Sage Blog

Podcast: Structural Variation Pioneer Evan Eichler on Moving Toward the Clinic

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.

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RAD-seq, High-Pass Sizing in Sunny San Diego

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!

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Farewell to 2016

As 2016 draws to a close, we’re taking a look back to capture some of the highlights of the year before it disappears into the blur of previous years.

One of the most exciting advances this year came from the realm of cell-free DNA. Whether it’s for tracking signs of cancer or detecting tiny signals from a fetus, accessing these circulating DNA fragments is allowing scientists to make some real progress in clinical applications. Because of the rarity of fragments of interest amid all the other circulating DNA, size selection has proven to be an extremely useful tool for isolating the target fragments for analysis. We worked with Rubicon Genomics this year on a protocol to enrich for cell-free DNA and reported results in a poster at the AGBT Precision Health meeting.

We also enjoyed seeing the science community continue to build on restriction-site associated DNA sequencing methods. From the explosion of new tools in this area, it may well have been the year of RAD! From a method to expand RAD-seq utility to low-quality DNA such as that in museum samples to an optimized protocol for plants, the approach has been embraced in areas of broad interest. We recapped several of the new tools and methods for RAD-seq.

2016 was also marked by the release of new and better human genome sequences. As sequencing and analysis technologies become more affordable and accurate, groups around the world are aiming for reference-grade assemblies for their populations. These projects have been made possible in part by landmark efforts from the Genome in a Bottle Consortium to improve the quality and reliability of variants called from these genomes. Two of the most impressive human genome assemblies released this year were the Chinese and Korean genomes. Both used an array of sequencing and other technologies and quickly became some of the highest-quality human assemblies ever generated.

We enjoyed some milestones here at Sage Science too. Our newest platform, the PippinHT sizing platform, was cited in its first publication. Meanwhile, we continued development of our next tool, the SageHLS for generating high molecular weight libraries. We described it in this blog and released more details at ASHG. The official launch is imminent — stay tuned!

It was also an honor to see that NGS and related companies continued to make use of Sage products to optimize results. Here are some examples and recommendations we noticed this year:

10x Genomics recommends BluePippin and PippinHT sizing
Oxford Nanopore data shows that BluePippin sizing increases read length
PacBio users report better results with SageELF and BluePippin sizing

Finally, we caught up with some customers to profile their great work. If you missed them, check them out now:

Hamid Ashrafi, North Carolina State University – blueberry breeding
Bruce Kingham, University of Delaware – genomics core facility
David Moraga Amador, University of Florida – ddRAD-seq and long-read sequencing

And now it’s on to 2017. From all of us here at Sage, we wish you a happy new year!

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HLA Typing Workflow Uses Pippin Prep for Size Selection

We’ve written before about the shift toward NGS-based technologies for the HLA typing market. HLA typing is used for everything from understanding autoimmune and infectious diseases to matching organ transplants to recipients.

But the HLA locus really make scientists and clinicians work for their answers. This region of the genome is one of the most polymorphic, with more than 14,000 recognized HLA alleles so far. The rise of affordable, high-throughput NGS platforms is an appealing alternative for labs responsible for typing these genes.

In a paper published in the December 2016 issue of Clinical Chemistry, scientists from The Children’s Hospital of Philadelphia describe an NGS-based HLA typing workflow that uses a kit from Omixon to report relevant class I alleles. We were pleased to see that our Pippin Prep system proved valuable in the pipeline; the team used it to select DNA fragments between 650 bp and 1300 bp prior to sequencing on an Illumina MiSeq.

Generation of Full-Length Class I Human Leukocyte Antigen Gene Consensus Sequences for Novel Allele Characterization” comes from lead author Peter Clark, senior author Dimitri Monos, and collaborators. In it, they describe their evaluation of the Omixon Holotype HLA assay using samples from 50 individuals. “HLA genotyping results and fully phased consensus sequences were successfully generated for all 50 participants using the Omixon Twin algorithm (300 total alleles) and were found to be concordant with SBT/SSP genotyping results,” the authors report.

Interestingly, the team found that 7.7% of samples featured novel alleles and predicted that this discovery rate means “there are likely to be numerous yet undiscovered alleles of unknown significance.” They add that “full-length gene characterization is paramount for unambiguous HLA genotyping and facilitates a deeper understanding of HLA gene polymorphisms and the eventual role they may play in the immune response.”

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Moving to the Clinic: Interview with BC Cancer Agency’s Marco Marra

Mendelspod’s recent interview with Marco Marra of the BC Cancer Agency and the University of British Columbia is well worth a listen. In the podcast, Marra describes his team’s use of genome and transcriptome sequencing for patients whose cancer is considered incurable.

Marra first captured attention in this area in 2009 when he reported his lab’s use of whole genome sequencing to inform treatment decisions for a patient with a rare adenocarcinoma. Genome and transcriptome analysis revealed that the tumor was driven by the RET oncogene. The patient, for whom there had been no clear therapy option, was treated with a RET inhibitor that was in clinical trials at the time — and the tumor shrank significantly.

Since then, Marra parlayed that individual project into a pilot study for how whole genome sequencing could be expanded to other cancer patients. That study was broadened again in 2014 and today his team has analyzed some 400 people who essentially have no other options for treatment. The scientists look for all sorts of mutation types, from SNPs to structural variants and more. One major challenge has been off-label use of drugs: in many cases, genome analysis points to a therapy that’s not indicated for the patient’s type of cancer, and gaining access to the therapy is hit or miss. As the cancer genomics program has expanded, Marra wrestles with questions like, “What is the meaning of having whole genome analysis that points you to a particular agent that you can’t get?” As he told interviewer Theral Timpson, “These are deep conversations that are happening within our environment and probably elsewhere.”

Marra is also keeping a close eye on how clinicians apply information from the genomic analysis. Doctors who just get a report of mutations tend to be less comfortable incorporating that data into treatment decisions. But a weekly conference that allows physicians, scientists, bioinformaticians, and pathologists to walk through case studies often prompts useful interdisciplinary discussions and frequently leads to increased implementation of genomic results, he said.

If you’ve got a little time, we highly recommend listening!

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