MiSeq Aims for Ion Torrent

     Illumina has released a MiSeq dataset for E.coli MG1655 on its website.  Accompanying the FASTQ and BAM files is a presentation, the first half of which compares the performance to big brother HiSeq.  The second half is an explicit comparison against the available Ion Torrent. 

A first peek at data from an Ion 316 chip

      Ion Torrent has made available an E.coli DH10B fragment dataset for the 316 chip, which is expected to be generally available early next month.  I've theoretically had access to the data since Saturday, but a series of events (computer-free weekend, a crashed home computer, a personal day & a good but unbloggable conference) have meant that ambitious plans to analyze it are still in progress.  So, what I discuss below is either from Ion's provided information or other sources, so take it with appropriate caution.

Two administrative notes

Two administrative items:

First, sometimes comments are getting moderated very late.  I do pledge this is not an attempt on my part to bury negative commentary.  Rather, it appears that sometimes when I thought I had moderated comments via my Android phone it hasn't succeeded.   

Second, I am trying out the new smartphone-friendly layout option for Blogger.  Please let me know if you are reading this on a smartphone and hate the new layout.

E.coli Outbreak Genomics

     The E.coli outbreak in Germany continues to be a major news item.  It is looking increasingly doubtful that the source of the infection will be conclusively traced, as the German authorities have already named and then backed off two suspects, Spanish cucumbers and German bean sprouts.  These activities have not been without repercussions; Spanish agriculture has been hard hit and exports of European produce in general are reportedly hurting.

     On the genomics front, the outbreak has demonstrated how quickly bacterial genomics can be run on the current class of instrumentation.  BGI Europe knocked off the sequence in a series of Ion Torrent runs in 3 days, and a group at University of Muenster worked at similar speed with the Ion setup as well.  Later, sequences have come in from the Illumina and 454 platforms.  The public release of this data has engendered a number of public analysis projects.

The Arsenic Bug, Revisited

As has been covered in many outlets, Science has released eight technical comments on the arsenic-loving bacterium issue along with a response by the authors, which I commented on when it appeared.  A good summary of the comments can be found at In The Pipeline, and Fejes.ca has a good take on it as well.  The quick summary is that various technical and theoretical issues are presented by the critics, and the NASA team dismisses all of them by standing by their data and interpretations.  

Paying a Painful 75% Secrecy Tax

In a post a while back, I mentioned that my Ion Torrent sequencing project was stalled because my service provider couldn't get some of the key kits, despite an Ion representative posting that no such shortages existed.  I've been remiss in updating that; last Tuesday the kits showed up and Monday I got my data -- and a bit of a shock.

MiSeq's First Light

Someone was kind enough to send me a copy of a poster by Illumina reporting results from the MiSeq.  Now, to be very upfront, by someone I mean "a person from a PR firm contracted by Illumina" and "kind enough" that she was doing her job.  I don't have illusions about motive here, the author list is all from Illumina or Epicentre, but this was a poster presented at the recent Cold Spring Harbor Biology of Genomes meeting.  It certainly isn't peer-reviewed data, but it is something. Of course, we can't know to what degree these are cherry-picked results.  If you want to be really cynical, call it messaging and not data.  Yes, I've taken some flak in the comments recently about how favorable my coverage of Ion has been, and I'm trying to adjust.  And don't worry; I have some new bones to pick in that area.

Forums: Open Beats Closed Hands Down

Around the Internet, there are a number of communities in which scientists can swap useful information.   SEQAnswers is a very useful site I frequent; BioStar is one I don't but probably should.  Life Technologies has set up a community around Ion Torrent, and the contrast between that and SEQAnswers is a useful one.

