Aside from the dubstep pumping out of the Roche and Agilent booths, the volume of AGBT has been somewhat muted.  There was no grand offering of new hardware or over the top promises of sequencing genomes on what now appear to be vaporware USB thumb drives.  This is my first in person experience of AGBT, so as a virgin it seems for the most part to be rooted in the science despite the ridiculous parties and “showgirl” casino nights.  The atmosphere here is unlike any other science conference I’ve attended.  It’s like the bastard child of a Gordon Conference and a Las Vegas Porn Convention.  I really hope that the deep pockets of Sequencing Centers are more influenced by the science than the free dinner parties and alcohol, but I have pretty low confidence in humanity.  Regardless, I think everyone in attendance today was overwhelmed by a stunning talk from PacBio and the dramatic advancements of their long read technology. 

The PacBio talk came on the heels of what felt like a warm-up opening act from Jeremy Schmutz of the Hudson Alpha Institute.  Schmutz has been working with a start-up that was recently acquired by illumina called Moleculo, which promises highly accurate long reads using short read sequencers.  I sat through two other talks, went to the Moleculo poster, and still do not have a clear idea of how the technology actually works. What I could deduce from the talks and the questions I asked at the poster session, Moleculo technology works by fragmenting DNA into 10 kilobase fragments.  These fragments are then diluted and clonally amplified in separate volumes.  How Moleculo does this clonal amplification of what have to be tens of thousands of small volume PCR reactions is beyond me.  When I asked this question at the poster session, Dmitry Pushkarev of Moleculo said that it was simple: they have a $4 piece of plastic about the size of a shoe box that does all of the hard work.  Right. So the first step involves magic.  Once the 10kb fragments are clonally amplified, they are again fragmented into even smaller pieces and Nextera tagged.  These Nextera tagged sequences are then sequenced on a HiSeq and the data output is sent back to Moleculo where they do the de-multiplexing and sequence assembly.  As before, when asked how the Moleculo assembler can accurately span long repeat regions greater than the 100bp short read output, Pushkarev offered, “We wrote the best assembler ever.”  Ok, I get it, you guys are going to be coy about how everything actually works.  From an early business perspective, I guess that’s a great idea, but some of us like to know the details of how techniques work so that we can do our own validation and be positive that the data we’re getting out is representative of what has actually been sequenced.  The sample to black box to data pipeline is not ideal to me, but others, like Schmutz, seem to be ok with this for now.  Hopefully, as the Moleculo technology matures, we get more information from them about how the technique actually works. 

Fortunately, Schmutz was at least able to show us some of his downstream data using Moleculo and also how it compared to other long and longer read sequencers such as Roche 454 and PacBio.  Schmutz works mostly with plants, and plants are tricky organisms to sequence because they’re full of repetitive information that cannot be accurately sequenced using the current short read technologies.  Schmutz showed that Moleculo reads appear to be highly accurate with an error rate of 1.2bp per 10kb of sequence; however, the Moleculo reads created a similar number of sequence gaps as shorter 800bp 454 reads and these regions had to be fixed with secondary MiSeq runs.  In contrast, Schmutz showed that PacBio RS reads provided the least “gappy” data which is important from a cost perspective.  It seemed like Schmutz final conclusion was that he liked the Moleculo technology the best because they did all of the hard work: they made the libraries and performed the read assembly without having to train staff on new techniques or machines.

After Schmutz had thoroughly fluffed the crowd over long read sequencing, Jonas Korlach from Pacific Biosciences took the stage to give a talk on the progress of assembling genomes using their long read sequencing technology.  If you’ve been in the sequencing game for any amount of time you’ve definitely heard of PacBio and their early promise of accurate, realtime, single molecule, long read sequencing technology and their subsequent absolute failure in rapidly delivering those promises.  PacBio has spent the last 2 years trying to dig itself out of a hole by aggressively working with early adopters to fix their original lapses in quality.  In an earlier talk in the week (Slides!), Michael Schatz, another plant geneticist and PacBio early adopter declared, “You don’t have to worry about the errors anymore.”  This is in part because of major improvements in PacBio chemistry and the introduction of a new more accurate polymerase.  PacBio has consistently doubled its average read length over the last two years and has made gains on the error correction front.  Last year, Schatz published a paper in Nature Biotechnology which showed that highly accurate illumina or 454 short reads could be used to error correct PacBio long reads to generate the most accurate long read data available today.  Korlach followed up on this short read correction scheme by showing that the illumina/454 step can now be eliminated completely and researchers can use the short reads generated during the PacBio run to error correct the much longer reads in a process called Hierarchical Genome Assembly (HGAP), with a base accuracy of 99.99965%.  That’s a far cry from the early real world production numbers of 85%!  Korlach then supported these tech numbers with an astounding amount of real world data.  Granted, all of the de novo sequencing presented was done in pathogenic organisms or bacteria with small circular genomes, but no one can dispute how impressive the data looked during the presentation. 

The big question now is: has PacBio finally weathered the storm and can it overcome its previous reputation as a failed sequencing company?  Time will really only tell.  It’s hard to predict winners and losers in this space, especially in light of the coming sequestration and dwindling research budgets.  PacBio may be peaking at the wrong time.  With the threat of Moleculo long read technology on the horizon, major sequencing labs may hold out on purchasing PacBio RS systems.  Why invest $700,000 in a sequencer if you can get “good enough” long reads off of your HiSeqs?