blots, and so on. But beginning with PCR in the early ’90s and continuing with short-read NGS since the early 2000s, life scientists have had the tools to do amazing things without the need for HMW DNA. Now, researchers are tackling repetitive genomic regions, long-range structural variation, and long-range phasing, and the need for high-quality, high molecular weight DNA has resurfaced.
Q: What are some of the long-range technologies that the SageHLS platform could be used with?
A: Really any system that requires DNA that is hundreds of kilobases to megabases in size. These include long-read sequencing platforms like PacBio or Oxford Nanopore, optical mapping technologies such as those from Bionano Genomics or Genomic Vision, and other long-range linkage analysis methods like the ones from 10x Genomics or Dovetail Genomics. The SageHLS can improve input DNA quality and size for all of these systems.
Q: What kind of sample prep is required before loading the SageHLS?
A: In general, very little. We have focused initially on sample types that 1) are important for biomedical research, and 2) work well in SageHLS. These include white blood cells from whole blood, tissue culture cells, or bacterial cultures. For these sample types, only a few brief centrifugation steps are necessary to wash the cells and resuspend them in an isotonic gel loading buffer. The cassette reagents do the hard work of lysis and purification without mixing or shearing the HMW DNA.
Q: How does the new system work?
A: Users load their samples into a gel. Then the platform automatically performs cell lysis and contaminant removal. This happens very quickly, leaving megabase-sized DNA stuck in the agarose. Next, the DNA is lightly cleaved with a non-specific nuclease and retrieved from the gel through an automated elution process.
Q: What kind of results have you gotten from the SageHLS internally?
A: From mammalian WBC and tissue culture cells, we routinely obtain DNA ranging in size from 200 kilobases to 2 megabases. From input cell loads containing about 10 ug of DNA (about 1.5 million human cells), we recover 1 to 3 ug of DNA of this size, which is sufficient for even DNA-hungry applications like optical mapping.
Q: You’re already working on new uses for the SageHLS platform. Can you give us a sneak peek?
A: There are several improvements that should happen fairly quickly after launch. Next up is a process we call HLS-CATCH, which uses CRISPR/Cas9 technology to excise and isolate a genomic fragment of interest in a targeted fashion. We’re also working on several methods for making NGS libraries directly in the HLS cassettes so that we can integrate DNA extraction directly with NGS library construction. It will be interesting to learn from customers what else they want to do with the system.
The Sage Science team had a blast at this year’s AGBT conference in Hollywood, Fla. We thoroughly enjoyed the parties, the beach, and especially the excellent talks. We’d like to thank all of the attendees who made their way to our suite to check out the SageHLS instrument for extracting and purifying high molecular weight (HMW) DNA, which is now commercially available.
Following a trend that’s been gaining ground in the last few years, many AGBT talks and posters focused on technologies that require HMW DNA. There was lots of data from users of Oxford Nanopore and PacBio sequencers, long-range methods such as 10x Genomics, and scaffolding tools like Bionano Genomics or Dovetail Genomics. All of these platforms need much longer DNA than scientists ever had to produce for short-read sequencers, kicking off a new era of enhanced sample prep for working with incredibly large genetic fragments.
That’s why we developed the new SageHLS platform, which can extract DNA as long as 2 Mb directly from a sample. Chris Mason from Weill Cornell, whose lab worked with a beta version of the new instrument, gave a presentation at AGBT and spoke about his experience with it. (For the record, we would have loved his fast-paced, entertaining talk about sequencing in space even without the SageHLS shout-out.) Mason used the instrument with the CATCH method — that’s short for Cas9-assisted targeting of chromosome segments, from this Nature Communications paper — and said the approach will be important for things like measuring telomere length with great accuracy.
For more information, check out our AGBT poster with results from the SageHLS instrument.
The Sage Science team is casting off the shackles of winter and gearing up for a trip to sunny Florida for the annual Advances in Genome Biology and Technology meeting. We look forward to this conference each year — it’s a great opportunity to catch up with friends while we learn about the latest technology innovations in the field.
AGBT has a long tradition of technology talks and posters, and this year’s meeting should continue the trend. We’re delighted to see two plenary sessions as well as a concurrent session focused on technology, giving us ample opportunity to discover promising new tools and biological insights.