SEQAnswers has a straightforward access policy.  Anyone can view content, but to post or reply you must register for a membership.  This approach appears to have been very successful, as there is a healthy number of individuals posting to the site.  You can browse around and figure out if the site applies, plus search engines such as Google can steer individuals in.  SEQAnswers boasts a number of authors of major second generation sequencing analysis packages, including Bowtie, Tophat, BFAST and Samtools, as regular contributors.  There is a significant network benefit to this; quality encourages quality and conversely such folks must be judicious in the number of forums they actively participate in.  The management of SEQAnswers applies a light hand, occasionally moving posts to more relevant forums and smothering all spam.  In addition to the forums, a key asset is a large wiki on second generation sequencing packages.

The Ion Community is set up on a very different basis.  It has two sections, each with its own membership restrictions.  PGM Users is open only to registered owners of the sequencing system; Torrent Dev is open to that group plus anyone registered for the Grand Challenges.  Each section has both discussion areas and documents.  The site is flashy, though more than a few links are indirect detours to what you really want.

Now, there are plenty of examples of how maintaining some control over a site can be productive.  I was recently trying to eradicate one of these nefarious fake antivirus viruses from our home computer, and on one major security software company's forums I found what looked suspiciously like a link to infect with one of these viruses.  Keeping a single point of origin for documents can be useful as well, to reduce confusion.  For example, if you Google around for information on 454 fusion amplicon design it is easy to find outdated information.  We also wouldn't want any forum to devolve to the level of the Biotech Rumor Mill, which by its very nature must allowed unregistered posters, and as a result is a mudpit of insults and near(?)-libel.

But, Ion's approach is in my opinion strongly self-defeating.  I won't go into detail here, but in the extreme form they have on two occasions (this thread and this one) argued for the suppression of information on PGM from SEQAnswers (I will try to tackle this soon, but after getting a chance to talk to at least one person with the Ion side of the story).  But it's also easy to argue from a purely practical standpoint, rather than a philosophical one, that this approach is not doing their platform any favors.

The first problem is that the closed access means that you can't lurk on the cheap there; either commit to being part of the community or stay out.  This prevents sucking people in slowly; for many the barrier of registering -- particularly since your registration does not become instantly active -- is too high a barrier.  Indeed, I would offer as evidence for this that the first major PGM-tuned software package not from Ion or a commercial partner was apparently not spurred by the Ion Community, but by Nick Loman's post on assembly of Ion data.  Nick's original post apparently received over 2K views, which must be at least an order of magnitude larger than the current Ion Community membership.

The second problem is the two layer design.  Okay, I'll admit it -- it's maddening to be excluded from the PGM Users forum.  How do I know that there are not discussions there I could either benefit from or contribute to?  If someone starts a discussion there better suited for Torrent Dev, will it get bumped up?  If so, who decides?  But, worse than that is that many technical documents that I would find valuable are beyond that access barrier.

The third problem is I find these rigid definitions completely at odds with scientific reality.  Just because I don't own a PGM doesn't mean I might try to do everything but run the instrument.  As an example on another platform, my colleagues & I have run SureSelect on Illumina where we did all the steps except shear the DNA (outsourced), prep the flowcell and run the flowcell.  Furthermore, in a modern collaborative environment, someone in one lab may own the machine but in another lab work up the samples. It's also not clear what any of this "security by obscurity" is buying; given the number of groups using Ion, whomever they're trying to hide the information from can certainly find a leak.  Ion should heed the example of the music industry, which failed to provide legitimate means to supply a growing demand for digital music, and thereby spawned widespread illegal file sharing.  Plus, many browsers are potential buyers -- people want to know what they are really buying in to and to start storyboarding what running an instrument would mean in terms of personnel and auxiliary equipment.

The fourth problem is discoverability.  How do I find information?  Well, for second generation sequencing stuff it is the trio of PubMed, Google and the SEQAnswers software wiki.  PubMed is great, but many packages show up online long before they are published.  Google is useful if you know what you are looking for, and SEQAnswers is great if someone has logged it there (and in general, once I see something I make sure it is logged there).

