A team of researchers at the Institute of Diagnostic Virology, Friedrich-Loeffler-Institute Insel Riems, Germany, has developed a simple and rapid method for preparing Avian Influenza samples of infected individuals for sequencing with the Genome Sequencer FLX system.
The highly pathogenic avian influenza A virus (HPAIV) of subtype H5N1 has caused global concern as a potential pandemic threat. The risk of sequence mutations, which cause an increase in pathogenicity (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.
Dirk Höper* et al. 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 seamlessly 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 GS FLX System with standard series reagents generated up to 500 Mb of raw sequence data in a single instrument run. The tremendous depth of coverage provided by the sequencing data enabled representation of every nucleotide in multiple independent reads.
“This method is simply not comparable with conventional sequencing and by far surpasses the single reads of ‘diagnostic’-relevant sites of the hemagglutinin gene of avian influenza as generated in former studies,” explains Höper. “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 8 complete viral genomes within only 3 days at an unprecedented depth and consequently, reliability of the sequence. The procedure has the potential to seamlessly integrate into the normal diagnostic routine.” The described procedure can be easily adapted to different HPAIV subtypes, allowing sequencing of full length genomes immediately after identification of the subtype by routine diagnostics.
“The high accuracy and quick run-time make 454 Sequencing ideal for medical applications. This study demonstrates the tremendous potential our technology has for future diagnostic applications in the field of virology,” says Chris McLeod, President and CEO of 454 Life Sciences.
* Höper D, Hoffmann B, Beer M. Simple, sensitive, and swift sequencing of complete avian influenza H5N1 genomes, J. Clin. Microbiol. 2009 47: 674-679.
Institute of Diagnostic Virology
454 Life Sciences