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

Another possible problem with this viral genome analysis is biased cDNA synthesis by quasi-random RT-PCR with the WTA kit. As shown in Figure S1 , a significant bias was found and its pattern was identical in all samples. TG (CA)-rich regions were selectively amplified with the WTA kit (Table S3) , probably due to nucleotide sequences of the quasi-random primer. Random RT-PCR amplification using the WTA kit was at least one log higher than that using the conventional random hexamer (data not shown). This suggests that further improvement is required for whole viral genome analysis, although our system is suitable for the comprehensive detection of viral genes. In addition, the TG (CA)rich bias was observed within the viral genome; therefore, it seems unlikely that the bias leads to quantitative differences of the detected sequences with respect to the original population. Almost all diagnostic NATs require viral genome information, and thus cannot be performed for novel or unexpected viral infections. In this study, we showed that a diagnostic system based on parallel high-throughput sequencing is useful for the direct detection of unknown and/or small numbers of viruses, as well as for the genetic characterization of major pathogenic viruses in clinical specimens. We plan to share this system domestically as well as with the Asian epidemic network (The Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases; http:// www.crnid.riken.jp), in order to enable the earlier identification of unknown pathogens in a novel outbreak or bioterrorism.