But with the Ion Community, those last two are problematic.  The objections Ion has raised to links going to the interior of the community mean that I don't dare put such in the Wiki; but conversely a link just pointing to the community is not terribly useful.  But worst, since Ion apparently won't let Google in their stuff is invisible to that valuable tool.  As evidence, at the moment if you Google for information on their TMAP aligner with "tmap source code ion torrent", nothing from the community comes up (but threads on SEQAnswers do!).

Ion isn't the first, and probably won't be the last, company to try to have its I proprietary control cake yet eat its Internet openness.  Life has SOLiD Community, Helicos has one for Helioscope, PacBio DevNet for SMRT sequencing and so forth.  It takes some real courage to relax some control and invite the whole world to your party.  Some folks would no doubt see view-without-registration as a loss of useful marketing data, forgetting that the openness of a site will itself draw in customers.  Anyone launching a new platform should really ask themselves, will I be better off trying to control a rare destination for a few visitors, or perhaps just cultivate a sub-community over at SEQAnswers.

Oh Would It Be Fun to Debug Strobe Sequencing!

Through the course of a month, I easily sketch out (on average) a dozen plus experimental designs in the context of my day job.  Of course, only a very few of these are ever executed; many never get shown to another soul.  By constantly pondering how I might tackle questions, I can keep in practice and present plans which are reasonably well thought-out.  It also helps to have thought through things; sometimes a rejected plan will suddenly look like a gem due to some other result or change in priorities.

Atop that, I also sketch up a few which have nothing to do with my day job.  Partly it is fun, and partly it is a way to exercise the process on areas I can be certain of my objectivity.  A related exercise is to sketch out a business plan; I end up doing that a few times a year.  Never have taken any further than a napkin; not only do I lack the necessary thirst for risk, but most are for businesses I'd be happier being a customer than an employee.

For example, after a recent lunch with a friend from graduate school I found myself again contemplating a question that had arisen at Codon in the context of gene synthesis.  We didn't have second generation sequencing there (if Codon had survived, we certainly would have launched into it at some point) and had seen the hint of an interesting phenomenon (and practical problem) but could have never collected enough data to really nail it down.  Now, with sequencing cheap on a large scale, and this being the perfect sort of problem for such sequencing (since one would need only very short reads), it was short-term obsession to work out how to run such an experiment.  For probably $5K and a tiny bit of molecular biology, a nice little paper -- pity I don't have a slush fund to cover it.   My apologies for not supplying any details; maybe I will someday have some found money to cover the project.

However, I did just get another idea that I might as welll be open about, as for one it will probably be solved in the near future and two there is no opportunity to actually work on it.  So it can be fun to speculate in the open on how to address the problem.

Blogger glitch

Blogger experienced an outage which I found out about today.  This caused my last post to disappear; any posts or comments in a certain time period were lost.

Thanks go out to the anonymous reader who posted a comment wondering where my last post went.  Blogger (Google) did not notify me of the issue.  The really disconcerting part is the Google cached version of the post was missing as well -- this suggested something very odd going on.

I often write posts directly in Blogger, so I don't have a backup.  This was an exception & I did actually have the rough draft, which I would have posted if the old one wasn't restored.

Ion's Growing Pains

A recent In Sequence article indicated that Ion Torrent is enjoying strong initial sales.  This bodes well for continued evolution and improvement of the technology, as LIFE will continue to smell revenues and opportunity.  Ion has announced a number of improvements, but most aren't scheduled to arrive until the near future.

The challenge is for LIFE to keep executing on their plan.  Already some issues have arisen; my own Ion experiment at a service provider is stalled due to a back-ordered template prep reagent (two weeks and counting!). This is a key reason to do pilots on emerging technologies with non-critical (but interesting) samples; I was bitten last fall by another backorder bug (that time, the paired end SOLiD reagents).  Of course, this time it is even more complicated, as Ion is making a major change to both the underlying kits and to the software to process the data.  It will be worth it if I get results anything like Ion's provided E.coli 314 dataset, which has about 4.5X the data of the original 314 chip spec.

