sequencing

When 10X Genomics launched their GemCode sequencing instrument at last year’s AGBT conference, what they offered seemed too good to be true. 10X was promising researchers a machine that could generate long reads using Illumina’s short read technology at a price lower than what PacBio could offer with their “real” long read instruments. A year earlier, Illumina had announced they were buying Moleculo, a company that promised to offer long read data out of the short reads. But good data with the Moleculo platform failed to materialize.

10X Genomics hasn’t had that problem of Moleculo, and was in fact declared the “winner” at AGBT this year when they presented de novo human data.

Today, for the first time, the CEO of 10X, Serge Saxonov, joins us to talk about their technology and the company’s stellar rise.

The question everyone wants answered from Serge is how well the 10X linked reads stand up to so called “real” long reads. PacBio has spent years co-discovering with their customers applications where their long reads provide significant advantage over short reads, at a price. And even though PacBio released a cheaper-faster-better machine, the Sequel, late last year, some researchers have been wondering whether 10X might come through and "clean house" with their inexpensive system?

“Now you can get the information that people were hoping to access in maybe five or ten years--you can get it now. And in fact you don’t need to make a tremendous new investment and change your workflow radically,” says Serge.

While 10X is enabling Illumina customers to generate long reads, are there still limitations of the short read machines that can’t be overcome?

Serge and 10X have already launched a second system, the Chromium, which offers single cell analysis. How big is the single cell market, and what are Serge’s thoughts on the future of sequencing?

Jonas Korlach is a natural storyteller—a rare trait in a scientist who is more comfortable presenting data than talking of himself. Jonas is the co-inventor of PacBio’s SMRT (single molecule, real time) sequencing, and we wanted to hear from him directly how it all got started, and also when the team realized that they had something big with long reads and close to 100X coverage. How many of us can boast of hitting it out of the park on our first try?

If you attended or followed the recent AGBT conference about all things sequencing, you probably saw a few BioNano Genomics t-shirts with the slogan, “Back to the Map.” They’re referring of course, to a genome map. Just like Google Maps, a genome map consists of landmarks that tell scientists where on the genome they are. But unlike Google Maps and more like the maps North America that were made by European explorers in the 17th century, the map of the human genome is quite incomplete, the map of a frontier.

Erik Holmlin is the CEO of BioNano Genomics which offers unique genome mapping technology. In today’s interview, Erik points out that content is not the only king, context is pretty important as well.

“You can go back and look at some of the early discussions that were happening around the beginning of the Human Genome Project. And in fact a lot of the leading scientists of the time, Maynard Olson, Bob Moyzis, and others, emphasized that as we’re doing this sequencing it’s going to be very important that we put the sequence in context of the physical organization of the genome. Otherwise we’re never going to understand it,” Holmlin says.

After the market has become dominated by “short read” sequencing with the race to the $1,000 genome—a drive many say has been steered by the NHGRI—BioNano is now cutting out some territory for their genome mapping technology. Their flagship projects have no doubt been their work on the reference genomes. Erik says that in a recent trio sequencing project of genomes of Ashkenazi Jewish descent, they were able to find “a lot of de novo variants,” or variants which had not been found with other sequencing technologies.

Though Erik has always had his eye on the clinic—in fact, he came to the tools space from the clinical diagnostics industry because he felt passionately that we needed better tools to develop clinically actionable genomic data—he admits at the end of today’s show that his time at BioNano has pulled him more into basic research.

“In some respects I underestimated the need for more basic research,” he says. “And what really needs to happen is we need to get the translational research efforts to focus on the structural picture much more because that’s going to break through and lead to many clinically significant discoveries.”

We’re all familiar with the announcement in the year 2000 by US President, Bill Clinton, and the UK’s Prime Minister, Tony Blair, that scientists had completed the first draft of the human genome. It was a big deal. But the actual publications didn’t happen until the next year, February of 2001. Which means that this February is the fifteenth anniversary of the publication of the first human genome. For our commemorative show we’re joined by Mike Hunkapiller, the CEO of Pacific Biosciences.

Mike and his team at PacBio are coming off a great year. Their stock is up. Their long read sequencing technology is used for over a thousand scientific publications. And last year they launched a new better, faster, cheaper instrument, the Sequel, which are sold out through the first half of this year. PacBio is cool again.

How much were tool makers in the driving seat of the genomic revolution? And how much further can sequencing improve? Before asking Mike this, we explore some of his memories of those wild days when sequencing the human genome got presidents and prime ministers on the phone with their speech writers.

OK, so we get it. Long read sequencing technology is cool. But how cool? Is it another great player on the field, or does it change the game altogether? 

The Mike Schatz lab at Cold Spring Harbor is well know for de novo genome assemblies and their work on structural variation in cancer genomes, so we were curious to hear how long reads have impacted their work. In todays show, lab leader, Mike Schatz, and doctorate student, Maria Nattestad tell of two new projects that include the de novo assembly of a very difficult but important flatworm genome and, secondly, making better variant calls for oncogenes such as HER2.

In the case of the flatworm, Mike says that the move to using PacBio’s long reads improved the assembly by more than a 100 times. That means the difference of looking at a super high resolution picture versus a fuzzy, blurry one, he says. With her work on cancer cell lines, Maria is seeing variants that just weren’t there with short reads. Will her work translate to lower false positive rates for HER2 in clinical diagnostics?

