Sequencer Archaeology

One of the frustrating side effects of outsourcing all your sequencing is that lack of connection to the actual machines.   Rarely have I gotten to visit sequencing labs, and the only time I've seen one with the cover open was when a Polonator was being shown off at a conference.  So it was a lot of fun to watch an almost hour-long YouTube video that Lex Nederbragt had pointed out in which someone who likes to disassemble complicated electronics performs his craft on a 454 FLX.

Roche Abruptly Breaks Off PacBio Partnership

This morning was a solid block of meetings, but in a pause I checked my phone and saw the shocking headline: Roche Diagnostics had suddenly terminated their partnership with Pacific Biosciences to commercialize the Sequel instrument for clinical applications. Based on the few things I've read and a conversation with Bio-IT World's Allison Profitt, I've formed a few ideas, but certainly still find this a bit mystifying.  Perhaps the first part of next year, with the JP Morgan Conference and AGBT, will see Roche revealing a bit more about why they decided to break up with their partner.  Particularly when Roche had already made all their milestone payments; going forward

Let's Stop Talking Consensus Accuracy

A common approach for comparing sequencing platforms and assemblies is to report the consensus accuracy, just as for the platforms themselves the raw read accuracy is often reported.  I'm going to go on record with my opinion that stating these as "99.9% accurate" is a terrible habit which must be kicked, as it interferes with proper comparisons.

Siddhartha Mukherjee's The Gene, An Intimate History & the Crafting of Scientific Stories

Back in 2011,  I read and reviewed Dr. Siddhartha Mukherjee's book on the history of cancer therapy, The Emperor of All Maladies.  I liked the book, but as is my character I also listed some criticisms.  It was a very pleasant surprise to one day discover an email from Dr. Mukherjee engaging me on my points.  A real author, writing me!  Fast forward to this fall, and I had some inexplicable inertia to reading his new book, The Gene, An Intimate History.  This time he drove the process forward, asking if I'd like to read and review the book and if so could his publisher send me a copy?  Wow!  Having just finished the book, here goes the full review.

Reversible Terminators: Not Just For Sequencing

Reversible terminator nucleotides lie at the heart of sequencing-by-synthesis systems such as Illumina.  These nucleotides in their original state cannot be extended, terminating DNA polymerization.  But with the correct chemical treatment, the block is removed and polymerization can continue.  A recent paper moves the concept from sequencing to making large single mutation libraries.  The authors have apparently also applied for a patent (according to the Conflicts of Interest statement accompanying the paper), though that does not turn up on Google.

Oxford Nanopore New York City Meeting, Day 2

The second and final day of Oxford Nanopore's New York User Meeting ran today.  I've again been mining tweets, since I wasn't on site.  Oxford itself has posted a summary of Day 1, which has the enormous benefit of the author being present! I'll make a few quick summaries.  The tweets for today can be found in two semi-thematic Storify entries: one gives general coverage and of ONT's demos, whereas the other covers ONT's technical talks and talks by users.

Oxford Nanopore New York City Meeting, Day 1

Oxford Nanopore officially kicked off its Community Meeting in New York City today; a training session took place yesterday.  Already there have been several interesting announcements and presentations, including a new prototype sample prep gadget, a new basecaller which improves homopolymer calling, a read-both-strands approach that isn't 2D sequencing and details on multiple human genomes run on MinION. A reminder: I'm working only from tweets; I'm not at the meeting.

Is PromethION a Strategic Error?

Be prepared for another burst of Oxford Nanopore coverage; their annual Community Meeting in New York City begins on Thursday (perhaps while you are reading this).  I won't be attending; only so many absences from the day job are ultimately feasible each year, and I've long since hit the limit.  Hopefully some prolific tweeters will keep everyone updated, and I can summarize from there.

As with most posts on Oxford Nanopore, my piece on the closure of the Illumina litigation captured some comments; I think it is reasonable to expect more on the piece on the opening of litigation by Pacific Biosciences.  How you perceive the ambiguity around whether they were using an MspA pore in the R6 and R7 chemistries or not tends close to a Rorschach test.  But with that behind us, attention can focus on their current state and progress. Now, I'm going to project some critical ideas around one of their platform pieces, but I have no delusions that they will affect Oxford's course.  It wouldn't shock me if Clive had a tart seven letter response, the only question being if the last three letters form a pronoun or a preposition. I don't have any inside information nor do I have any direct financial interest in the company, in case you are wondering.  As my title suggests, what I'm going to argue is the case that pursuing the launch of the PromethION instrument is an unnecessarily risky detour.

Revisiting Mendel

In yesterday's post, I flagged a small factual error in Siddhartha Mukerherjee's The Gene.  I really liked Mukherjee's prior history of cancer, The Emperor of All Maladies, and was thrilled when Dr. Mukherjee wrote a thoughtful response to the criticisms I did make.  So it was another happy moment recently when he asked me if I'd like a copy of the book so I could review it.  I'm only about a third through the book, but it is definitely worth reading (why did I wait so long? no good reason).  Since I like it, when I get to a full review I'll probably be mostly in "this is what I would have suggested if I were an editor" mode.  I'm not ready for that yet (finishing the book is a pre-requisite!), though cryptic notes are piling up in my Evernote on the topic.  However, there is a specific part of history covered in The Gene which warrants separate treatment, using the book more as a springboard than as the central subject.  That concerns the amazing man widely regarded as the founder of the science of genetics, Gregor Mendel.

Nostalgic for Fly's Eyes

Kumar Thangdu's mission of prodding me has contributed to a bout of nostalgia for one of my graduate student rotation projects.  He asked in a tweet how I'd allocate funds if I was given stewardship of a billions in grant money.  If you want to get some big results but are willing to be very patient, then a great way way to invest is in model organisms.  As a rotation student at Harvard, I spent several months pushing Drosophila melanogaster, the not-so-humble fruit fly.

RNA-Sensing USB Stick: Promise Despite the Hype

Earlier this month the newsfeeds were abuzz over a new USB-stick style nucleic acid testing device. Based on technology from the United Kingdom firm DNA Electronics, this device is intended to make testing for infectious diseases portable and inexpensive.  Since the pulse of news was triggered by a publication in a journal, I dove in to see where things really stand.  The device is interesting, but alas the paper describes a prototype far from ready to deploy.  Also interesting to ponder is how this device might stack up against other devices emerging for the portable diagnostics marker, such as Oxford Nanopore's MinION.