Ion Torrent's Data Quality Is Pretty Good (and Better Than Ion Claims)

One of the key questions around Ion Torrent, as with all new platforms, is what is the sequence data quality like.  Now, that can be a loaded question but I'll ask a slight variant on it: how truthful (or accurate) is Ion at estimating base quality?

Quality scores are a useful adjunct to sequencing data and are commonly expressed as phred scores, which are the integer part of -10*log10 of the error probability.  Any base caller needs to estimate these and many downstream programs, from aligners to assemblers to variant callers, rely on these quality values for their operations.  In many cases, the individual quality scores are combined to generate some joint estimate of the error (I built one such model at Codon).  These error probabilities come not from an infallible source, but are rather estimated from aspects of the raw data.  

Which numbers did I use this time?

Noted screenwriter William Goldman's second memoir on the film industry is titled "Which Lie Did I Tell?".  The title is not a quote from Goldman, but rather what another movie industry said after getting off a long phone call he had taken in Goldman's presence.  I'm a bit nervous I inadvertently strayed in that direction in my last item on Pacific Biosciences.

I was relying on memory for my numbers & in the course of writing things I think I also revised those downwards, not out of malice but rather an attempt to be conservative.  As a correspondent pointed out, the first pass accuracy on the first commercial system is claimed to be 85%, not 80% as I stated.  On the number of reads, I failed to update for the newer SMRT cells which are out; I said 10K and it's probably at least 3X that.  However, I do have a bone to pick.

How Many More Machines will PacBio Sell This Year?

Amongst last week's news is the item that Pacific Biosciences has officially launched their SMRT sequencing platform.  I'd too eye-deep in various projects to figure out how off schedule that is (I think nearly a year from their original target), but now it is launched.  

Better Template Prep for Ion Announced

One of the perceived weak points of the Ion Torrent, in contrast to the Illumina, has been the use of emulsion PCR for template preparation.  The original template prep protocol was apparently around 10 hours of wall clock time, with a substantial amount of hands on time.  Improved template prep is the subject of one of the Grand Challenges.  A new kit being released today along with a new instrument announced today (but not generally available until late summer or sometime this fall) go after this issue; the new kit providing an inexpensive but substantial immediate improvement and the modestly priced template prep instrument providing a very low labor solution once it arrives

Vostok & Columbia

It has hardly gone unreported in the media that today marks the 50th anniversary of the manned spaceflight and the 30th of the first space shuttle launch.  It was an accident of scheduling delays that put that flight on the 20th anniversary of Gagarin's, but what an appropriate synchrony that is!  My own contribution herein is to pen two quick capsules of two books that deserve longer reviews: Two Sides of the Moon and Riding Rockets.  If you have a strong interest in the history of spaceflight, you should consider reading each of these if you haven't already.  The only caveat I'll throw in is that if you have a young friend who fits that category, but you do not, you should at least skim Reading Rockets before passing it on; some of the content (and most of the humor) is of a mature nature.  While neither book is primarily about either of the events commemorated today, both bear on it.

Gnoteworthy But Gnot Gnoticed

A commenter on yesterday's piece on Intelligent Biosystems scolded me on not mentioning GnuBio and said they had released data.  This had totally slipped my my notice, and indeed it seemed to have slipped past the GenomeWeb and BioIT worlds as well, judging from some Google searches.  There is certainly a press release out there and covered by several outlets, but amazingly stealthy for public relations.  Strange!  But many thanks to my anonymous correspondent for flagging me on this!

But, it does make a set of stunning claims.  When GnuBio launched last year and announced they would have alpha instruments in collaborator's hands by the end of 2010, I was skeptical.

Another Low Cost Sequencer on the Horizon?

An article in GenomeWeb's In Sequence (which, alas, requires a subscription for which I've never sprung) has a piece on Intelligent Biosystems (IBS), which iat the X-Gen Congress meeting apparently announced a plan to launch the "Pinpoint Mini" sequencer.  The box would come in at $85K, putting it smack in the middle of Ion Torrent PGM and Illumina MiSeq pricing.  The hope is to have boxes shipping to early access customers by the end of the year.

