title: Deep sequencing approach for genetic stability evaluation of influenza A viruses.
abstract: Assessment of genetic stability of viruses could be used to monitor manufacturing process of both live and inactivated viral vaccines. Until recently such studies were limited by the difficulty of detecting and quantifying mutations in heterogeneous viral populations. High-throughput sequencing technologies (deep sequencing) can generate massive amounts of genetic information and could be used to reveal and quantify mutations. Comparison of different approaches for deep sequencing of the complete influenza A genome was performed to determine the best way to detect and quantify mutants in attenuated influenza reassortant strain A/Brisbane/59/2007 (H1N1) and its passages in different cell substrates. Full-length amplicons of influenza A virus segments as well as multiple overlapping amplicons covering the entire viral genome were subjected to several ways of DNA library preparation followed by deep sequencing using Solexa (Illumina) and pyrosequencing (454 Life Science) technologies. Sequencing coverage (the number of times each nucleotide was determined) of mutational profiles generated after 454-pyrosequencing of individually synthesized overlapping amplicons were relatively low and insufficiently uniform. Amplification of the entire genome of influenza virus followed by its enzymatic fragmentation, library construction, and Illumina sequencing resulted in high and uniform sequencing coverage enabling sensitive quantitation of mutations. A new bioinformatic procedure was developed to improve the post-alignment quality control for deep-sequencing data analysis.
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA.
authors.forename: Bella
authors.initials: B
authors.lastname: Bidzhieva
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Zagorodnyaya
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Karagiannis
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA.
authors.forename: Vahan
authors.initials: V
authors.lastname: Simonyan
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA. Electronic address: majid.laassri@fda.hhs.gov.
authors.forename: Majid
authors.initials: M
authors.lastname: Laassri
authors.affiliation: Center for Biologics Evaluation and Research, US Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville 20852, MD, USA.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Chumakov
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D004285:Dogs; D042822:Genomic Instability; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000254:growth & development; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation
keywords: Illumina Solexa sequencing; Profiles of mutations; Pyrosequencing; Sequence heterogeneity; Sequencing libraries preparation