News from Old Neighborhoods

Five years ago, the initial band of scientists at my current employer had just moved from the offices of our venture capitalist sponsor, Third Rock Ventures, into lab space in Cambridge sublet from Blueprint Medicines.  The Athenaeum Building is a large brick-faced building which once held the publishing house by that name.   Eleven Biotherapeutics was trying to engineer new immune-modulating proteins, initially focusing on a treatment to dry eye.  Some of their IP I understand was pulled from the wreckage of Codon Devices.  Also on the floor was Verastem, a company spun out of work from Robert Weinberg and Eric Lander on cancer stem cells. In the last few months, major news has been announced by Third Rock, Blueprint, Eleven and Verastem which illustrates many of the forces that make life in early discovery interesting (as in "may you live in interesting times").

Will Liquid Handling Robots Ever Join the 21st Century?

In the course of this blog, there are many topics I've thought about writing that I haven't touched.  Sometimes it is due to the problem of the topic being too revealing to what I am working on, sometimes it is because I'm not satisfied with the result, but far too often I procrastinate so long that it no longer seems fresh.  Or I'll just do it another time when the moment is right, which it never is.  But a recent Twitter exchange reminded me of a long-suppressed lament on some expensive, finicky and problematic -- but very useful -- denizens of a modern lab: liquid handling robots.

HGP Counterfactuals, Part 7: Wrapping Up

It's been interesting revisiting a bunch of now ancient history of the Human Genome Project with the goal of exploring other possibilities.  I started by considering the entire concept of alternative histories, then reviewed the construction of physical maps, strategies which were considered for sequencing the clones comprising the minimum spanning map of the genome and the actual sequencing technologies employed, then considered scenarios in which no HGP is launched or the project is given a much smaller budget and forced to focus on technology development.  Tonight, I'll close this out by trying to summarize some of the ideas that came out through this process, as well as some further thoughts on the whole exercise.  Plus some references to the two megaprojects to which the HGP is often compared, the Manhattan Project and Project Apollo.

HGP Counterfactuals, Part 6: Ax Sharpening Only

At the beginning of this series, I promised two alternative histories on the Human Genome Project.  Yesterday I explored a timeline in which opponents of the HGP successfully kept it from ever being funded.  Today, I'll try to imagine what would have happened if the project had been funded only to develop new sequencing technology.  One warning: as part of this I will show the most reviled plot in genomics, but to make something other than the usual point.

HGP Counterfactuals, Part 5: HGP Stifled

Okay, enough procrastination. I've outlined the general idea of counterfactuals and then obsessively detailed the generation of physical maps, planning the sequencing of BAC clones and the rapid winnowing of sequencing technologies during the early stages of the genome project.  Time for a main act: what if the public Human Genome Project never happened?

HGP Counterfactuals, Part 4: Sequencing Tech Landscape Circa 1992

In this series leading to a pair of Human Genome Project alternative histories, I've been warming up with a trio of analyses of the technology landscape.  At first I couldn't decide on the order to post these in, but then it was clear to me: a progression of scale from physical maps of the genome to how to organize the sequencing of the BACs, and now to the actual sequencing technologies which were in play.  In particular, how there was a very rapid evolution from 1992 (when I first was deeply exposed to this angle by attending Hilton Head) to 1997 (when I defended, but also the outcome was clear).  In this time period, a large number of possible options essentially compressed to a single one: automated fluorescent dideoxy sequencing.  That outcome was not clear in 1992

HGP Counterfactuals, Part 3: BAC Sequencing Strategies

I introduced this seven-part series with an exploration of the values and challenges of counterfactual histories.  Yesterday, I looked at the "forgotten maps" which laid out the genome ready-to-sequence as a minimum tiling set of BACs, made sure that those BACs faithfully represented the genome and that this set was tied at regular intervals to the genetic markers and cytogenetic locations which were the linga franca of human geneticists.  Today, I'll look at strategies that were considered for sequencing all those BACs.

HGP Counterfactuals, Part 2: The Forgotten Maps

Yesterday's post explored the concept of alternative histories, or counterfactuals and laid out why they might be a useful way to think about the value of the Human Genome Project.  In this installment, I'll explore what I will call the forgotten maps, the critical elements of the HGP which are all too easily forgotten.  These were both critical and expensive components of the project, so forgetting them is a mistake.  Their prominence has faded as new technologies have come in and subsumed them, or they were mostly means to the end of a first human sequence, but understanding the project requires understanding these forgotten maps.  And I will admittedly cover only a few; I'd invite anyone familiar with the ones I don't illuminate to remedy my failings (a careful reading of the Nature paper on the physical maps wouldn't be bad either).

HGP Counterfactuals, Part 1: An Introduction

My new correspondent, Kumar Thangdu, has posed some challenging questions with regard to the Human Genome Project.  He's been good enough to capture my initial tweet stream over at his blog, which was my initial defense.  I then followed up with my note on my one paper in proteomics, which I received favorable feedback on  from one of my co-authors.

Oncology: The 10Km View

My Twitter correspondent Kumar Thangdu keeps throwing interesting but difficult questions my way, far faster than I can keep up.  Not sure I'm even particularly skilled at many of these.  But, one must try.

@OmicsOmicsBlog The best thing we can do to cure cancer, might be to try not to cure cancer, but just fund basic research? accurate?

— Kumar Thangudu (@datarade) November 8, 2016

Homopolymers and Other Recurring Topics in Pore Taste

Some interesting comments showed up on my piece covering Pacific Biosciences trade action launch against Oxford Nanopore.  Alas, some silly comments showed up as well.  Life on the Internet.  In particular, Mohan Chennupati asked a series of questions that can be seen as more friendly to PacBio and less so to Oxford Nanopore than my analysis.  They're all good questions and worth digging into, and you'll find some other commenters addressing them.  I'll quote from his comments but rearrange the order a bit.  I believe I've not changed their meaning or damaged his argument, but please check me.  The main reason I've rearranged is that one of the comments is the one I find most interesting, so I'm saving the long answer on that for the end.