To be honest, I'm guilty of mentally writing off IBS, as they had been around a long time and very quiet.  Indeed, in my defense their website looks like it hasn't been updated since it first went up, and you'd think now that they made a big announcement it would be updated, but apparently not yet.  On the other hand, while stale websites can indicate fading companies, the fact that it still is working suggests some life.

In any case, I was apparently hasty in my thoughts.  The box as described has some interesting features.  Supposedly it will crank out data at $75/Gbase.  The claim is that one exome could be sequenced (reagent costs only, mind you) at 30X for $150 in about 1.5 days.  No word on read lengths.  The system will mount 20 flow cells, each of which can be run independently.  Chemistry is based on reversible terminators that they exclusively licensed; if I understand it correctly the big advantage of their chemistry is simplicity.  There's also a curious bit in the publication from the founders is using a mix of unlabeled reversible terminators with labeled dideoxy terminators; the same cleavage reaction removes both the terminator and the label.  This was touted as a way to reduce the discrimination of the polymerase against the reversible terminators.  Of course, an alternative would be to generate mutant polymerases which are more amenable to being fed terminators.  Having four pairs of complicated compounds wouldn't seem to be a route to low cost, but perhaps the gains are worth it (or this chemistry isn't being used any more; very hard to tell from what I was able to read).

Sounds great, but of course there's a lot to do before such a machine can launch.  There's no word about sample prep method, which probably means it will use emulsion PCR, since necessary licenses for that can apparently be obtained.  There's also the problem of manufacturing the instrument and the reagent kits.  Te fact that IBS apparently planned to launch a PinPoint large-scale sequencer a few years back and couldn't get it out the door is not going to help them compete in the expectations market with the other boxes.

One solution to some of these issues would be a strategic partnership with (or outright acquisition by) a major reagents and/or equipment player. It's not hard to come up with a list of candidates, based on nothing more than that description.  Perhaps at some point Affymetrix will decide to move into next-gen.  Roche could always decide to go for something cheaper than 454, but I doubt it.  Agilent seems quite happy supplying picks and shovels, but perhaps they'll go for the big time.  Perkin Elmer, GE (which is working on blue sky sequencers) or a host of others.  Picking the right partner will be key; Illumina has the advantage of an enormous installed base and thriving ecosystem of associated vendors, whereas Ion Torrent has a lot of buzz and serious marketing muscle (not to say Illumina is lacking there either).

It's also interesting to see this machine being touted as an exome sequencing workhorse for clinical use.  The issue really deserves its own detailed post, but such an application brings some serious issues.    Library preparation requires a lot of labor and a bunch of other instruments, or a bit less labor and some more instruments specialized for prep.   Right now, the market for exomes on HiSeq using either SureSelect or EZCap  is quite competitive; I've recently gotten quotes ranging from $2K-$5K (the lower quotes tend to be from new entrants; promised coverage varies a bit too) .  For 50Mb capture at 50X coverage, it would seem you could get around 50 exomes into one HiSeq flowcell, which at $10K each means about $200 in sequencing (feel free to correct my math in the comments).  That would suggest that very little of the cost of these exome captures is sequencing reagents; the majority is labor and the EZCap or SureSelect kits.

While some elegant library-free methods  (really, methods which add the sequencing adaptors as they are capturing the targeted DNA). for exome sequencing have been published, none of these are commercially available on an exome scale.   RainDance requires an expensive ($200K+) box and isn't quite up to exome scale,. .  Halo Genomics. has announced a library-free  prep for "1000s of exons", so perhaps this will break this problem open.  Whether this is really whole exome, or something smaller, remains to be seen.  A safe rule, though, is that these methods are only efficient if read lengths are significant.  At a minimum, the first part of a read is burned on getting through the targeting primers, and with very short reads the size of each targeted amplicon must be small, meaning for a fixed number of amplicons (cost) you can capture a lot less DNA than with a longer read technology.

