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Driven by the impact of influenza A viruses on human and animal health, much research is conducted on this pathogen. To support this research, we designed an all influenza A-embracing reverse transcription-PCR (RT-PCR) for the generation of DNA from influenza A virus negative strand RNA genome segments for full-length genome deep sequencing on a Genome Sequencer FLX instrument. For high reliability, the RT-PCRs are designed such that every genome segment is divided into two amplicons and for the most variable segments redundancy is included. Moreover, to minimize the risk of contamination of diagnostic real-time PCRs by sequencing amplicons, RT-PCR does not generate amplicons that are amenable to RT-qPCR detection. With the presented protocol we were able to generate virtually all amplicons (99.3% success rate) from isolates representing all so far known 16 hemagglutinin and 9 neuraminidase subtypes and from an additional 2009 pandemic influenza A H1N1 virus. Three isolates were sequenced to analyze the suitability of the DNA for sequencing. Moreover, we provide a short R script that disambiguates the sequences of the primers used. We show that using unambiguous primer sequences for read trimming prior to assembly with the genome sequencer assembler software results in higher quality of the final genome sequences. Using the disambiguated primer sequences, high quality full-length sequences for the three isolates used for sequencing trials could be established from the raw data in  de novo  assemblies.

A Comprehensive Deep Sequencing Strategy for Full-Length Genomes of Influenza A