Next Generation Technologist

Next Generation Sequencing, Marketing, and the Genomic Revolution

April 22, 2015
by Dale Yuzuki
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Highlights from the American Association for Cancer Research Meeting, Philadelphia 2015 #AACR15

The AACR Meeting Philadelphia PAThe annual AACR (American Association for Cancer Research) meeting was held in Philadelphia PA from April 18-22, and being able to attend several sessions is a privilege. (Yes I’m writing this the morning of the last half-day, knowing that there’s still a full morning of sessions including the last poster session.)

Over the years there have been major themes that are reflected in the major plenary talks, and one can also gauge interest in which of the parallel sessions are well-attended, and which of the posters have a crowd around them. In a meeting that has historically had from 15,000 to 17,000 attendees, this year the number was estimated to be over 18,000; with other duties for my own employer Thermo Fisher Scientific there was a lot of running around.

Not that I’m complaining – the feet are doing fine, even though it was probably a good 1/3 mile to walk from the farthest points (the Philadelphia Convention Center has one huge hall above the Reading Terminal Market, and another very large section called the Terrace Ballroom that was the main ‘hike’ to undertake). In particular, my ‘official’ company duties were interviewing people for Behind the Bench, such as this video of Dr. Kara Norman from the Acrometrix brand of control materials talking about the need to well-defined NGS controls given all the variables involved with the entire process.  But the video interviews were not only of internal Thermo Fisher Scientific employees; this video of Dr. Shintaro Iwata from Chiba Cancer Center Research Institute, Chiba Japan discusses his work (presented in one of the poster sessions) on pediatric osteosarcoma, a rare bone cancer, where he discovered a dozen novel coding SNV’s and used the Ion AmpliSeq™ Comprehensive Cancer Panel in conjunction with the Ion Torrent Proton™ System.

So after that explanation why my own intended schedule of presentations I was planning to attend didn’t quite work out the way I had anticipated (I wrote up a post detailing the talks I was planning to attend here), nonetheless for me there were a few major take-away themes that I’ll be thinking about. The first is the importance of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) for early detection and on-going monitoring.

A presentation by Victor Velculescu (Johns Hopkins Kimmel Comprehensive Cancer Center, MD) yesterday did not disappoint, as he’s an outstanding speaker; he gave a great overview of his work on cell-free DNA, and if you’d like to dig into the details he presented work from this 2012 paper and this 2014 paper on cell-free DNA and its relationship to therapeutic course and outcomes across cancer types. This 2010 paper laid out their ‘PARE-Seq’ method, where copy-number alterations were shown from cfDNA. (Oh, by the way, that paper used Applied Biosystems’ SOLiD sequencing.) And there was this 2008 paper accompanied by the statement that cfDNA picked up alterations that were not detected in tissue; something that makes sense when you think about the inherent heterogeneity of cancer along with the ongoing processes of apoptosis and cfDNA being constantly cleared from circulation.

I now realize I’m falling into the trap of recapitulating favorite talks and the notes I’ve taken. I’ll try from here to refrain from delving too deep into my notes and share these details later when I have the luxury of time, which I do not have this morning.

On the topic of CTCs, an early ‘meet the expert’ session from Mehmet Toner (Massachusetts General Hospital, MA) was a highlight of the meeting for me. An engineer by training, he laid out the three generations of the microfluidic technology behind Johnson & Johnson’s CellSearch™ technology and the latest iteration of the technology called i-Chip™. The CellSearch technology uses positive-selection for the EpCAM surface adhesion molecule, while the third-generation i-Chip uses negative-selection (taking out the platelets, RBCs and WBCs using three physical properties called Deterministic Lateral Displacement, Inertial Focusing, and Magnetophoresis). For details here’s the 2013 reference, and a 2014 one to boot. And naturally he introduced the topic of CTC isolation with the huge technical challenge involved, with 1 Billion to 10 Billion cells that are normal, and looking for the 1 or 10 individual CTCs that may be lurking within it; imagine the challenge of having all the inhabitants of Earth going through turnstiles while looking for one or ten individuals.

And of course he had to end his presentation with data – that there are cells that are CTCs but have the same size as WBCs (there are methods that separate out CTCs by size), and other data indicating there are CTCs that are EpCAM-negative. Here’s a 2008 reference laying out the methods utilized for CTC isolation.

Yet another indication of the popularity of both cell-free DNA and CTC isolation was the interest in the Thermo Fisher Scientific booth about the LiquidBiopsy™ Platform. (I interviewed the CEO and CSO of Cynvenio Biosciences here.)