PacBio's Quixotic Patent Litigation

I'm feeling very glad I closed out the Illumina/U. Washington litigation vs. Oxford Nanopore the other night, albeit very belatedly, as now Oxford is facing a similar set of legal actions, but this time initiated by Pacific Biosciences.  PacBio has filed a complaint with the U.S. International Trade Commission (ITC) alleging that Oxford is infringing on an issued U.S. patent, 9,404,146 (aka 146 Patent).  Surely PacBio's management thought this was a good idea, but from this perspective

Tidying Loose Nanopore Patent Threads

A commenter on my recent piece on Genia made a number of assertions with regard to nanopore patents.  A more general one was a complaint that I had covered the opening salvo of the battle from Illumina and University of Washington  and Oxford's formal response and feinting to the CsgG/R9 pore, but failed to cover the campaign's termination.  It's a fair charge and I'll rectify this here.  My (rather lame) excuse is the news broke while I was on vacation in the Pacific Northwest and trying to avoid any remotely professional activities, other than reading Luke Timmerman's Lee Hood biography.  But, that doesn't excuse not writing something on my return.  If you're going to enjoy flame-broiled steak, one must clear the vegetables from one's plate.

10 Years of Omicing!

Today marks a special day - it was 10 years ago that I launched this endeavor.  I'm going to take time today, and perhaps a few times in the near future, to look back on the journey.  Ten years of blogging also happens to be around my anniversary of starting work at Millennium, which means I've been a professional in biotechnology for two decades.  So I must be old; indeed, this summer I celebrated birthday 30 (numeric pedants might make the base charge that I am misleading by not writing that 0x30) and about a quarter century since I launched into bioinformatics full time.  So a bit of nostalgia seems not out of place.

Segmental Duplications and Deletions in Books of History and Life

I have long had a historical interest in the U.S. Civil War.  In fifth grade we had an all-day field trip to the Gettysburg Battlefield, which is very well preserved, and it enthralled me.  A few years later I would hike all over the battlefield with my Boy Scout troop, which is probably even closer to experiencing a taste of what it was like to be soldier. Of course, we had good hiking boots - a proximate cause of the battle occurring there was an attempt by the Confederates to raid a shipment of shoes which had just arrived in the town.  My great-grandfather served in the Union Army, though entirely on garrison duty.  However, his two older brothers saw action and one lost an arm at Chickamauga.  I just failed for arguably the fourth time to get to that site, but I've toured a few other key fields (Antietam and Petersburg).

How Genomes Enabled Proteomics

My recent piece on the folly of moonshot projects caught the eye of a Silicon Valley blogger named Kumar Thangudu (@datarade), who gave me some nice praise. But along with that were some notes of his on other big science projects, and one he targeted was the Human Genome Project, with a sarcastic comment about the paucity of drugs coming from the HGP.  Them's fighting words!  So I launched a tweet stream his way outlining a number of impacts of HGP on biomedical science, and he was very nice and suggested I write more on that. I'm thinking of a number of angles on the topic, but here goes a bit of nostalgia that I think illustrates a number of points on the topic.  Except I'm first going to talk about Escherichia coli, and worse, talk about it before its genome was complete.  But that's part of the point.

Messaging Counts

My recent piece on SeqLL's reboot of the Helicos sequencing technology attracted a number of helpful comments either on blogger or via email.  Several of which pointed out the high suitability of this box for counting applications such as expression profiling, ChIP-seq and copy number analysis. Julia Karow of GenomeWeb graciously provided me with a copy of her piece, which underscored the value of GW in having real reporters dig into a subject.  Much of what she dug up concerns SeqLL's future plans and for the near term mostly confirms suspicions that the technology will mostly look like the previous Helicos box, just scaled down.  But Julia also got out of SeqLL some of the numbers that are shockingly absent from the press release, such as the throughput (though I don't see the number of channels, a key aspect of this technology as covered below). My correspondents also pointed out a number of careless errors in the piece: I sliced $100K off the price of the PacBio Sequel (doesn't mean you can't try to get them to honor it!) and the In Sequence brand is long-gone from GenomeWeb's site.

SeqLL: Helicos van Winkle

Helicos was the first company to launch a single molecule DNA sequencing system. They never sold many systems (my estimate is fewer than 20), but some of those sites really loved their machines. One beauty of the system was a very simple sample prep: fragment your DNA, add terminal transferase and dATP and the sample was ready-to-hybridize.  Helicos demonstrated a number of interesting applications, such as direct loading of RNA onto the system and performing capture on the flowcell.  But with anemic demand and mounting losses, the company faded, finally filing for bankruptcy in November 2012.  One true believer bought up key IP and hardware and kept the torch burning as SeqLL, headquartered just down the road from me in Woburn, Massachusetts (best known for the book and movie A Civil Action, but also the birthplace of thermodynamicist Count Rumford ).  Now SeqLL is re-launching the technology in a new box, or is it similar technology? That's the big question, poorly addressed by their beta test announcement or their website, and that website seems to be positioning the new SeqLL box against the state of competing sequencing technologies of back when Helicos folded.

IGenomX Launches New Linked Read Chemistry

A hot concept in the genome world is how to capture long-range information, enabling assembly through repeats, resolving haplotypes, resolving copy number variation and many other applications in which short reads from short fragments are just not sufficient. A number of approaches have been proposed and published over the years, with some hitting commercial markets and significant use.

Genia Publishes Polymerase-to-pore docking scheme

Back in April, I wrote up a PNAS publication from Genia and collaborators that described the modified nucleotides they have developed for their sequencing-by-synthesis with nanopore detection sequencing scheme.  I had the opportunity to chat with George Church (who is an adviser both to my company and Genia, and a co-author on the paper) just after that and he remarked that there was a companion paper in the works describing the polymerase engineering for the system.  That paper is now out in PNAS (and Open Access) and I'll take a little walk through it.

