long read sequencing

January 2018 Review Show with Nathan and Laura: CRISPR vs The Immune System, Biotech Math, and MinION's Big Test

Some stocks are up on news of big biotech mergers, but others are down on hearing of the latest difficulties of gene therapy. One thing’s for sure—blood diseases are where it’s at.

Speaking of the latest difficulties, we start our January review by going back to that paper out of Stanford about a new obstacle to using CRISPR as a new drug platform. It’s called the human immune system. Major roadblock or small warning light?

“Smart people have been thinking about the wrinkles in CRISPR and Cas9 for a long time. This is one of them, and it’s not going to stop the technology from being used well in people in the long run,” says Nathan.

Neither of our commenters are happy with Luxturna’s pricing of $425,000 per eye. But who is happy with drug pricing these days? How are drug companies supposed to recoup their investment on roughly 2,000 patients?

“We’re gonna have to price these drugs based on the whole platform, but also we’ll have to look at creative things like . . . how successful it is,” says Laura.

Last, but not least, we finish up with the new paper out in Nature showing the sequencing of a reference genome using the MinION handheld nanopore sequencer. Laura says it’s a snooze, but then she comes around.

Sharon Begley of STAT News

Sharon Begley joins us for our last show of the year to look back over some of the year’s top stories. She’s the senior science writer at STAT News where she covers genetics, cancer, neuroscience and other fields of biomedical research. Prior to joining STAT, Sharon was the senior health and science correspondent at Reuters, the science columnist at the Wall Street Journal, and the science editor at Newsweek.

If you’re in genomics, you’ve no doubt found yourself reading one of Sharon’s columns. Her range is astonishing, her depth shows years of insider knowledge, and her output prodigious. She managed to write an article on George Church this year that no one had written before. Not easy.

Sharon says her audience has a big industry component and certainly includes people with special interest in the life sciences but it’s also for “ordinary human beings . . . people who go to doctors, who’s friends and loved ones get sick.” That Sharon’s articles can be read by anyone, but are of interest to insiders, makes her a great guest here on the program to see how many of our stories make it out to a larger audience. For instance, in this year when 23andMe’s test rivaled the InstantPot for top seller at Amazon, what does the average person think of genomics in 2017?

In answer, Sharon says that the typical American tends to be a genetic determinist, gullible for any genetic association that comes along--this despite being overwhelmingly religious. Does that mean she proactively takes on the role of pushing back with skepticism?

A New “Middle Way” for Genomics, with Physical Chemist, Yuval Ebenstein

“I love low tech,” says today’s guest.

It’s not your typical catch phrase for 2017. But then today’s guest is not your typical genome scientist.

A professor in the Department of Chemical Physics at Tel Aviv University in Israel where he runs the NanoBioPhotonix Lab, Yuval Ebenstein came to the genome from an unusual direction. As a physical chemist he started working with DNA as “just a material.”

The low tech is the method of visualizing genomes with microscopy, such as the old FISH or cytogenetic experiments. However, with the advances in imaging and single molecule analysis, he can now go far beyond these dated methodologies and "take dense chromosomes and stretch them out and read information along them in a very sensitive and informative way that is not accessible to other established genomics techniques."

“I love low tech and then giving it a little twist and turning it into high tech," he adds.

Yuval calls the twist a new “middle way” in genomics, between the large structural cytogenomics of the past and all the specific base calling going on now with next gen sequencing. Will his lab’s work turn into a new instrument able to be commercialized?

He says that PacBio, Oxford Nanopore, and BioNano are making headway in filling in this third or middle way, but that yes, there are new techniques that everyone should be able to use.

One specific paper Yuval’s group has recently preprinted is a method for isolating and cloning very long fragments of DNA using Cas9, or what his group calls CATCH (Cas Assisted Targeting of Chromosome Segments).

“This is a nice demonstration of taking low tech and reviving it,” he says.

It’s also an example of what Yuval says is the problem today with NGS, which is too much data.

To look at certain regions of the genome, such as BRCA, one does whole genome sequencing, or exome sequencing and ends up with a confusing amount of data. With CATCH, he suggests one can take advantage of CRISPR to isolate just the target DNA one is looking for. As our audience will know, most of the methods we use today, say in cancer diagnostics, are looking “under the lamppost,” using templates of known mutations rather than being able to discover what’s actually there.

“You could PCR out large pieces of the genome, but it’s hard and tedious, because you need a lot of primer sets. If it’s a very variable region like BRCA, you may have problems with your primer design, which won’t fit. This is another, hopefully more elegant way of taking out the intact region of interest of the genome and analyzing it very deeply,” he says.

Yuval’s lab is one to keep on our radar.

