long read sequencing

Will there be a fourth surge of COVID here in the U.S.? Already that we’re asking the question and it’s not an inevitability is a good sign. It’s become a race between vaccination clinics and viral variants.

The U.S. was a bit slow to this race, but we are catching up. Viral surveillance has become a key part of any nation’s pandemic strategy. This past month, PacBio and Labcorp announced a partnership that brings the tool of long read sequencing to this effort.

Jonas Korlach, CSO at PacBio, and Brian Caveney, President and CMO at Labcorp, join us to discuss their joint surveillance work. Their partnerhsip is not only national but is putting in place what they call a “global pan-pathogen surveillance network."

Jonas compares the network to the kind of close surveillance that global weather systems have been doing for many years. Brian says that Labcorp has an international footprint. He's optimistic that those countries that have been viral hotspots around the world will be working together in close scientific collaboration aided by new laboratory tools to prevent the next pandemic.

Since this podcast was recorded, the Biden administration has announced huge investments in innovation and research as part of their new $2 trillion Jobs Plan. We can hope that there is money marked specifically for the pandemic preparedness infrastructure Jonas and Brian are calling for today and that some version of this plan passes.

"Welcome to the era of T2T genomics,” tweeted UCSC’s Karen Miga on August 16th of this year. Then she linked to a paper on bioRxiv that begins:

"After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist.”

That would be soon fixed by Karen and her cadre of notables listed at the outset. The paper goes on to present a de novo human genome assembly that “surpasses” the GRCh38 and offers the first gapless, telomere-to-telomere assembly of a human chromosome.

Calling herself a satellite biologist, Karen has been the co-lead of the Telomere to Telomere Consortium. Her passion for quality science along with her up-to-date knowledge of the latest tools (when you google her name, the word "nanopore" often comes up beside it) make her today's torch bearer for the finest in DNA sequencing. She is the the most recent of a number of biologists and bioinformaticians to update us on the “completeness” of the human reference genome.

When will it be finished? What does finished mean? How far does this latest phase get us? And who is paying attention?

It’s the kind of plot that makes great science.

There are genes that have been hiding in plain sight, undetected until now. They’ve gone unseen, that is, by short read sequencing. Today’s guest and his colleagues call them “camouflage genes,” and a couple in particular may play functional roles in Alzheimer’s disease.

Mark Ebbert is an Assistant Professor of Neuroscience at the Mayo Clinic where he is using long read sequencing technology and computational biology to study neurodegenerative diseases, including Alzheimer’s and ALS.

“For years, the field has know that there are regions of the genome that remain dark when you are using short read sequencing data,” says Mark. "But up until recently, there had been little work characterizing how big the problem was. And as we were working on some studies on Alzheimer’s disease and ALS, we started to bump up to some genes that completely surprised me that they were dark, or what we now call camouflaged. It turns out that 26% of CR1 is camouflaged. I’ve been in Alzheimer’s research for seven years now, and in all that time I hadn’t noticed, and I’ve never heard anyone else mention that CR1 is camouflaged.”

Which leads us to ask, again, why wouldn’t all scientists doing discovery work use long read sequencing? What is the cost of missing an important gene?

If you’re not on the long read sequencing train, you’re not landing in the world of genomics.

A new paper out begins, "Structural variants contribute greater diversity at the nucleotide level between two human genomes than another form of genetic variation.”

If arrays and short read sequencing lead to the discover of many point mutations, or SNPs, which no doubt advanced genomic science a long ways, long read sequencing is now in its heyday with the contribution of structural variation detection. And according to the aforementioned paper, said structural variation is no small matter.

Mark Chaisson is an Assistant Professor in Quantitative and Computational Biology at USC. He cut his computational chops in the lab of Evan Eichler and is now etching his own name into the genomic literature. We discuss two such papers today.

Storylines repeat in genome science every decade or so. The human genome is complete. No. Now it's complete. Or, in the 90's, it was first announced that the first chromosome was sequenced. We have the same story for you today--breaking news from a paper that has not even been published yet: the first “complete” assembly of a human chromosome, end to end, telomere to telomere.

So what’s going on?

As every bioinformatician will tell you: There are levels of completeness. It is these levels of completeness that have kept folks busy at the NHGRI for many years and will for years to come. For in some of the incomplete areas, the "holes", lurk compelling secrets.

“These genome assemblies come out of very complex software, and they often contain numerous errors. And so it's key to go back into the wet lab and validate in any way that we can that our reconstruction is accurate."

That’s today’s guest, Adam Phillippy, who has been at the forefront of bioinformatics for over a decade at the NHGRI and has been an important contributor to the problems of genome assembly. He is the head of the Genome Informatics Section, which he founded.

We jumped at the chance to talk to Adam about his upcoming paper on the now complete X chromosome and the chance to hear his thoughts on the “completeness” of the human reference genome. Adam goes on to tell us that the energy at NHGRI is now shifting toward the Human Pan Genome, an attempt to represent all variations of humanity into the reference genome.

What are the challenges for such a project? And hey Adam, while we have you on, please give us your thoughts on sequencing technologies in 2019 as only a bioinformatician can.

We can all recognize that PacBio has laid down the railroad tracks in the frontier of long read sequencing. What many are asking is just how close on their caboose is Oxford Nanopore? And just what exactly will be the differences between the two technologies?

Chia-Lin Wei is the Director of Genome Technologies at the Jackson Laboratories. When we called her up for today’s interview to talk about how she is using nanopore sequencing, she said, “I’ve been using nanopore for years, why the interest this year by the media?”

Well, there are the milestones of first her own lab’s recent paper out on structural variation which shows nanopore sequencing doing what no other technology can. Then there is the Nature paper out earlier this year demonstrating the sequencing of a human genome using only nanopore technology.

But hey, wait a minute. Aren’t we supposed to be asking the questions?

She chuckles and then gives today’s interview covering her lab's paper and peeking into the future of cancer research and clinical diagnostics. Coming in at 23 minutes, the show ends with her describing the 4D Nucleome Center at JAX.

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.