Sequencing, not random probes, are future of microbiological diagnostics

Okay, I'll admit I take requests.  Throw a topic at me by Twitter or email, and if it piques my interest and I feel like I can say something intelligent, then I'll take it on -- but not necessarily instantly.   That's the genesis of today's item, a tweet from Kyle Serikawa directed at me, asking if a new paper  from groups at Rice and Baylor College of Medicine in Science Advances on a proposed microbial diagnostics (a paper highlighted by Eric Topol) had any legs.

Huh. Well, intellectually clever, but isn't #Nanopore already leading the way in direct ID? @OmicsOmicsBlog, have any thoughts? #pathogens https://t.co/Ac2JFrbGNT

— Kyle Serikawa (@kyleserikawa) September 30, 2016

A novel uniform microbial diagnostic platform @ScienceAdvances https://t.co/EPHZrh3Ffx pic.twitter.com/dPEHMci8Jf

— Eric Topol (@EricTopol) September 30, 2016

I'll fully confess that I realized upfront that this paper cuts across my usual biases, so I resolved to try to read it with that in mind.  That's not really an excuse in the delay; I did read the paper right away, but the usual conflicts stretched out writing it up.  The paper, in brief, proposes a non-sequencing approach to an area which I had previously thought was going to be totally dominated by sequencing, and I inherently like sequencing.  After reading the paper, I would agree with Kyle: I'm not convinced that this method has legs.  It doesn't help that the paper has some serious issues with describing the methodology, as well as utterly failing to make its computational methods available.

DNA: How Clean is Clean Enough?

Nick Loman has a new blog post nicely covering the current state of affairs for DNA preparation in the field.  Earlier this year I had some thoughts about DNA preparation and the degree to which our current methods reflect history rather than some ideal.  Even before Nick's post I had some new thoughts on the topic, but seeing his exposition helped me consolidate my own musings.

Bill Gates Succumbs to Moonshot Madness

Another day, another misguided call for a moonshot in human disease.  This time, it's Bill Gates laying out four goals that he believes can be attained in the next decade, given the correct amount of dedication and determination.  Among these goals are a vaccine for HIV and a cure for neurodegenerative diseases.  I'll focus my comments on the neurodegenerative diseases with a few comments about fellow Blue Hen Joe Biden's cancer moonshot, but many of the criticisms apply to the HIV or just about any serious disease.

ONT's Wafer Thin Update

Oxford Nanopore's Clive Brown gave an hour-long update on their platform last week.  A busy social schedule featuring (on different nights) GMO beer and challah - plus six hours travel each way to the challah-fest. Add atop that a fast-moving upper respiratory tract infection, and I'm even more behind in blogging than usual for such events (plus I gathered another post idea away -- and had another suggested to me). Time to get working on the backlog!

Directed Panspermia Doesn't Belong In Schools (and Probably Never Will)

Opening to the Ideas section of the Boston Globe this past Sunday, I was immediately faced with a grotesque site.  Nothing to do with the appalling terrorism of fifteen years ago, but instead the Globe putting front-and-center in the section a truly awful idea, which the subhead trumpeted "Creationism can have a basis in science - if aliens are involved".

Portrait Of A Genomics Instrumentation Impresario

Veteran biotech reporter Luke Timmerman's new book, Hood: A Trailblazer of the Genomics Age, is a valuable exploration of one of the leading figures in the early development of genomics and proteomics.  This in depth look at a key scientist covers not only his achievements and glories, but also his less than stellar moments and tendencies. Timmerman has combined his own interviews and research with nuggets pulled from prior news articles and oral histories of scientists who crossed paths (and sometimes figurative swords) with Hood.  While the book has issues, I would recommend it to anyone interested in the history of biotechnology.  For the full review, including numerous spoilers, read on.

Four Things You Might Be Surprised Can't Be Done On An iPhone or iPad

In well under a year, I've been sucked into Apple's mobile ecosystem.  Partly this was due to lobbying from TNG (he fervently denies receiving any commissions, but I'm still suspicious).  Partly it was due to the Android world not really locking me in; there were just too many things that weren't quite right.  First, the family caught me ogling an iPad Pro and talked me into getting one.  Once I had that, there was confusion over which text channel messages had been sent and getting an iPhone to consolidate them (since iMessage would go to both devices) started making sense.  Plus my Samsung phone was chewing through batteries; that is one area where Apple seems to have a huge lead.  Also the problem of periodically forgetting which device I was on and using a gesture from the wrong operating system.

Wikipedia, and other non-evil things

Tommorrow, TNG will head off for his penultimate first day of grade school.  And unfortunately, that almost certainly means another attempt to indoctrinate him in a catechism I find both depressing and infuriating.  It goes something like this: "Wikipedia is not a reliable source. Wikipedia is not citable. Don't use Wikipedia".

Where the bleep did my pores go??

Another afternoon spent this week in the lab, and another awful MinION run.  I figured that the last run was eminently beatable, given that I ended up with exactly one read aligning to my reference (though perversely, re-calling the data via Metrichor ended up with two reads in the pass folder -- one of which had zero bases in it -- I guess it is a passing null read).  But no, despite coming close to being botch-free on the routine lab stuff, dark clouds came early and the experiment yielded not a single usable read.

Sometimes It's More About the (1D) Journey

I will confess that I had a post planned in advance of which this post is but a faint echo. A grand scheme was going to yield a triumph worthy of brass bands.  But instead, it's barely kazoo territory, but sometimes even a small victory must be celebrated, if only in moderation.

Math Toys, Much Enjoyed!

Last week, I attended a free public talk at the Society for Industrial and Applied Mathematics (SIAM) here in Boston.  This is a wonderful public outreach concept which too few conferences sport.  The speaker, Tadashi Tokieda of University of Cambridge, illustrated a number of fascinating phenomena which can be demonstrated with simple toys or household objects.  Tokieda didn't lecture from a bunch of slides; most of the talk was in the form of live demonstrations -- and demonstrations made with overt glee!  There's a Storify of the entire meeting, in which mine and others of the Tokieda talk can be found.

Infinity's Sad RUD

Almost five years ago, I wrote of voluntarily leaving Infinity Pharmaceuticals.  Particularly due to the novelty (for me) of the voluntary part, at times I've wondered how long I would have stayed there.  Indeed, the offspring asked just that question just over a week ago. I do know now when I couldn't have stayed any longer, as to my great dismay Infinity announced last week it was discontinuing its Discovery operations due to a disappointing drug trial result.