Huh? 30 Million Americans Have a Rare Disease? Howard Jacob on the State of Clinical Sequencing

Here’s a title for you. Chief Genomics Officer. Today’s guest is also the VP of Genomic Medicine and a faculty investigator at the HudsonAlpha Institute for Biotechnology.

He launched the world’s first genomic medicine program becoming the first person in history to use genome sequencing to diagnose, treat, and cure a patient. Few people exude the sheer force and vision for the future of genomic medicine that comes from Howard Jacob. We’re very pleased to have him on Mendelspod for the first time to talk about progress with rare disease, sequencing technology, and how he would teach genomic medicine to young people today . . . And of course, that genomic age old question: the exome or the whole genome?

We've Become Too Single Variant Centric, Says Deanna Church on Genome Analysis

From 1999 to 2013, Deanna Church was a staff scientist at the NCBI where, for a time, she headed the Genome Reference Consortium. This was the effort to continually update, improve and maintain the reference genome. Then Deanna went into private industry, first to Personalis--a genome interpretation company, and now she’s Director of Applications at 10X Genomics--the tools company offering linked read sequencing technology. Deanna's work in the public and private genomics domains has given her a comprehensive and even profound knowledge of the human genome and an authoritative ease in communicating about it.

When we asked about the recent paper out by the 1000 Genomes Project—which includes her name as author—that brings to light hundreds of heretofore unknown structural variants, she says this:

“What I think would be really great is to see the community move toward the integration of structural variant calling and short variant calling. These still tend to be very separate. This paper, of course, only dealt with structural variant calling because it's a very challenging problem. Many times the [different] variant calls end up in separate files. What you’d really like to do is have a wholistic view. Analyzing the whole genome and thinking about how all the variants go together will be an important step for the community.”

Many of the scientists we talk to often begin at a tools company and then move on to an institution where they can work with an array of tools. Deanna has gone the other direction. But she says that working at 10X has “expanded her inner scientist.” There she has access to a lab which wasn't the case at the NCBI and is challenged by an array of hard scientific problems brought by customers of their linked read technology.

So what is new in the world of linked reads? What are Deanna’s thoughts on the incredible uptick in single cell sequencing applications? And in an age when the NIH’s budget has been threatened, how does she see the roles of private and public genomics institutions playing out?

It’s Deanna Church for the first time on Mendelspod.

Charting the Dark Matter of Cancer Genomes with Jim Broach

We’ve heard a lot this year about the search for new structural variants and the hope that scientists will find new causal linkages for diseases such as cancer. But will the genome still yield dramatic genetic signatures such as KRAS, BRAF and EGFR that have been so helpful in cancer treatment?

Today’s guest says, yes, and he’s on the trail.

Jim Broach is the Director of Penn State’s Center for Personalized Medicine. He and his team have come up with the highest resolution genomic data to date on certain cancer cell lines using sequencing and mapping tools. In some cell lines his research has revealed 150-200 more structural variants than had previously been discovered.

“There are a whole set of structural variants which haven’t been taken into consideration to date,” he says in today’s interview. "For the next couple of years, this is the dark matter of the cancer genome. We’ve got to sort out which of these structural variants are going to be relevant in understanding how best to treat the patients. Once we generate that information, I think these structural variants will be just as relevant as the point mutations or as large scale translocations."

Jim mentions paired end reads and PacBio’s new long read technology, but the main tool he talks about is Bionano’s optical mapping technology. Previously the field used karyotyping to look for variants of this size, but he says Bionano has got their technology to the quality and price point where it will now replace the older technology.

How will Jim’s research impact treatment in the clinic? He is doing de novo sequences of cancer cell lines. Does he envision the need for de novo sequencing of a patient’s cells as part of a commercial assay?

Will This New Nano Technology Be the Microarray of Genomic Structural Variation? Barrett Bready, Nabsys

Barrett Bready is back on the program. He’s the CEO of Nabsys, a company with some new technology for genome mapping.

Originally Nabsys had been working to develop nanopore sequencing, but after a recent reboot has become focused on scaling up scientists' ability to read structural genomic information. Barrett compares Nabsys’ new multiplex technology for genome mapping to the improvement of arrays over single nucleotide (SNP) detection.

"When we first started we were using solid state nanopores. And we realized that there were limitations to nanopores. Nanopores don’t multiplex well. If you have two nanopores very close to each other and a DNA molecule goes through nanopore number one, the signal in nanopore number two will be effected. So we developed our proprietary nano-detector that can be multiplexed at really high density.”

With long read sequencing now gone mainstream coupled with a growing interest among genome scientists in structural variation, Barrett says Nabsys has a chance to enter the marketplace competing on price and throughput and will have their instrument ready for beta testing early next year.

Cardiologists Love Genomics: Euan Ashley, Stanford

Euan Ashley is one of the big names in genomic medicine that has been missing from our guest list. We’re happy to correct that today.