Another major theme to emerge from this year’s meeting was tumor heterogeneity, and its implications for tumor resistance. As an example, Jennifer Pietenpol (Vanderbilt Univeristy TN) gave a great talk on triple-negative breast cancer and its heterogeneity, with its poor prognosis, no targeted therapy, and 35K cases/year in the US. Analyzing extensive genomic datasets of TNBC and looking at gene expression profiles, she was able to molecularly classify TNBC into six subtypes (2 basal-like, immunomodulation, mesenchymal, mesenchymal/stem-cell-like, luminal/androgen receptor, and unknown), and develop a tool for its classification (the 2012 reference for the TNBCtype tool is here).

While on the topic of heterogeneity, in one well-attended session on data analysis Gad Getz (Broad Insititute MIT MA) shared extensive datasets with unique types of systematic artifacts where he developed new software tools to correct for them. One was caused by 8-oxoguanine formation during the process of ultrasonication (a standard library prep method for shearing), and a software tool to correct for these errors. Panel of Normals filter (PoNfilter), removing Tumor in Normal effects with the deTiN tool, and MutationValidator for TCGA data, it was rewarding to hear about all the effort he’s making to solve these problems (and making the tools available here, with the latest ones described to appear in the coming weeks). And the theme of his talk (and two subsequent ones) was repeated again and again – from an informatics standpoint, “somatic mutation detection is not a solved problem”. Or, to reverse it, “somatic mutation detection is still a problem”.

The last theme is related to the prior one – drug resistance in cancer. Bert Volgelstein (Johns Hopkins Kimmel Cancer Center, MD) made a clear case that inherent tumor heterogeneity at the molecular level. I won’t forget the amazing video illustrating graphically with both size and increasing colors the growth of a tumor, going dark grey with the first round of chemotherapy and then another small cluster growing size and different colors again from the side. And because of this heterogeneity, combination therapy will need to be adopted sooner in treatment rather than later, and he had convincing data to show in the case of pancreatic cancer. He also had cell-free DNA data that tracked treatment (he and Victor have worked closely together for many years), and made the statement that early detection need not to be very early; just the need to treat tumors when they are still small.

Well it’s time for me to run off now. The video interviews finished up yesterday, so no running back-and-forth except to catch the last poster session. By my count there were fifteen videos we were able to film (with me as the host and additional ones were filmed as well), some already available on Behind the Bench. I just stand there in front of a camera asking questions to people, so it really isn’t that bad if you haven’t done it before. A special ‘thank you’ goes out to the entire team (video crew and colleagues I work closely with), as well as with my co-worker friends (you know who you are!) who had to control themselves from ‘photo-bombing’ me while on-camera. And for all those who stopped me to say ‘hi’ while I was running around I thank you – those interactions meant a lot to me, and like other large conferences, may be the most significant ‘highlights’ of all.

April 1, 2015
by Dale Yuzuki
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Complete human diplotypes & Google X Life Sciences podcast

PodcastTheral Timpson produces a weekly podcast at Mendelspod.com, and in his latest edition he interviews me about Google X Life Sciences (I wrote about them before here), long-read sequencing (in particular Pacific Biosciences) which then I’m able to discuss the value of complete human diploid sequencing, and my involvement with the Behind the Bench blog.

It is produced with a ‘Charle-Rose’ style, and he let me get ‘deep into the weeds’ in terms of technical detail. I normally don’t listen every week, especially if it is a corporate type. He usually has CEOs, nothing against CEOs but even if they are scientists their job is focused on the business and its related topics (sometimes the product, other times the market), and will also have research scientists on. It his in this area where I believe there’s an audience.

Anyway having this interview was a good experience, to summarize where we are in 2015 on a number of topics.

Here’s the link to the podcast – 22 minutes of audio to keep you entertained while to cook, do errands, rake leaves (this is where I listen to podcasts) or exercise.

March 2, 2015
by Dale Yuzuki
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A few observations from Advances in Genome Biology and Technology

150302 Marco Island 1 Crop

View from the Thermo Fisher suite

In the closing session of the Advances in Genome Biology and Technology meeting recently concluded in Marco Island, Florida, the main organizers Dr. Eric Green (National Human Genome Research Institute Director, Bethesda MD) and Dr. Elaine Mardis (Co-director with Rick Wilson of the Genome Institute of Washington University, St. Louis MO) asked an interesting question – whether they should set-up an area where blog posts could be written (and recorded) for the benefit of others. I think it is a good idea – to have a forum by which participants can share their thoughts and impressions, as over the years these get scattered as certain blogs appear (and disappear as circumstances change).