Writing Big

For the past few decades, since the early stirrings that led to the Human Genome Project, technology for reading DNA has gotten immense investment and attention.  Particularly noted, perhaps ad naseum, is the rapid decline in sequencing costs which outpaces Moore's Law for microprocessors.  The converse activity of writing DNA has generally played second fiddle, but these past few weeks have seen a flurry of headlines on that topic.

London Calling 2016: Further Thoughts

London Calling was two weeks ago, and I still haven't written anything beyond the write-up of Clive Brown's talk (note that many of the talks are now view-able via Oxford Nanopore's website).  I did finally make some headway on Storifying the tweets.  After several self-inflicted wounds (starting with failing to read my write-up of the previous experience of trying to post to Storify from the command line, but also from failing to record the details of that final critical command), I did succeed.  This time I decided to group the tweets into several broad categories -- with tweets potentially showing up in multiple categories, though these are a bit rough-and-ready and probably most folks will want to check multiple or all of the stories.  One major value I derive from this exercise is having them to refer back to, so I'll probably feel the pain of erratic characterization the most (though I also have the tweet database, so I could reslice these in new ways).  Anyway, the categories are: de novo Assembly, Bioinformatics and R9, Real Time and Read-Until, PromethION, Microbiomes, Clinical/Human (except Infectious Diseases), Infectious Disease and Biowarfare, Field Uses plus finally Library and Sample Prep (another one that failed initial upload; includes Zumbador and VolTRAX tweets).

There's supposed to be a final one encompassing everything that was left over, particularly general tweets about the conference.  Unfortunately, Storify balked at that one -- have to look again and perhaps break the set into at least two pieces.  (nope: worked with one)

London Calling: Notes on Brownian Commotion

I'm behind on writing up London Calling.  I can partly blame a failing computer -- though rebooting it seems to have righted it for the moment.  A bigger challenge is that I had the luxury of staying in London thru the weekend, and have been trying to pack as much in of England as I can.  To really do justice to everything, I need to scan all the tweets  -- and that will take some time.

But I have dug into everything around Clive Brown's talk (kudos to NextGenSeek for Storifying that portion of the meeting's tweets!_ about the current and future state of the Oxford Nanopore platform, so I will focus on that, with a few side-trips on closely related topics. A few gaps on topics I previewed but didn't show up in the presentation were filled in with chats with Clive.  Plus, the indescribably huge advantage of actually going to a conference are the tidbits gleaned from late night chats over drinks (and no, I didn't ply anyone to get them to spill -- all was coughed up out of pure free will).  I'm going to roughly divide these by the announced timeframe: now, imminently, later this year, perhaps next year and unspecified.

London Calling Preview

ON Thursday and Friday this week Oxford Nanopore will be holding their second annual London Calling meeting.  I successfully defended my schedule this year, so I'll be on the ground there. If you follow me on Twitter and don't want to be buried in nanopore tweets, mute the hashtag #nanoporeconf (a rather large hashtag for talking about nano stuff!)  LC is OxNano's premier event, so what might we see from the company?

Inconstant lines

If you order chemicals, then the supplier provides a certificate of analysis, which shows the amounts of impurities or their limit of detection.  Fir physics experiments, one can purchase components which have been carefully cast or machined to precise dimensions. Barring errors by the manufacturers, these reagents and components can be relied upon, as their consistency is known.  Alas, for biological systems, such constancy is often a mirage.

Sickle Cell Anemia: An underprioritized disease?

The Sunday Boston Globe today had a front page piece by STAT's Sharon Begley that asks some challenging questions about prioritization of disease research.  Poking around the STAT site, I found that the original article was even longer and better, but between the important issues it raises, some interesting peripheral stuff and at least one gaping hole, there's plenty to discuss.

Kendall Square Tech/Biotech/Biopharma Needs to Get Vocal About Transit!

Earlier this week, the current big Boston-area mass construction transit project, known as GLX, went through a near-death experience.  The project, having been mismanaged to be over budget and behind schedule in the early going, was approved to survive in a stripped down form.  Numerous political types were quoted supporting the project, albeit complaining about contributions their towns were making to keep the project alive. What wasn't heard was any sort of support from the tech, biotech and biopharma companies which crowd Kendall Square.

Exploring Critiques of Siddhartha Mukerjee's The Gene, An Intimate History

My finely tuned skills in the art of procrastination KOed my plans to see Siddhartha Mukerjee's talk tonight at a local bookstore (apparently with Henry Louis Gates) to promote Mukerjee's new book The Gene: An Intimate History -- the event sold out.  Perhaps I could have found a scalper, but I decided I'd just head home.  Mukerjee's first book, the cancer history The Emperor of All Maladies, was very well received (even spawning a PBS series), and I was impressed that Mukerjee took the time to contact me after I wrote a review in this space.  The new book has been taking quite a bit of criticism, and even more so his New Yorker piece that preceded it (and I assume is derived from a portion of the book).

Around the World in Amino Acids

This post is pure whimsy, growing out of killing time on a train ride. The not-so-serious question: what is the geography of amino acids?  If I search for them by name in Google Maps, what will I find?  With just Google Maps, plus some Google Translate thrown in, I found a few surprises.

Genia Publishes Platform Progress

Nanopore sequencing developer Genia published in PNAS last week a study demonstrating the basics of their current approach to sequencing.  I say current, because Genia has gone through a number of iterations and on at least two occasions promised to be going into beta in a 6-9 month timeframe.   The paper demonstrates the basic concepts of a sequencing system and generates some short reads, but also suggests that Genia won't be hitting beta sites in the near future either.

Protein Homeostasis: Has it Hit The Classrooms Yet?

I wrote a piece earlier this year suggesting that introductory Biology textbooks should emphasize protein complexes more.  My basis for assuming that they generally don't isn't very good: a single textbook in use in TNG's high school class, which sports a copyright date from a decade ago. I also remember what I was taught in high school and college courses, which I would rate as not bad and truly excellent (respectively), plus I was a teaching fellow for one semester of intro bio at Harvard.  I now have another suggestion to cram into every biology course: an overview of ubiquitin-proteasome system.

