A team of researchers has developed a simple and rapid method for preparing Avian Influenza samples of infected individuals for sequencing with the Genome Sequencer FLX system from Roche.
The highly pathogenic avian influenza A virus (HPAIV) of subtype H5N1 has caused global concern as a potential pandemic threat, killing millions of poultry and fatally crossing over to humans in a number of documented cases.
The risk of sequence mutations that cause an increase in pathogenicity, meaning an escalation in the health threat of the virus, demands fast and reliable methods for in-depth full-length sequence analysis to prevent spread of the disease.
Researchers at the Institute of Diagnostic Virology, Friedrich-Loeffler-Institute Insel Riems in Germany designed a simple and sensitive method for the preparation of sequencing libraries from HPAIV H5N1 RNA samples for sequencing with the Genome Sequencer FLX instrument.
The presented method integrates high-throughput 454 sequencing into analysis without necessitating additional equipment or molecular biological techniques besides standard PCR and the Genome Sequencer FLX sample preparation and sequencing pipeline.
According to the authors, their approach is the first published method enabling sequencing of complete HPAIV H5N1 genomes directly from individual human samples using the Genome Sequencer FLX.
Massively parallel sequencing of shotgun libraries with the FLX system with standard series reagents generated up to 500Mb of raw sequence data in a single instrument run.
The depth of coverage provided by the sequencing data enabled representation of every nucleotide in multiple independent reads.
'This method surpasses the single reads of diagnostic-relevant sites of the hemagglutinin gene of avian influenza as generated in former studies,' said Dirk Hoper.
'Our method allows for sequencing of complete HPAIV H5N1 genomes from routine samples, neither requiring previous virus propagation in eggs or cell culture nor cloning and amplification of cDNA in vectors.
'Our approach permits sequencing of up to eight complete viral genomes within three days at tremendous depth and reliability.
'The procedure has the potential to seamlessly integrate into the normal diagnostic routine,' he added.
The described procedure can be adapted to different HPAIV subtypes, allowing sequencing of full-length genomes immediately after identification of the subtype by routine diagnostics.
