By Julia Karow
This article was originally published Feb. 27
MARCO ISLAND, Fla. — Pacific Biosciences presented its commercial sequencing instrument, the Pacbio RS, publicly for the first time last week, along with a 10-kilobase read generated by its technology and a number of applications, ranging from influenza virus sequencing to structural variation analysis in humans and mutation detection in cancer.
The company also provided an outlook of how the system is expected to improve over the next few years.
At a company presentation during the Advances in Genome Biology and Technology meeting here, PacBio CTO Steve Turner showed a video of how the new instrument is operated. The 1,800-pound machine, about the size of an airport X-ray machine with a dishwasher-sized blade server by its side, was also on display to potential customers in a suite at the conference hotel.
"Yes, it is big, yes, it is heavy, but yes, it does work," said CEO Hugh Martin during the presentation.
The machine, which has a large touchscreen on its front, has two drawers: one holds an array of SMRT cells, each with 80,000 zero-mode waveguides, which are loaded in up to 12 rows of eight, as well as pipette tips; the other holds the sequencing samples and consumables. Once both drawers are loaded, an automated fluid handling system picks up a single SMRT cell, fills it with sample and reagents, and transfers it to the camera area, where four liquid-cooled cameras start collecting signals from labeled nucleotides as they get incorporated into DNA in real time at a speed of 1 to 3 bases per second. The adjacent compute cluster processes these signals in real time.
In collaboration with a number of scientific collaborators, PacBio has tested the technology in a variety of projects, using its prototype instruments, which differ from the commercial version mainly in the number of ZMWs – 3,000.
An influenza virus sequencing project, for example, in collaboration with the J. Craig Venter Institute and others, demonstrated that the time to conduct an experiment on the platform — about nine hours, including both sample prep and sequencing — is compatible with clinical requirements for obtaining fast results.
By sequencing transcripts from a well-studied breast cancer cell line, MCF-7, company scientists showed that reads on the order of several thousand base pairs were able to span entire transcripts and pick up several alternative splice forms.
The system is also capable of producing very long reads, although these represent only a small percentage of the overall reads: for example, Turner showed a 10,351-base E. coli read that incorporates nine elements with regulatory of structural functions. Customers will be able to produce reads of that size, too, he said, adding that PacBio has seen reads as long as 20 kilobases in house.
Eventually, he said, the company expects to increase the read length to 50 kilobases. As a proof of principle that a DNA fragment of that size would actually be viable in the ZMW, company scientists prepared intact DNA from the 48.5-kilobase lambda phage genome and sequenced it on both ends. The majority of fragments, Turner said, remained intact in the ZMWs.
In an example of "strobe sequencing," where the instrument generates several reads on the same DNA molecule that are separated by "dark" stretches, PacBio analyzed a human fosmid with well-known structural variants — a collaboration with Evan Eichler at the University of Washington. The company has generated "strobed reads" spanning as much as 20,000 bases, putting it "in a very nice position for de novo human genome assembly," Turner said.
The technology has also helped with the assembly of difficult-to-sequence organisms, for example Rhodopseudomonas palustris, which is of interest to researchers for fuel production. Sanger sequencing had only been able to produce a genome with 58 contigs, but a hybrid assembly with PacBio reads led to a single contig.
The company readily admits that its technology suffers from poor single-read accuracy, similar to other single-molecule sequencing approaches, but it offers circular consensus sequencing as a remedy, where the same molecule is sequenced several times over. Individual molecules can reach quality values of Q40, Turner said. In a separate conference talk, Elaine Mardis from the Genome Center at Washington University showed how this method can be applied to detect mutations in regions of tumor genomes.
At launch, PacBio plans to support three applications: standard sequencing, strobe sequencing, and circular consensus sequencing. Methylation sequencing will follow shortly, and the company is already working on additional applications, such as direct RNA sequencing and ribosome translation analysis.
Turner showed that based on changes in the enzyme kinetics, the technology is able to identify not only methylated cytosine and adenosine but also hydroxylmethyl cytosine and a number of other modified bases.
Over time, PacBio expects that the throughput of its instrument will increase severalfold: the number of ZMW wells occupied by a single polymerase will triple from 30 percent to about 90 percent through "molecular nanotechnology techniques," and the number of ZMWs per SMRT cell will double, to 160,000. In addition, the company plans to use faster polymerases that generate DNA at up to 15 bases per second.
In 2014, the firm plans to come out with a second-generation instrument that will multiplex more than a million ZMWs (see In Sequence 2/23/2010).