Mosquito Genomes: Chance for Long-Range Companies to Shine

Friday's New York Times carried a front-page illustration of the current status of the Aedes aegyptii genome, accompanying an Amy Harmon story on efforts to improve the currently highly fragmented state of this genome

Hey @DrKatHolt @rrwick, there's a Bandage plot on the front page of the @nytimes today! #NoFooling @MarkKunitomi pic.twitter.com/a1n6QyQWr3

— Adam Phillippy (@aphillippy) April 1, 2016

The pice has seen a lot of opinion on Twitter with regard to its value and other issues (such as calling an assembly a map -- which to me is correct as the perfect genome sequence is the ultimate physical map!)

This whole thread. My science peeps keep me here. ❤️ https://t.co/96mifMD0da

— a muse (@_a_muse) April 3, 2016

Reflections on And The Band Played On

Fellow blogger, colleague and science history buff Ash pointed out to me recently that Randy Shilt's And The Band Played On for Kindle was on sale.  I hadn't read the book, nor seen the miniseries, so I snapped up a copy.  It's a good read -- though at times a hard one - I don't believe I've ever read another work of non-fiction where such a high fraction of the named individuals are dead by the end of the book

Who Wants To Write A Review Article?

Yes, this is a solicitation.  I'm on the Editorial Board of the journal Briefings in Bioinformatics,.  I'm looking for authors who would like to write high-quality, compact reviews.  If you are interested, or you want a little back-story, then keep reading.

At the Edge of The Cloud

I've used cloud computing at Amazon Web Services (AWS) off-and-on now for over five years.  The cloud has all sorts of handy advantages -- flexible access to large amounts of compute, inexpensive access to any flavor of Linux you wish, the ability to guiltlessly kill a huge server you just fatally cratered with the wrong command.  And until now, I''ve always been able to find machines that fit my needs -- perhaps sometimes just fitting or with a bit of compromise   But, now I've hit the wall: nobody at this time offers a really serious cloud machine with 500Gb of RAM.

Selective sequencing: A Programming Opportunity!

I ask a bit of indulgence from my regular readership for this piece, as I am going to explain a number of things in depth that probably will be very familiar to them.  My hope, perhaps fantastic, is that this piece will get out to some who are not so familiar with such topics, as I think the problem at hand might be very fascinating.

PacBio's big splash

[18 March 2016 -- my original inclusion of the Pac Bio marketing image 6 years ago was claimed to be a DCMA violation -- I've simply removed it, though I do think this would fall under fair use ]

The Pacific Biosciences instrument is officially unveiled now, with those lucky/smart (or SMRT?) enough to go to Marco Island filling in all of us not in that position. Sounds like a great lot of hoopla, though they didn't drag the Hornet for the splashdown.

First of all, it's a beast. "In this corner, weighing in a nearly an imperial ton...". Too bad their marketing picture has nothing good for judging the scale --
it's apparently 6.5 feet wide.

Kevin Davies at Bio-IT World has a wonderfully detailed article and there is a lot of nuggets in the Twitter feed. Anthony Fejes has two different sets of notes out -- one from a workshop and one from another speaker; Dan Koboldt has some good notes too (and if I haven't shouted out your notes, it's probably because I'm oblivious -- leave me a comment pointing to them). There was also a little bit of PacBio science in Elaine Mardis' talk (she's on their SAB) -- Anthony's notes & the twitter feed.

Okay, besides worrying about the capacity of floors & freight elevators, what's new? Well, not much on error rates from PacBio (apparently in the Q&A their presenter executed a jig, tango, waltz & rumba when asked) -- though the Mardis talk described resequencing samples of PacBio that had been done before by Illumina -- and the results are quite good. Another important note is that their system doesn't seem to have much bias in terms of composition -- bias against hi/lo %GC has been noted in all of the amplification-based systems and can be a serious problem.

There's also a lot of talk about being able to distinguish various modified bases by their effects on polymerase kinetics. PacBio has also demonstrated direct RNA sequencing (substituting a reverse transcriptase for DNA polymerase) and is talking about watching proteins being made. I haven't quite figured out why you'd want to do that last one, but presumably it's for more than a cool Nature cover.

Read lengths decay exponentially -- but with lots around 1Kb and quite a few around 5K. The big problem is apparently oxidative damage to the polymerase triggered by the laser -- so they are working on both getting the oxygen out of the system and engineering hardier polymerases (the sort of biz I used to be in). Their strobe sequencing mode -- in which the laser is turned off to enable elongation in safe darkness -- enables multiple reads separated by long gaps.

The instrument definitely raises the bar on sample prep -- it's apparently entirely automated within the monster. YEAH! A machine I can delude myself into thinking I could run it! One drawer takes the SMRT cells and another the DNA samples -- 500 ng of each. That doesn't sound like much (it's at least better than the 5-10ug most library prep protocols call for -- except the ones looking for 20-30ug), but it seems you don't get a lot from each sample.

The number of reads per cell isn't huge -- but you're still getting about 2 E.coli genome equivalents by my calculation. This is a bit undersized for a lot of applications -- but grand from many others. Mardis' talk discussed using PacBio for sequencing PCR amplified resequencing samples -- this would appear to be right in the PacBio sweet spot. Perhaps a few hundred long PCR products could be packed into one SMRT run and still get many hundreds of reads per sample -- well, maybe pack fewer amplicons.

What might be other good uses? Clearly metagenomics and similar. I just saw a posting on a professional board of someone pondering multiplexing hundreds of samples for an Illumina run (the current barcode schemes are for a few orders of magnitude fewer samples). Blitzing each sample through the PacBio instrument would seem to be obvious -- if the error rates are acceptable. Folks doing whole genome sequencing of small genomes will love having PacBio to generate scaffolds. For bigger genomes, it may just still be too expensive to get much coverage ($100 a SMRT cell sounds cheap, until you start multiplying that out for the numbers you need) -- but perhaps not (much too fried to do that calculation at the moment).