Notes from AGBT 2010

As one example, Anthony Fejes shared a lot on his old blog, and if you want a snapshot on how things have changed in five years you can access his notes from AGBT 2010 here. (His 2009 observations are summarized in a single post here, dated Feb 9.) And then in 2010 he moved over to the Nature.com blog network but it was short-lived, and is now blogging under a subdomain (here’s a link to his AGBT 2011 notes). Not being able to attend since (the most recent post was an invitation to ‘guest blog’ from the 2012 meeting but there were not any takers), he’s moved onto other topics in bioinformatics, and notably hosted a Reddit AMA a few months ago (I admit that I missed it, even though /r/bioinformatics is an interesting sub). He even received ‘Reddit Gold’, which is a way readers can reward useful contributions with additional Reddit superpowers.

Anyway, back to the topic of guest blogging and AGBT: there were a number of new faces on Twitter using the #AGBT15 hashtag, and the prior notables that blogged about AGBT in prior years were absent, such as Lex Nederbragt (his ‘In Between the Lines of Code’ is widely read and last year was his first AGBT) and Keith Robison. Keith’s ‘Omics! Omics!’ site had a number of notable #AGBT15 posts, including a nice description of the just-announced 10X Genomics platform, and this review of Thermo Fisher Scientific’s progress with the Ion Torrent platform. Another blogger that comes to mind is Dan Koboldt of MassGenomics.

One simple fact is that at AGBT the intersection of public communication via this new media platform and attendees will change from year to year. This year perhaps other voices will start to share their opinions online – but for Lex, Keith and Dan they all have limited travel funds (who doesn’t?) and like anyone else has to be selective on which conferences to attend in a given year. (Of course there were commercial media present, such as reporting from GenomeWeb which will be sure to be written up and published over the next few days, as well as a number of Wall Street analysts who will be writing up their research reports for their respective audiences.)

The challenges of getting blog posts written

And blogs quickly become dormant – I see that the excellent GenomesUnzipped hasn’t posted since March of 2014 – for a wide variety of reasons, the primary one is that everyone is busy. The opportunity cost of writing down thoughts versus doing ‘regular work’ or other special projects or fulfilling other commitments is completely understandable.

So if AGBT as an organization does setup a guest-blogging platform (which I do plan on supporting, for what that’s worth), there is a bar it must hurdle – that of taking the time and effort of sharing opinion and feedback ‘on the record’. I hope that in the coming year there will be more people who can summarize their thoughts and impressions – it seems like it was only James Hadfield and his excellent Core Genomics blog who was there in-person.

And thus the value of in-person (“IRL”) interaction at a conference – the social component of asking for opinions and perspectives is invaluable. No dependance on getting people to write a guest post – they can simply share what they think. (And at all those – ahem – after-session informal get-togethers colloquially known as ‘parties’, there were plenty of opinions shared over a wide variety of topics, for sure.)

Okay, it does leave me as a blog-post writer who attended as well – and I want to thank all those who came up to me during AGBT to introduce yourselves. If I’ve learned anything through almost three years of blogging and tweeting, is that there are many people who find out who you are through your writing that you may never meet. (And thinking about it, we all have benefited enormously from the writings of people we will never meet, in particular the many writers of the Great Books that have so shaped Western civilization.) Thus the reach of the written word – even if it is in an ephemeral media such as an online blog – is a powerful thing.

Impressions from AGBT

So onto what I thought about AGBT: I’ve written up already (for the Thermo Fisher Scientific blog Behind the Bench) a post of my favorite talk at this year’s AGBT, Carlos Bustamonte (Stanford) talking about a unique application of forensic DNA analysis combined with his expertise on human population genomics – tracing the trans-atlantic slave trade from 1721 through several centuries (his data goes through 1910). His presentation was entitled “PhenoCap: A Targeted Capture Panel for Comprehensive Phenotyping of Forensic DNA Samples”. So keep an eye out for it, it should go live in a few days. (There already are some 5 video interviews from AGBT 2015 up on the website, with many more to come.)

For sure I would be remiss to not mention 10X Genomics; they launched their product at this conference, and made quite an impression. A sample-prep device that takes 1ng of >100kb-length genomic DNA, partitions it into 100’s of thousands of pL-volume emulsified droplets (although to be frank the word ’emulsion’ has never been used by them in any of their presentations nor in my conversations with them), and uses one unique barcodes per microreaction (picoreaction?) to individually label that >100kb strand, so that afterward the 100’s of thousands of uniquely-labeled sequences can be deconvoluted and aligned together. I won’t re-iterate the details – Keith Robison wrote them all up here.

Shortly after the afternoon presentation, which I described to a friend as ‘pitch perfect’, I was minding my own business at dinner when John Stuelpnagel (a founder of Illumina as well as of 10X) joined us. (It brought back fond memories of my own cubicle at their old building on Towne Centre Drive, where I was only around the corner from John’s cube. I found out quickly that he was an early-riser like myself, and found his input always insightful and so valuable. Good times.) After talking about Poisson distributions and clarifying species specificity and other minutiae, he told me that they had mathematically modeled the numbers their engineers needed to design the system to (you can imagine how expensive it is to synthesize and manufacture many hundreds of thousands of oligos, but that is how Illumina started way back in 2000-2001), and that once they built their system the experimental results matched perfectly with their calculations! This can be called ‘optimum company management through mathematics’.