So, another player in the field -- but with a far off beta release and a lack of a track record.  They'll be fun to watch (assuming they go out of possum mode), but probably won't be a real factor in the market for over a year.

Can we treat the kinase du jour?

For the second time in just over a week, the Boston Globe Sunday was discussing protein kinases in the context of cancer.  A group from the Broad has just published a sequencing study (Sanger!) identifying mutations in the protein kinase DDR2 in about 4% of squamous cell carcinomas of the lung.  This is a common form of smoking-induced non-small cell lung cancer (NSCLC) and one for which many therapies useful in lung cancer are contraindicated.  The prior study by another group at the Broad was published a bit over a week ago in Nature detailing an extensive look at myelomas by sequencing, and found mutations in the kinase BRAF in 4% of myelomas.

The myeloma study is quite a watershed and in some ways raises the bar for cancer genomics publications.  Whereas most papers have published a single cancer genome and a few have published single digit genomes, this one looked at 38 myelomas.  Now, not to overstate things, as only in 23 patients were matched normal and myeloma whole genomes sequenced; for the other 15 patients just the exomes were sequenced (one additional exome pair was run in a patient with whole genome sequencing, to enable comparison).  Clearly this is a serious scaling up of effort, enabled by dropping costs.  By sequencing multiple genomes, the possibility exists both to discover rare variants as well as get some rough mutation frequency information.

The myeloms study is curious in one aspect: the results were first discussed about a year ago at AACR, the big pre-clinical meeting going on right now, and were described as submitted at a conference I attended at MIT last June.  Indeed, the paper states "Received 11 June 2010; accepted 17 January 2011".  While there is a bit of functional investigation of one gene (siRNA vs. HOXA9) and some Western blots of coagulation factors, this is primarily a genomics paper.  Is Nature becoming reluctant to publish such papers?  What really held this up for so long?

In any case, the primary finding in both of these papers is low but measurable frequency mutation of protein kinases in human cancers.  This should come as no surprise, as a previous paper from the same groups in lung adenocarcinoma (the other major class of NSCLC), multiple kinases were found to be mutated beyond the relatively high frequency EGFR, again including BRAF but also a host of other kinases.  The new DDR2 paper also found mutations in multiple kinases, though any follow-up was focused on DDR2.  Another 5%-or-so slice of adenocarcinoma carries a fusion protein of the kinase ALK, which can be treated with inhibitors developed against ALK.  It also may be an opportunity to target ALK by a different strategy, one which my company has explored (yes, I have a financial interest there!).. The challenge is to determine which, if any, of these mutations are driving tumors and which are just passengers.

In the case of the DDR2 paper, the authors built a pretty nice story.  One big bonus to protein kinases is that there has been extensive efforts in the last 30 or so years to study them, with many inhibitors available.  A raging argument in the field is whether clinically useful inhibitors need to be exquisitely specific or can be as subtle as a wrecking ball, and the truth is that clinically approved kinase inhibitors run the gamut.  Imatinib (Gleevec)  is quite specific, though it still hits multiple kinases and that has proven useful as it has enabled targeting multiple cancers.  For example, some gastrointestinal stromal tumors are driven by c-KIT mutations and others by PDGFR mutations, but luckily imatinib hits both.  Other inhibitors such as sorafenib and suntinib  are less discriminating, but still tolerable.

In the case of DDR2, the approved inhibitor dasatinib turns out to be effective, and the new paper shows this first in cell lines.  Cell lines carrying DDR2 mutations are more sensitive to dasatinib than those which do not, but the trend continues both in mouse xenograft models and finally in a single human patient carrying a DDR2 mutation in her tumor.  Alas, the patient apparently had to discontinue therapy due to side effects.

Now that genomics has demonstrated the ability to find these low frequency mutations, the question is quite open as to how to move them into clinical practice.  One model would be to simply sequence extensively and treat each patient by the best guess for their mutations; this approach has been published and is apparently being used in the case of author Christopher Hitchens.  While whole genome or exome sequencing might be too costly or slow for routine use, targeted mutation panels are another possible approach (though honestly, exome sequencing is getting down in the $2.5K range these days).  Such targeted panels can attempt to focus on the most frequent and actionable mutations, though DDR2 in squamous cell carcinoma appears to not have any one mutation particularly favored.