RNA-Seq might be a bit trickier. If you need 500ng of input material, that's an awful lot of ribosome-depleted or poly-A RNA. Plus, getting only tens of thousands of reads, making it hard to see lowly-expressed messages -- but very long ones, perhaps priceless. But, if you can get tons of RNA, then 100 SMRT cells would be about $10K and offer similar depth to what you can get today with Illumina but with those super long reads.

Now, who is this going to crimp the most? The instrument is clearly a ways from really threatening Illumina & SOLiD for the large genome market. 454 is a likely candidate to see growth pressured -- though between the new lower-priced "junior" and both PacBio's $700K price tag and their inability to flood the market with instruments, this will be ameliorated.

PacBio might have almost as much effect on the surrounding sequencing ecosystem. Making library prep reagents for this system is not going to make you lots of money! But, there will be a serious niche for targeted sequencing -- though with the scale it will probably require some rethought. Stuffing the whole exome into this doesn't really make sense -- if there are ~250K segments of the genome to read & you want 40X coverage of each, that's a lot of SMRT cells. But, intelligently chosen gene sets totaling about 500 regions (or around 20-50 genes) with pre-validated reagents -- now that might be a market (though one which might have 1-2 years of life -- better get cracking!). Simpler library prep will also go nicely with some of the enrichment systems -- a bugaboo of hybridization systems can be "daisy-chaining" of fragments via the amplification adapters -- but, on the other hand you don't get 500ng off an array or in-solution system without amplification. As with many disruptive technologies, it won't fit a lot of bills but will nibble off various parts of the business that are individually small but significant in aggregate. As noted above, RNA-Seq might be an initial success story for PacBio -- when RNA is abundant.

IMHO, PacBio does need to get some papers out on applications (Mardis' group apparently is close to having one) and make sure that the next tranche of installations not only includes the Sanger & BGI, but that there are also some core labs or commercial providers. Also, they need to start pumping data into the public domain -- while they signed a bunch of commercial software providers up, it is definitely out of academia that you find the most radical advances. Plus, there are a lot of now well-entrenched open source tools that need to be tested with the new kid. Even simple things like the semi-standard SAM/BAM format are going to need tweaking -- SAM/BAM stores all sorts of information on read pairs, and the strobe sequencing can generate many more than 2 tags per DNA fragment.

Of course, we have to wait another half day plus to find out what Ion Torrent is really delivering. That could really shake up the landscape -- at least the mental one.

A huge thanks to all the bloggers & twitterers for pouring out so much information. I'm still getting used to scanning past the retweets (is there a way to condense them) and there is the occasional shock-to-the-system (how could anyone in the field not have heard of Rodger Staden?!?), but that's a tiny price to pay for such fascinating stuff.

A Mosquito ExAC?

Okay, there's a scheme for a crazy big genomics project has bitten me, infecting my brain.  It's definitely not something I'm in a position at all to execute on, but I throw it out as an idea in case anyone finds it useful.  And admittedly, it is pretty much stealing straight from the ExAC human exome aggregation project, which contains huge numbers of human exomes.  Behind all those is a lot of phenotype data.  Now, inspired by recently re-reading Laurie Garrett's The Coming Plague and also faced with daily news items on the Zika virus epidemic, I've had this question: what if the same approach were applied to key disease vectors?

Oxford's Riposte To Illumina Trade Action

Along with the "No thanks, I've already got one" online session, the other big Oxford Nanopore news is the public release of Oxford's response to the trade complaint filed by Illumina which was attempting to exclude all Oxford Nanopore devices from the U.S. markets.  Nature News' Erika Check Hayden has posted the document on Dropbox, which was a big help.  While no documents from Oxford's side concerning the simultaneous patent lawsuit have yet surfaced, it is reasonable to expect that it will use many of the same arguments.

Oxford's "No thanks, I've already got one"

Oxford Nanopore today hosted a Google hangout titled "No thanks, I've already got one".  Only this morning did it occur to me I could have re-watched Monty Python and Holy Grail and scored it as blogging-related time! Oxford CTO Clive Brown went through a number of interesting (and in many cases, long-awaited) announcements on the release of multiple key upgrades to the platform (note: unless otherwise specified, images swiped from ONT).

Digging into the Illumina Lawsuit vs. Oxford Nanopore

Illumina's and University of Washington's filing of a patent lawsuit and related trade complaint against Oxford Nanopore made big news yesterday, with nice coverage from Mick Watson, GenomeWeb, Nature's Erika Check Hayden, Technology Reviews' Antonio Regalado,  BioIT World's Aaron Krol, and venture capitalist Vishal Gulati. Each of these covers the onetime partnership between the two companies and their acrimonious parting of ways.   Oxford Nanopore released a short and pithy response. Having failed to get an early jump on things, the ground is already well plowed.  So my sloth and inertia have forced me to take an unpleasant route I usually spend great effort avoiding: actually reading the complaints and the two key patents licensed from Jens Gundlach's group at University of Washington (US8673550 and US9170230 ) they cite.

Amplification-free, library-free sequencing? NanoString wants to be It

Perhaps the most unusual new technology to be unveiled at AGBT16 is NanoString's new approach to sequencing, which is in very early stages of development.  Called Hyb And Seq  the process is remarkable in being a purely hybridization-based single molecule method -- absolutely no enzymes are harmed during the operation of the system. That's remarkable -- the only enzyme-free (or nearly so) sequencing approaches to deliver serious amounts of data into Genbank are Maxam and Gilbert approaches (including Church's genomic sequencing and multiplex sequencing), and even those typically required restriction digestion of the target.

AGBT16 Storify Completion & Rate Limits

AGBT16 ended a week ago, but for various reasons I'm just now catching up on my Storify project.  A vacation was in there but also some tool building.  As I was griping about the pains of organizing the tweets manually, Brian Krueger suggested what was already dawning on me (but it helps to be poked -- professional embarrassment is often a stronger motivator than pure annoyance) -- I needed to stop doing this purely manually.  So, off to deal with pulling in Tweets automatically and at least doing some organization programatically.

10X Launches Chromium (#agbt16)

10X Genomics launched their approach to obtaining long-range genomic information last year with a big financing and some exciting preliminary data at AGBT15.  Now they are back at AGBT16 with an upgraded instrument, improved biochemistry, new software and new applications, along with a trio of major co-marketing agreements and a splashy hire and a raft of both published and unpublished data from academic collaborators.

