Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

Nakamura, Shota; Yang, Cheng-Song; Sakon, Naomi; Ueda, Mayo; Tougan, Takahiro; Yamashita, Akifumi; Goto, Naohisa; Takahashi, Kazuo; Yasunaga, Teruo; Ikuta, Kazuyoshi; Mizutani, Tetsuya; Okamoto, Yoshiko; Tagami, Michihira; Morita, Ryoji; Maeda, Norihiro; Kawai, Jun; Hayashizaki, Yoshihide; Nagai, Yoshiyuki; Horii, Toshihiro; Iida, Tetsuya; Nakaya, Takaaki

We previously demonstrated the direct detection of a bacterial pathogen from a patient sample using 454 high-throughput DNA sequencing [20] . Here we report the design and diagnostic validation of an unbiased high-throughput sequencing method for the direct diagnosis of viral infections in clinical specimens. Patient samples were obtained during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. cDNAs, as templates for the GS FLX platform, were prepared by random RT-PCR using RNAs extracted from clinical samples. High-throughput sequencing yielded 15,298-32,335 sequences, of which 7-15,260 represented the targeted viral sequences. Furthermore, sequences of two recently identified human viruses, WU polyomavirus (WUV) and human coronavirus (HCoV) HKU1, were detected from the nasal and fecal samples, respectively.