What will be the major headline for sequencing and informatics in 2016?

Mike says we’ll see many more reference genomes done, that the term “reference genome” itself is changing as we go from the one standard reference genome to multiple reference genomes representing the broader population. These new reference genomes are pushing bioinformaticians to come up with new ways to visualize and compare the genomes. Maria details her work into using “graph” assemblies as opposed to the linear approach made popular by the Genome Browser. She says that already a new generation of informaticians are rethinking genome visualization using graph assemblies. (Included below is an image representing her work.)

Neither mentioned it, so we ask at the end, what about Oxford Nanopore’s tech?

(The spectral karyotype of the Her2-amplified breast cancer cell line SK-BR-3. The original chromosomes are different colors, so this genome is a complex mixture of various chromosomes. The total number of chromosomes has also jumped from 46 to 80, and there is approximately twice as much DNA as in a normal human genome. Maria Nattestad and Mike Schatz are studying this genome to see how oncogenes like Her2 became amplified while all these changes took place in the big picture of the genome.)

Today’s story is one of a personal quest, of groundbreaking science, and the creation of a new movement in human genomics.

Jim Lupski is a professor at Baylor College of Medicine where he’s on the frontline of incorporating genomic research into everyday clinical practice. The story begins with Jim’s own genome, which is perhaps the most sequenced genome ever. Jim's life as a leading genomic researcher has been driven in part for a strong personal reason. He has a rare genetic disease named after three researchers who first defined it, Charcot Marie Tooth Neuropathy.

What began as a personal journey to uncover the source of his own disease led Jim to seminal work that launched the field of structural variation. Working first in the gene-centric mindset of the 90’s, Jim's team discovered the first gene known to be associated with CMT disease, PMP22. But while this gene is related to 70% of the cases, it wasn't the mutation responsible for Jim's own version of CMT. His discovery of that would be some years later, and from a much better picture of his genome.

Find out in today’s interview where Jim thinks we are now in genomic science, and why he says the goal in the clinic should be a de novo assembly.

Cliff Reid, CEO of Complete Genomics, is back on the conference circuit, touting a new product. After years of building his company to do sequencing as a service, Cliff presented data at last week's ASHG meeting on Complete's first sequencer as a product, or what they are calling the Revolocity supersequencer.

Cliff was a pioneer in developing the service model, offering only whole human genome sequencing. But after being bought out by BGI, who already had a service business in China, he was compelled to shift his business model to that of selling sequencers.

So where does this position Complete in an already crowded and mature sequencing tools market? And how does Cliff see the future of clinical sequencing? Cliff says that the new Revolocity offers the highest quality of any of the other sequencers. This is an impressive claim in a world where PacBio customers are saying they can get up to 100% accuracy with deep enough sequencing. As for throughput, the new supersequencer is similar to Illumina’s HiSeq X Ten system, producing about 10,000 whole human genomes per year.

However, Cliff says that high quality and high throughput are not the important part of the story here. “That's historically what people expected from us,” he says in today’s interview. "The most important thing we did with the Revolocity system is that we packaged it for clinical researchers and clinical use. The packaging is end-to-end.”

Strangely, in an age when longer sequencing reads have enabled genomic research to go to further heights, such as with HLA typing, the new Revolocity does not incorporate Complete’s LFR or Long Fragment Read technology. Long read sequencing is still a highly specialized effort, Cliff argues. Rather, he says, “we haven’t used the technology that we have today [short read technology]. What we need to do is sequence a million genomes."

One of the popular questions on the program this past year is how those doing sequencing decide between the quality of Pacific Bioscience's long reads and the cheaper short read technology, such as that of Illumina or Thermo Fisher. Today’s guest provides the most clear and dramatic answer yet: use the PacBio system exclusively.

We heard this from Steve Marsh, the Director of Bioinformatics at the Anthony Nolan Research Institute in London. Established in 1974 by the mother of a boy with a rare blood disease, the Anthony Nolan Institute is a world leader in blood crossmatching and donor/patient registries. Steve and his team at the Institute have dramatically improved the resolution of HLA typing, one of the methods for matching a donor’s blood tissue with that of the transplant recipient.

Thirty years ago when Steve entered the field, HLA typing was performed with serology and there were just 119 HLA antigens that were known. “We thought 119 was a lot of diversity,” says Steve. With the advent of genomic tools in the 90’s, HLA typing moved to the level of the genetic allele, done first with PCR and then with sequencing. “We knew that the HLA molecules were polymorphic, but now we know they are hyper-polymorphic. . . For example, 'A2' is a specificity, and serologically we recognized the specificity 'A2,' and that was it. We now recognize that there are over 500 different variants of A2,” Steve explains.

Knowing more about the incredible diversity of blood types can make achieving a donor/patient match seem all the more prohibitive. More variables mean fewer candidates. But research at the Anthony Nolan is now paying off and is robust enough to make a difference in the clinic. Ideally blood registries will provide precise matches and do so immediately.

All this explains why Steve is so keen on the PacBio system. In a field where the quality of the sequencing makes the difference between the right match and not, the increased price of the longer reads is worth it.

Finally, it should be said that we recorded this interview with Steve just before PacBio announced their new higher throughput Sequel Sequencer. This new smaller footprint instrument is promised out in 2016 at half the price, seven times the throughput and with the same high quality long reads. The decision between quality and price in the world of sequencing will soon be easier.