Influenza genome analysis using pyrosequencing method: current applications for a moving target

Abstract

Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.