The alternative is to try to run clinical trials to carefully appraise the clinical utility of these approaches.  When I mentioned the BRAF in myeloma story a while back to a co-worker (who happens to have developed multiple drugs, including an effective one in myeloma) and expressed the opinion that it is a slam-dunk to use a BRAF inhibitor (which is near approval in melanoma) in such cases, he took a more cautious view.  How do you know these are really the important mutations?  How will you know how long the treatment lasts?  Perhaps the BRAF mutations in myeloma help the tumor but are not critical.  How will you know the correct dose schedule?  Combination therapy?  Whether drug is getting to a very different tumor?  To truly answer these questions rigorously, trials are needed.

But the difficulty in running such trials cannot be underestimated.  For example, a company thinking of running a clinical trial looking at BRAF in squamous cell carcinoma faces quite a task.  Now, the market is not small: according to Wikipedia (an easy lookup late at night, though perhaps with large error bars) there are about 500K new cases yearly, and a quarter of those are squamous.  Presumably at least a quarter of those cases are in the U.S., so around 70K new cases per year.  Four percent of 70K (error bars growing with each estimate) is 2.8K patients, which is possibly attractive but getting small..

However, to get the trial going you are going to need to screen to find that 4% of patients.  Squamous is a standard diagnosis, so you can start there, but will still need to recruit, consent and screen to get that small fraction.  In the mean time, you are competing with every other trial out there to recruit, consent and screen patients.  Sure, once they miss another trial they might come to yours -- or might not.  To top it off, a lot of patients either are never offered or will never consent to a trial; the farther you are from a large academic cancer center, the less likely you will have a trial available to you.

Now, if oncogenic mutation screening becomes a standard part of cancer care, as it has at MGH and probably some other leading institutions, then if these mutations are in the panels it may be that many patients will know their mutation status before you recruit them into your trial.  But until this becomes widespread, and only if your gene of interest is sufficiently covered, will this method work.

Yet another approach is to design trials which test multiple therapies.  One prominent example in lung cancer is the BATTLE trial, which is trying 4 different therapies with an adaptive design which uses molecular testing as part of the therapy-assignment scheme.   Designs such as BATTLE are quite complicated (well beyond my expertise to critique) and get only more so with more drug regimens; if lung cancer is driven by a dozen or so kinases suggesting a slightly smaller number of therapies, can a trial to test these therapies be designed, patients accrued and useful results out?  In such studies, will they be judged by whether the study overall improves survival, or can each treatment be viewed as a separate study?  

For the sake of patients, these issues need to be tackled.  They'll be hard in lung cancer, and far worse in a disease like myeloma.  If we ballpark myeloma at 15K new cases per year in the U.S., 4% of that is getting to be a small group (600 patients).  Any sizable trial is going to need to recruit a huge fraction of these patients.  Now, with patient advocacy groups and publicity it may be possible to find that small population, but it will certainly be challenging.  Indeed, the Multiple Myeloma Research Foundation (which sponsored the sequencing) is already talking about how to support such efforts.

So, in closing, these sequencing studies are suggesting very real therapeutic options for patients.  However, driving these findings to routine clinical use, even when drugs are available off-the-shelf for the kinases of interest, will continue to challenge all of the scientists working on translational oncology research.

What might a PGM2 look like?

The New England chapter of the Laboratory Robot Interest Group (NE-LRIG) had a nice meeting tonight over at Astra Zeneca's beautiful Waltham campus (woodsy borders, with a view of the nearby reservoir).  The meeting was sponsored by Ion Torrent & they gave one of the three talks.  All three talks were quite good, with my friend & former Millennium colleague Sunita Badola from Amgen leading off with 454 amplicon sequencing in oncology clinical trials, followed by Mark DePristo from the Broad Institute talking about the 1K genomes effort and finally Jason Affourtit from Ion Torrent (he oversees all their field applications scientists) about the Ion platform.  The Ion talk gave a bit of the standard overview, followed by some slides summarizing the talks at Marco Island.

