By Julia Karow
As Pacific Biosciences is shipping its first sequencing instruments to beta customers — as of earlier this month, it had installed them at eight sites — the company has been testing its real-time single-molecule technology for a variety of applications, both in DNA sequencing and other areas.
At a company seminar during the American Society of Human Genetics annual meeting earlier this month, PacBio scientists presented projects focusing on pathogen detection, bacterial genome assembly, RNA modification, splicing, and translation.
Sample prep and sequencing on the PacBio platform can be completed in a day, which has advantages for applications such as rapid pathogen detection, according to PacBio chief scientific officer Eric Schadt.
In a collaboration with Gen-Probe, which struck a partnership with PacBio and invested $50 million in the company this summer (IS 6/22/2010), PacBio scientists recently tested how well their system can distinguish between subtypes of hepatitis C virus, of which more than 70 are known. Minority populations of one subtype often become drug resistant, so it is important to be able to detect them. In a pilot study, the researchers mixed two subtypes provided by Gen-Probe at a ratio of 1:100 and found that the technology was able to detect the relative abundance of both forms. A single read covered the haplotype containing the variation that distinguishes the two subtypes. They were also able to detect unknown variants of the virus in two clinical samples.
In order to assess the technology for disease surveillance applications, PacBio scientists also constructed "disease weather maps," where they looked for human pathogenic viruses in different locations and how their distribution changes over time.
In a collaboration with Joe DeRisi at the University of California, San Francisco, for example, they analyzed viruses in samples taken from two sewage substations in San Francisco. In addition to human respiratory viruses, they picked up a variety of plant viruses that allowed them to draw conclusions about the diet of residents in the area. The project also used short reads generated by Illumina's sequencing technology, which could not be mapped on their own but yielded hits to database entries when they were combined with PacBio's long reads, Schadt reported.
In another project, the company analyzed samples from high-traffic areas in its own buildings, for example door handles, as well as nasal swabs from employee volunteers, at different time points, looking for viruses "of public health concern." They were able to track different strains of influenza virus in individuals, and detected varoius viruses on the commonly used areas. The hope is that in the future, the appearance of viruses can be connected to disease outbreaks in order to take appropriate public health measures.
PacBio also believes its technology can help with the de novo assembly of genomes that cannot be solved using short-read technologies alone. For example, its long reads — currently averaging 1,000 to 1,200 bases, with some reaching more than 10 kilobases — and strobed reads have helped to order contigs from the genome of the hydrogen-producing bacterium Rhodopseudomonas palustris, resulting in a single contig.
The company has shown previously that chemical modifications of DNA bases, such as DNA methylation, slow down the polymerase, and that these kinetic changes allow it to track epigenetic modifications while sequencing DNA. Company researchers have now applied this concept to the R. palustris genome and have found that genomic regions in which they observed kinetic variation correlated with regions of gene activity, Schadt reported.
Looking beyond DNA sequencing, the company has also been exploring new applications for its technology. For example, company co-founder Jonas Korlach showed that he and his colleagues have been able to sequence synthetic RNA using reverse transcriptase instead of DNA polymerase. There is still some development work ahead, he said, because each nucleotide incorporation currently requires more than one binding event.
PacBio's technology could also be used to analyze RNA modifications, such as RNA methylation. These are known to exist but their function is almost completely unknown because there is currently no technology to study them, Korlach said. In a collaboration with Tao Pan from the University of Chicago, PacBio scientists have already attempted to study modified RNA and have found that like with DNA, the modifications slow down the enzyme. Based on these results, Korlach said he believes the technology will be able to analyze RNA methylation.
PacBio has also started using its technology to study the activity of macromolecular complexes, like the ribosome and the spliceosome. Earlier this year, in collaboration with Jody Puglisi at Stanford University, PacBio researchers published a study on the transit of tRNA on ribosomes. According to Korlach, they have now improved the assay by increasing the number of tRNA labels from three to four. Also, they can now label both tRNAs and ribosomal proteins, allowing them to visualize macromolecular dynamics for the first time. They are interested, for example, in studying the effect of antibiotic drugs, such as aminoglycosides, on ribosome dynamics.
In another example of investigating the mechanisms of drug action, they have studied, in a collaboration with Ken Johnson at the University of Texas, the kinetics of HIV drugs binding to HIV reverse transcriptase.
Recently, PacBio scientists have also studied the spliceosome, which consist of five RNAs and more than 150 proteins, Korlach said. In a collaboration with David Rueda at Wayne State University, for example, they tethered pre-mRNA in the wells of their zero-mode waveguide chip, added cell extracts from actively splicing cells, and recorded signals that indicated splicing activity. They are now planning to study different steps of the process.
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