long read sequencing

Hanlee Ji is the Senior Associate Director of the Stanford Genome Technology Center as well as an oncologist at Stanford. He’s also a clinical geneticist. In other words, he doesn’t need to take off his glasses and spin around in a phone booth to be able to do about everything.

“I was in fellowship for a long time,” he says in todays interview.

Long reads have been an important theme in the genomics community of late, and Hanlee’s lab recently developed a new method for isolating long fragments of DNA that rivals the long reads of PacBio and Oxford Nanopore. The new method is the first we’ve featured that uses 10X Genomics’ linked reads. The new method also uses CRISPR and CATCH (a new sample prep system from Sage Science), and because it’s done with digital PCR, it offers the nice advantage of only requiring very small sample sizes.

Applications? Hanlee says he’s most excited to use it to identify 're-arrangements’ such as those in congenital disorders or oncogenic drivers.

Hanlee’s lab is also involved in a new clinical trial using precision cancer vaccines that is pulling him headlong into the immuno therapy space.

With a foot deep in the world of genomic technologies and another foot in the clinic, what does Hanlee the oncologist want to tell the technologists? And what does Hanlee the technologist want to tell his colleagues, the cancer docs?

It’s some big questions, and he takes around 27 min to get around them. Enjoy.

Sequencing geeks are fresh off the trail from AGBT, and it’s time for our annual look at the sequencing tools space. This year we sit down with the longtime Omics! Omics! blogger, Keith Robison, who not only can answer all your questions about the topic, he even knows which sequencer you’re using right now, and in which department.

Keith jauntily runs through the Big 3--Illumina, Pac-Bio, and Oxford Nanopore--and has a few odds and ends to say about the "niche developers."

We finish by asking Keith what new trends and new instruments he's looking at. He says his son is a senior in high school where Keith has offered to go in and demonstrate the MinIon nanopore sequencer.

"Imagine if the next generation of kids all learns sequencing on this little device. It starts becoming a practical reality where kids in high school--even middle school--learn sequencing and then learn data analysis."

The Flongle Generation, anyone?

Nanopore sequencing has arrived. Passing test after test this past year--including one we discuss today--this technology which was being hyped decades ago is delivering on its promise.

Winston Timp joins us today. He's an assistant professor at Johns Hopkins and one of the leaders on a recent large scale project to directly sequence RNA on an array of nanopores. Winston's is the first in a series of shows we've lined up with users of Oxford Nanopore's technology.

Why RNA-seq? Hasn’t this been done for years?

Yes, says Winston, but in the past no one has been looking at the RNA itself. They’re usually making cDNA from the RNA and sequencing that.

“One of the big advantages to nanopore sequencing, is that you can characterize any polymer you can put in the pore.”

Nanopore sequencing is polymer agnostic.

So what good does it do to look at the RNA directly? Ever heard of epitranscriptomics? Winston has worked for a while on DNA methylation in his lab. Now he’s looking at RNA methylation. Not only is he seeing basic biology that we've never seen before (unique isoforms, exon connectivity that has been imputed by informatics but never seen directly), he’s coming up with new translational questions for health and disease.

When we started Mendelspod seven years ago, the next gen sequencing race was in full swing. It was all about the push to bring down the price of sequencing. The lower cost brought an excitement to the world of biology with all the new projects it made possible. But we found out there were was a major limitation to the technology. Read length. Over the past couple years, PacBio paved a whole new world with their long reads that enabled many new genomic projects. BioNano gave us the big picture with their optical mapping. Today users of nanopore sequencing are generating reads of 1 megabase and the versatility of the nanopore is giving scientists even newer views of biological activity. The race is still very much on.

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 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?

“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.

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?

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.

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?

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.