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

One-step real time RT-PCR (qRT-PCR) was also performed on the extracted RNA. The estimated copy number of norovirus in each fecal sample is shown in Figure 1C . The copy number of norovirus in sample #N1 with qRT-PCR was 3E+03, whereas those of other samples ranged from 1E+06 to 5E+07. The relative copy numbers of norovirus in samples #N1 to #N3, which were isolated in 2006 (October to December), were almost consistent with the semi-quantitative PCR results, although the sensitivity of the semi-quantitative PCR was 10-fold lower than that of qRT-PCR ( Figure 1C ). By contrast, the real-time RT-PCR and semiquantitative PCR were ,1,000-fold different in samples #N4 and #N5 ( Figure 1C ), which were isolated in May 2005 and January 2006, respectively. In these samples, RNA isolation for 454 pyrosequencing was performed in February 2007, whereas RNA for qRT-PCR was isolated just after sample collection. Thus, the above inconsistencies might be due to the storage periods of the samples (at 4uC).