Application of the New Generation of Sequencing Technologies for Evaluation of Genetic Consistency of Influenza A Vaccine Viruses

Abstract

For almost half a century, Sanger sequencing has been the conventional method for sequencing DNA. However, its utility for sequencing heterogeneous viral populations is limited because it can only detect mutations that are present in a significant portion of the DNA molecules. Several molecular methods that quantify mutations present at low levels in viral populations were proposed for evaluation of genetic consistency of viral vaccines; however, these methods are only suitable for single site polymorphisms, and cannot be used to screen for unknown mutations. Next-generation (deep) sequencing methods have enabled the determination of sequences of the entire viral population, including minority components. They enable not only sequencing, but also accurate quantification of mutations. This technique has great value for monitoring the genetic consistency of viral vaccines. Recently, a number of new deep sequencing platforms were introduced (MiSeq, Iron Torrent, etc.) that made such an analysis quite affordable for individual research labs. Here, we review the use of current deep sequencing approaches for influenza virus studies, focusing on the evaluation of the genetic consistency of influenza A vaccine viruses. We also describe a new bioinformatic tool to analyze deep sequencing data and identify artifacts from the true mutants.