In 2010, he led the team who did the first clinical interpretation of a human genome--that of his Stanford colleague, Steve Quake. Since then Euan, an MD PhD, has been driving to make the use of new genomic tools and discoveries a routine part of medicine at Stanford, particularly in his own discipline of cardiology.

A regular speaker on the conference circuit, Euan titles his talks, "Genomic Medicine Is Here."

"There were these one off examples of great stories that captured everyone’s imagination,” he says at the outset "but somewhere in there, what happened is it just became routine. And we started sending exome and genome sequences on patients and using that information to help find a cause, and in some cases, treatment for their condition. We were all waiting for it to happen, but it just happened under our noses.”

At the same time, Euan acknowledges that he “loses sleep at night” over “dark corners of the genome.” What are these dark corners? What recent findings were made by new long read sequencing? How has genomics impacted cardiology?

We begin with the question, if genomic medicine is here, why are there still so many skeptics?

Join us in our first interview with one of the few jazz saxophonists in our field, someone who knew he wanted to be a doctor at age four but wasn’t inspired by science--that is, until a high school teacher handed him a copy of Richard Dawkins' “The Selfish Gene” after class.

People Told Us It Was Impossible: UCSC’s Mark Akeson on Nanopore Sequencing

Mark Akeson has been working on nanopore sequencing at UC Santa Cruz’s biophysics lab for twenty years. Up until the past few years with the launch of Oxford Nanopore’s sequencers, that work was mostly the methodical toil of the quiet inventor.

Today it is quite ordinary to see a sequencer the size of your wallet being taken out into the field for DNA work. But for years, the naysayers dominated.

“Back in the day, the skeptics outnumbered the proponents 99 to 1,” Mark says in today’s show.

In his beginning-of-the-year blog, NIH Director, Francis Collins, called nanopore sequencing one of the four breakthroughs of 2016. And the NIH deserves some credit.   Mark says they were constant in their funding and belief in the technology.

With the success of nanopore sequencing technology has come legal battles to secure the IP.   Both Illumina and PacBio have sued Oxford Nanopore—the Illumina suit is now settled. And at the end of last month, Akeson’s lab (meaning the University of California) sued Genia, claiming that they owned the patents for Genia’s technology.  Genia was founded in 2009 and we have interviewed them several times since 2011.

“There's the old adage about once something succeeds, there’s all sorts of people who claim to have invented it,” says Mark.  

So what’s next for Mark? Is he on board the “long read train?” How much more can sequencing improve?


When Long Reads are Double the Price of Short Reads, Short Reads Are Dead, Says Evan Eichler

Each year at this time, sequencing tools leader, Illumina, generates another round of sequencing buzz in the industry, this year by announcing the $100 genome is around the corner with their latest boxes. But more and more, people are asking just what they will get with that $100. Indeed, what do they get today with a $1,500 genome?

Illumina sells short read sequencing technology which is unable to characterize much of the human genome, particularly complex regions which are responsible for many of the known and unknown diseases.

Today’s guest has made his career studying structural variation of the genome. He’s done it with the rapidly improving long read sequencing technology, mostly on instruments produced by Pacific Biosciences. He says researchers have been seduced by the ability to sequence thousands and tens of thousands of genomes as opposed to understanding five or ten genomes really well.

Evan Eichler is a professor of genomics at the University of Washington and first made his name known back with the original Human Genome Project. In the final days of the project, he was brought into the NIH to analyze the genome for structural variation repeats. Neither the private Venter enterprise nor the public attempt had the ability to see them at the time, and with what Evan calls his “young, stupid naivety," he waded into the project. He was able to compare data from the two groups without getting too caught up in the politics and ended up making an important contribution to the final output. Today Evan has established himself so well in the structural variation space that it is said no project into structural variation can be conceived without him.

“Work that we have done over the past couple years has shown that if you apply a new sequencing technology like long reads, you basically uncover 90% of the structural variation that is missed by short read sequencing technology.”

That’s a big number.

“That is a big number,” says Evan, “so the question is, how important are structural variations? That’s open to debate.”

Evan says there is data which shows that structural variant level changes are likely to be more impactful than those of single nucleotide variants (SNVs). He compares SNVs to little tremors and structural changes to earthquakes when it comes to regulating the genome.

As with his mentor, Jim Lupski, (featured on the program here), Evan is adamant that we must stop using short read technology and aligning to a reference genome. Rather, he says, we must get to the place where we are doing de novo assembly of each genome. We can do that in the research setting now, but we must do that clinically as well.

“If we’re still aligning sequences to a reference genome, and that’s our only way for understanding genetic variation ten years from now, clinically we’ve failed. 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."

New to Mendelspod?

We advance life science research, connecting people and ideas.
Register here to receive our newsletter.

or skip signup