Now, when I've run previous items on Ion Torrent they have garnered a lot of comments.  Some of these are positive, others (and some of my posts) not so much, with some of the commenters most charitably described as downright cynical.  I won't have any data of my own for at least a month (and a machine on my own is still no more than a great desire), but I must say that if Ion Torrent is all smoke and mirrors, as some comments have insinuated, they have an awful lot of good people in on the ruse.  A friend of mine who is a very experienced genomics lab head was raving about her new machine and I happened to talk to another site today and their first four runs have all come in with greater than 2X the number of reads in the specification and very good quality.  About the only thing I've heard that is less than raving is that the quality drops near the end of the reads in a way that the effective read length is sometimes more like 80-90 rather than 100.  Still, if you know this going in you can adapt to it.

A star before and after the talks was a PGM which was available for viewing, along with 314 and 316 chips being passed around (which I was sorely tempted to have disappear into my shirt pocket, but morals prevailed).  These, for example, brought home the fact which I hadn't appreciated before that the 316 has significantly more actual surface area than the 314 chip, though it fits in the same carrier.  The difference between the chips is quite visible in your hand.  The 316 would appear to essentially max out the form factor, so the 318 can't keep up this trend. [corrected 3/25 per Rick's catching the typo]

Something that was emphasized tonight that I hadn't appreciated before is that there are no pumps in the PGM.  Reagents are propelled by argon gas pressure, managed by valves which are themselves actuated by the argon (some electrical widgets ultimately control the valves).  Also, the case apparently encompasses a lot of empty space (the Ion folks were open about this, though the machine was not open to view the innards).  Presumably some of this was a conservative design leaving space for late additions (or perhaps the server), but some had to do with wanting a visually striking design.

Since the PGM instrument itself has little to do with performance of the instrument, there isn't a need to redesign it to address sequencing performance.  However, there might be other reasons.  While it is a relatively small benchtop instrument, space is often at a premium.  A group of us from Infinity visited an academic site nearby today, and their lab made ours look spacious -- every square inch of bench was crammed with machines.  

Furthermore, seeing the machine in person made it clear that in placing it, a lab must leave some space on the right side to access the wash and waste bottles along that side.  Hence, if you really wanted to cram a lab the effective footprint of the instrument is a bit larger.
So, to engage in rank speculation utterly uninformed by any hard facts, I might imagine that a focus for a PGM2 would be an even smaller footprint.  The four tubes in the front, which hold the nucleotides for sequencing, could be rearranged in a manner still artful (perhaps a diamond?) yet far more compact, enabling the side bottles to move to the front.  Perhaps the screen could move down below the instrument -- or become an off-machine accessory capable of driving multiple instruments.  Alternatively, perhaps the screen would be mounted behind the flowcell access hatch.  This hatch on top (dark grey) for placing the flowcell also seems larger than necessary.  So, if you really could combine all these, it could yield an instrument with an effective footprint about one half as wide or maybe better.

A question I didn't think to ask tonight is how sensitive is the instrument to the flowcell being level.  I'm guessing (but certainly not a confident guess) that such devices mostly don't care; at these scales gravity isn't a dominant force.  In that case, the hatch might be turned to open outwards, enabling a redesign to improve the ability to stack the instruments vertically.

Another obvious dimension would be a multi-flowcell instrument ala HiSeq 2000.  Could most of the mechanical simplicity of the system be retained while enabling multiple flowcells to be run in parallel?  That would be the key question.

Of course, the key driver for many of these would be if groups wanted multiple machines to drive very high throughput.  I think there will be a market for this, but it is premature to think it has developed.  And, it will be critical for such operations to have the promised emulsion PCR improvements (or replacement) which is promised.