The H1N1 influenza A virus that emerged in 2009 spread rapidly around the world, infecting millions and killing hundreds. Although this epidemic was not as lethal as some had predicted, the possibility remains that highly virulent and drug-resistant new variants of this virus will emerge.
Viral genomes can be analyzed using standard dye termination DNA sequencing reactions, but although accurate, these methods are not practical for studying virus outbreaks because they are slow, costly and labor-intensive. They can also produce ambiguous results and false-positive base pairings. This is problematic when sequencing new virus variants that contain DNA mutations that differ from known sequences.
Now, Wing-Kin Sung and co-workers from the A*STAR Genome Institute of Singapore have developed a new kit capable of analyzing the H1N1 genome and accompanying software called EvolSTAR.
The kit reduces the ambiguities inherent in results obtained by conventional DNA sequencing, because EvolSTAR uses novel algorithms to automatically identify and re-analyze ambiguous results. This two-step process makes the kit highly sensitive to mutations in the DNA sample, enabling the identification of new mutations with a high degree of confidence.
The kit assesses other critical regions of the virus’s genome, such as drug-binding sites and regions that encode structural components of the virus particles. It is also quick and cheap: 24 samples can be sequenced in just 30 hours, at just one-tenth the cost of the commonly used 454 sequencing method.
The researchers field-tested the kit by analyzing the H1N1 genome in patient samples. They used an advanced DNA microarray chip containing approximately 120,000 short DNA ‘probes’, which covered more than 99% of the virus genome twice and with an accuracy of 99.99%. The probes also provided extensive, repeated coverage of 36 mutation ‘hotspots’ and ten drug-binding sites within the virus genome.
“For standard sequencing techniques, the researcher needs to do a lot of work purifying H1N1 from the sample prior to the sequencing,” says Sung. “With our technique, we can directly sequence the virus in human samples, cost-effectively and rapidly. This is a good tool for detecting early virus variants in an outbreak, and potentially preventing the spread of the virus.”
Sung and his co-workers are now developing a new version of the microarray chip that can detect multiple viruses in patient samples. They are also working on a ’pan virus’ chip that can rapidly sequence any new strain of flu virus.
The A*STAR-affiliated researchers mentioned in this highlight are from the Genome Institute of Singapore.