Rapid genome sequencing and characterization of novel avian-origin influenza A H7N9 virus directly from clinical sample by semiconductor sequencing

Background Recent outbreaks of severe pneumonia or acute respiratory distress syndrome have attracted much public interest. Rapid and accurate diagnosis of the causative agent is key for an adequate response to suspected outbreaks. Objectives We report a case that highlights the potential of semiconductor sequencing to rapidly determine the novel virus genome sequences. Study design We have developed a method for rapid de novo assembly of the novel influenza A H7N9 virus genome directly from the tracheal aspirate of a patient using semiconductor sequencer without culture and prior sequence information. Further, characteristic amino acids were analyzed and phylogenetic analysis were done for key genes of the influenza A virus. Results Deep sequencing yielded 435,239 reads assigned to H7N9 viruses, with an average length of 172 bp, accounting for 18.6% of total reads (2,339,680). Complete genome of the virus was obtained by de novo assembly method within 2 days. Genomic average depth of coverage of the Ion Torrent PGM was up to 5679 fold. Selected characteristic amino acids were observed, and phylogenetic analyses showed that the novel H7 virus was genetically close to 2011 duck H7N3 viruses in Zhejiang. The novel N9 sequences were most closely related to gene sequences of N9 derived from ducks H11N9 in 2011 in Jiangxi and H2N9 sequences from Hong Kong in 2010, in China, and therefore they may share a common ancestor. Conclusions The sequence-independent semiconductor sequencing is a powerful tool to investigate outbreak of a novel pathogen.