From the applications perspective, friends I spoke to (several core facility directors, who I just might see at the upcoming Association for Biomolecular Resource Facilities meeting in Saint Louis MO March 28-31) were eager to give this platform a try. They all agreed that for cancer applications Levi Garraway’s plenary talk was noteworthy (yes they had a plenary speaker in the morning session have several data slides featuring 10X Genomics results).

One other notable item regarding long haplotyping (for background on diploid phasing, I wrote a piece called ‘The Unexplored Diploid Landscape’ here), was that several presenters said they had optical mapping data (in reference to BioNano Genomics), but no data from that platform. It seemed as if optical mapping was only used in a validation function, to confirm findings from either Pacific Biosciences’ long reads (average on the order of 10kb, up to 47kb long) or short-read method (i.e. Moleculo).

Another impression from AGBT

Craig Venter presenting at the Pacific Biosciences workshop; Deanna Church (Personalis) in the foreground

Craig Venter presenting at the Pacific Biosciences workshop; Deanna Church (Personalis) in the foreground

On the note of Pacific Biosciences, it seemed like they were everywhere. (By ‘everywhere’, I meant in the sessions.) Certainly The Gene Myers spoke about his next step – development of a local aligner, with other tools making much-improved human genome reference assemblies with far lower compute requirements. (It was not unusual to hear 60x improvements in speed, just by looking at these problems in a different way – thus my own admiration for people who have the freedom and ability to choose the problems they want to work on.) If you want to see what a splash The Gene Myers made at AGBT last year, see here.

By getting fully-phased human genomes first with CHM1, Mike Hunkapillar (CEO of Pacific Biosciences) showed metrics for the CHM1 hydatidiform mole (a haploid human genome), with N50’s far longer than any human genome reference assembly to-date (for HuRef-1, it was an N=50 of 10.5Mb). In addition there are some 18 projects at four collaborators worldwide working on other CHM samples (i.e. derived from different individuals). Important work here – an overarching theme of phased haplotypes, and what the scientific community can learn about the role of genetic variation in an allele-specific manner.

A final impression from AGBT

The splash from prior years about single-molecule sequencing from newer technologies (i.e. Oxford Nanopore, Quantum Biosystems, ZS Genetics, and others) seems to have died down. There were a few presentations that included Oxford Nanopore data but they had no error rate metrics, talked about how it felt like an ‘alpha or beta’ version, using His-tag metal chelating purification resin (hello QIAGEN!) to enrich for hairpin-containing library molecules, so the MinION platform is still something of a work-in-progress. I myself am amazed that they can get 512 single-molecule pores as a manufacture-able item and read electrical signals as nucleotides pass through. (For additional context and background, here’s a post from AGBT14 about ONT.)

A parting thought

One observation (from another friend, yes he was another core facility director) noted that there was no data or other findings from the X-10 whole-genome sequencing platform. Naturally J. Craig Venter said he was planning to purchase many more of them for his Human Longevity Institute to fulfill their goal of 1M whole human genomes and that he wouldn’t share any results that he will be commercializing. And other than a poster (or two?) about technical aspects of the X-10 platform, it was something of an omission that did not go unnoticed.

February 22, 2015
by Dale Yuzuki
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Marketing Precision Medicine Pt2

Borrowed from this infographic: http://www.nih.gov/precisionmedicine/infographic-printable.pdf

Borrowed from this infographic: http://www.nih.gov/precisionmedicine/infographic-printable.pdf

After writing up ‘Marketing Precision Medicine’ it turned out that I spent so much time in giving the background and context, that I only was able to mention briefly the marketing challenge that the Precision Medicine Initiative brings.

So here is Part 2, getting deeper into the Marketing challenge of precision medicine – a concern about privacy and data security.

Again via LinkedIn, and of course will need to come up with a Part 3 next week – what government can do to help.

Marketing Precision Medicine Pt2.

February 16, 2015
by Dale Yuzuki
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Marketing Precision Medicine

Borrowed from this infographic: http://www.nih.gov/precisionmedicine/infographic-printable.pdf

Borrowed from this infographic: http://www.nih.gov/precisionmedicine/infographic-printable.pdf

Last week I listened in with interest on the Precision Medicine Workshop held at the NIH, and wrote up some thoughts about the need for them to market it effectively. You can access this post on LinkedIn – something of an experiment in posting in various places.

We are living through a remarkable period in the history of healthcare, implementing precision medicine on a population-scale of a million individuals.

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