Yesterday the Pacific Biosciences commercial instrument (photo) was at last unveiled to a packed room of conference attendees. The road to this third generation sequencer’s release has been paved with nearly $300 million of investment capital since leaving a basement at Cornell University. PacBio, in addition to becoming something of a media darling, has quietly swelled to a several-hundred-employee company.
Since last year, PacBio claims to have achieved read lengths of up to 10.3 kbp, although I haven’t spoken to anyone outside the company who has seen reads that long. Even so, a few vignettes presented in the workshop told of how PacBio has been applied to influenza strain identification and detection of stuctural variants (SVs).
Strobe Sequencing in Real Time
Of particular interest is the “strobe sequencing” mode of the instrument, in which the detection laser is turned off for precise amounts of time to generate mate-pair-like reads spanning large fragments. This feature relies on the real time sequencing, which occurs at a very consistent per-base rate. In fact, it’s possible to infer sequence insertions and deletions as spikes or dips (respectively) in the time required to sequence a template of known size.
Kinetic Variation Applications
The kinetics of real-time sequencing offer an informative new dimension of information from the PacBio data. In a talk today, Eric Schadt of PacBio showed that the kinetics of sequencing vary significantly for “modified” bases, i.e. methylated residues. In a collaboration with Carrie Harwood (UW), PacBio is sequencing the genomes and transcriptomes of 132 isolates of a hydrogen-producing species of Rhodopseudomonas. It turned out that kinetic variation exists at many bases as a “mixture” of sequencing times; by mining these, they identified thousands of methylated bases that caused up to 12-fold variation in sequencing kinetics.
Burning Questions Unanswered
Personally, I was not entirely satisfied with the PacBio workshop. When it opened for questions, I asked the first: whether PacBio had improved any upon the “dark bases” that go by undetected in single molecule sequencing. The presenter — Stephen Turner of PacBio — first gave me a nice 2-minute lecture on why there are no such thing as “dark bases” on PacBio’s sequencing platform due to its inherent awesomeness (sarcasm mine). There is still a problem with “missed bases” but Turner was almost comically evasive (as Daniel MacArthur put it) in stating how often they occur. The next question concerned read lengths, a second topic on which Turner refused to provide concrete information.
Thus, I find myself cautious in my excitement about this new platform, and will reserve judgment until later this year, when the first of the golden-ticket early access partners begin generating data on their own PacBio SMRT sequencers.