#AGBT16 Day 2: How is AGBT On Twitter Like Sequence Assembly?

I spent a bunch of time yesterday going through the Tweets from AGBT.  For me personally it is a useful exercise, plus I'll have it as a resource to go back to for future posts.  But the time and pain involved definitely had me sometimes questioning the wisdom of attempting this.

AGBT Begins (with bonus Storify Jeremiad)

Just finished my last Storify for tonight from AGBT16, and boy am I wondering how sustainable this will be.  The "problem", which is wonderful to have, is that the number of tweeters has grown substantially, and so there is a wealth of material to attempt to distill down. There's also the desire to make sure I don't further propagate the spam which sneakily re-tweets the occasional item. The other problem has been my tools

AGBT16 Preview (aka The Non-Attendee's Lament)

AGBT16  starts this today but I'm again not there. The usual complex set of personal constraints (or imagined ones) kept my hat out of the ring this year, and now I'm again torn between wanting to be there and why it would have been hard.  Easy would be leaving our most recent snow and ice storm and the general cold weather.  A bit harder is it is early in the school term, and back-to-school night is Thursday -- plus I spent last night chatting with a candidate for a local office (School Committee) at a low-key campaign event.  The big, and unforeseeable, challenge is that the other half of Starfleet's bioinformatics group is out on paternity leave, and while I'm proud of how much quotidian work I get done during conferences, it still isn't the same as being on full duty.

Why do we purify DNA the way we do?

An interesting conversation on Twitter on means for purifying DNA for PacBio and the risks of phenol-chloroform extractions restarted some pondering on the historical contingency of experimental techniques.  Or, as the title says, why do we (today) purify genomic DNA the way we do?

First on an occasional series on high school biology: Complexes

TNG has biology this term, so I will be (at erratic intervals, of course) sometimes venturing my thoughts on the teaching of that specific subject.  I think I never quite got around to venting the last time he had a biology unit, back in middle school, which among its sins was still teaching the seven kingdoms system of classification, which for the love of Woese is absurd.

Does any analytical program really care about the order of paired end files?

I was recently experimenting with C. Titus Brown and company's khmer package and hit an interesting little snag.  First, I had my usual problems with installing a Python-based program, which were solved by the totally counter-intuitive absurdity of actually following the installation directions precisely. Armageddon is certainly near if random shortcuts and assumptions can't be relied on to get the job done.  But once I had it working for a simple test case, I crazily tried to build something -- and that's when a new, maddening bug cropped up.

Illumina's MiniSeq Giveaway

This morning, Illumina announced a Scientific Challenge program as part of the launch of the MiniSeq sequencing instrument.  Three prizes will be given away, with 3 sequencing runs on MiniSeq as 3rd prize and a MiniSeq plus reagents for 3 runs as second prize, and a MiniSeq plus reagents for 3 runs plus a Mini Cooper automobile as the grand prize. Entrants in the contest will submit a proposal for how to they plan to use the instrument (but not the car; if you support future reagent purchases by being an Uber driver, that's your business). There will also be a set of iPad mini giveaways based on recommending colleagues to enter the contest on social media; if your recommendation results in an entry, then you are entered in the iPad giveaway.

Illumina's Unveils Firefly

Illumina third big announcement around JPM is to unveil Project Firefly, a semiconductor sequencer which will use existing SBS library preparation and a derivative of SBS chemistry.   Slotted with a price point ($30K), physical size (small pizza box ish?) and data yield (4M reads, 1Gbp data)  below the just announced MiniSeq , Firefly would be two small boxes which could stack: one for library preparation and one to run single channel sequencing.  The flowcell would use ordered arrays, layered atop the semiconductor sensors.  Launch is proposed for the second half of 2017.

MiniSeq!

Okay, third (and last) post for tonight.  Time for the victory lap -- and a reminder of the limits of educated guessing.  On Sunday I threw out a prediction of a hypothetical Illumina MiniSeq based on ordered arrays, NextSeq chemistry and optics, but with only one optical unit and a price point of about $50K -- and today Illumina announced precisely that.

Grail will change oncology & society, but how?

Second big genomics news this weekend around the JP Morgan is that Illumina is spinning out a new company called Grail, backed also by Jeff Bezos, Bill Gates and some VCs, to pursue mass cancer screening via liquid biopsies.  Given that cancer is most treatable when caught early, it's an exciting idea.  But, the devil as always is in the details, and they could be quite diabolical.

Affymetrix Assimilated

With JP Morgan Conference starting, there's lots of big news in the genomics world, and for 2016 I'm throwing out my previous internal policy of one post per day; if the news warrants multiple, then write multiply! Plus, too often in the past I've jammed topics together, and if nothing else it makes it hard for me to find my own posts on a topic! The first big news this weekend is the announcement that Affymetrix has been snared in a $1.6B tractor beam from Thermo Fisher.

Illumina JPM/AGBT Predictions

The JP Morgan conference is next week, which for several years now has been Illumina's venue for making major platform announcements.  So naturally, based on not even anything as substantial as scuttlebutt or rumor, I'll venture some predictions. 

When it comes to Nanopore, am I too GAGA?

In the comments to yesterday's piece on the reboot of Nabsys, a commenter used a truly colorful epithet in inquiring why I am so bullish on Oxford Nanopore, and in particular whether I am paid to do so.  Whether I've lost objectivity on this (or any) subject is something I take seriously, and I think it is worth a look.  To deal with the more serious allegation up front: I have never been paid by Oxford Nanopore, and if I ever do write on a company which has paid me in money or substantive gifts or in which I held stock I would disclose that. My company has been a member of the MinION Access Program (MAP), and one could argue that Oxford has provided materials worth far in excess of the $1K entry fee.  On the other hand, I and my co-workers have sunk quite a bit of time trying to use bad flowcells and unstable kits, so we've also sunk a lot into that project, so there's hardly a windfall there to cloud my judgement.

Nabsys Reboots

It's the beginning of the year, so new beginnings are a natural subject (though to be honest, spring works for that too).  The holidays brought word of an effort to reboot a company that seemed to have expired: Nabsys.