A major theme in recent AGBT meetings, and one that’s apparent again this year, is a focus on alternatives to short-read sequencing. While NGS platforms offer a great deal of useful information, there’s a growing awareness that they miss important genomic elements that are too long to be spanned by 300-base reads. We’ve seen loads of interest for long-range technologies that can extend information from NGS systems, genome mapping approaches, and of course single-molecule, long-read sequencing platforms.
With that in mind, we’ve developed our newest sample prep system to help scientists generate the high-quality, high molecular weight DNA required as input for these long-range methods. The SageHLS (that’s short for HMW Library System) will be on display in our AGBT suite #3281 for anyone who wants to check it out and learn how it can be used to purify DNA from 50 Kb to 2 Mb. We look forward to seeing you in Florida!
We’ve been big fans of the structural variation work coming out of Evan Eichler’s University of Washington lab for years, so when he was the subject of a Mendelspod interview, we couldn’t wait to listen. If you missed the podcast, check it out here.
In his chat with Mendelspod host Theral Timpson, Eichler begins with an overview of structural variation and a really interesting perspective on how duplications were handled during the Human Genome Project by both the public and private initiatives. It was a nice reminder that structural variants have dogged the genomics community from the beginning!
Eichler’s career has included a focus on segmental duplications, the hotspots of rearrangements that tend to be home for most of newly acquired genes that make us uniquely human. These areas are replete with structural variants, which is why Eichler put such effort into resolving those elements. The segmental duplication regions are dynamic, frequently implicated in disease, and “move at light speed compared to most regions of the genome,” he tells Timpson, calling them “crucibles for evolutionary change.”
SNPs, Eichler says, are kind of like tremors in the genome, while structural variants are full-blown earthquakes. He notes that while NGS technologies allow us to observe SNPs directly, we’ve had to infer structural variants from this data, which limits our ability to detect these variants accurately and comprehensively. While he believes that short-read sequencing was revolutionary for genomics, he made the case that for optimal clinical utility, we have to move toward a de novo assembly approach for each person’s genome that will fully phase maternal and paternal haplotypes.
Eichler is looking to long-read sequencing to discover and catalog existing structural variation in humans as well as to produce de novo assemblies. This technology can uncover 90% of the structural variants that have been missed by short-read sequencers, he says. Today, long-read sequencing is too expensive to replace short-read tools, but Eichler suggests that could change. “I think if we had a long-read sequencing technology that was even double the price of Illumina, short reads would be dead,” he tells Timpson. But in addition to lowering costs, he says, these technologies have to produce megabase-scale reads while also increasing capacity in order to make these platforms better suited for clinical use.
Eichler also sees room to improve sensitivity and characterization of structural variants. “What we need to think about is how to do this right, and that means understanding all the variation from stem to stern in these genomes,” he says.
We are shaking off this northeast winter and heading out to San Diego for the 25th annual International Plant and Animal Genome meeting! This event is always a great way to kick off the year, surrounded by cutting-edge science and cool technologies.
The Sage team will be in booth #422, so if you venture to the exhibit hall, we hope you stop by to say hello. We’ll have our SageHLS instrument available for a sneak peek — it’s not officially launched yet, but we’ll be happy to tell you about its performance for isolating and purifying high molecular weight DNA.
At PAG, we’re looking forward to seeing studies involving two of the most popular applications of our DNA size selection technology: RAD-seq and high-pass sizing. The original double-digest RAD-seq protocol was based on our Pippin Prep instrument, and new iterations of the method have continued to rely on Sage platforms for highly accurate, high-yield DNA size selection. Since ddRAD-seq and related methods allow for massively parallel genotyping of organisms without a reference genome, it has been widely adopted within the plant and animal research community.
High-pass sizing, which allows scientists to keep all fragments larger than a chosen cutoff, has been particularly helpful for PacBio and Oxford Nanopore users looking to extend the average read lengths they can generate with their sequencers. By removing the smaller fragments ahead of time, these sequencers can focus their attention on only the fragments most likely to produce ultra-long reads.
If you’ll be at the Town & Country, we hope to see you there!