The next-generation sequencing technology that was harnessed to assemble the entire sequence of James Watson’s genome has been put to a new and potentially life-saving use: identifying novel pathogens. After several other identification techniques failed, the new sequencing approach was used to discover a never-before-seen virus that was likely responsible for the deaths of three transplant patients who received organs from the same donor.
The technique, called unbiased high-throughput pyrosequencing, or 454 sequencing, was developed by 454 Life Sciences, owned by Roche. This is the first time it was used to probe for the cause of an infectious-disease outbreak in humans, and experts say that it could ultimately usher in a new era in discovering and testing for agents of infectious disease.
“This is going to begin to allow us to understand the etiology of infections that had previously gone undiagnosed,” says Richard Whitley, professor of medicine at the University of Alabama at Birmingham, who was not involved with the research.
Last spring, several weeks after receiving organs from a single donor, three Australian transplant patients became ill with fever and encephalitis; within six weeks of the operation, all three had died. When traditional methods failed to identify the cause of the patients’ deaths, the Victorian Infectious Disease Reference Laboratory turned to W. Ian Lipkin, director of the Laboratory for Immunopathogenesis and Infectious Diseases at Columbia University’s Mailman School of Public Health, for assistance.
To find the mystery pathogen responsible for the deaths, Lipkin’s team extracted RNA from the tissues of two of the patients and prepared the sample by treating it with an enzyme that removed all traces of human DNA; this enriched the sample for viral sequences. The researchers then amplified the RNA into millions of copies of the corresponding DNA using a reverse transcriptase polymerase chain reaction (PCR). Usually, PCR requires some advance knowledge of the sequence in question because it relies on molecular primers that match the string of code to be amplified. But 454 sequencing avoids that problem by using a large number of random primers.
The resulting strands of DNA were sequenced using pyrosequencing, which determines the sequence of a piece of DNA by adding new complementary nucleotides one by one in a reaction that gives off a burst of light. Pyrosequencing allows for fast, simultaneous analysis of hundreds of thousands of DNA fragments. Although traditional pyrosequencing generally produces relatively short chunks of sequence compared with earlier sequencing techniques, 454 Life Sciences has improved upon the technology such that longer reads are possible.
When 454 Life Sciences used this technique to sequence James Watson’s genome, its approach was nearly identical. Lipkin’s modification was to eliminate human DNA so that only the mystery pathogen’s genetic material would remain.