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

To remove bacteria and human cells present in the feces, 15,000 rpm centrifugation was performed and the supernatants were used for RNA isolation. The norovirus sequence was detected from all five samples in 15,298-32,335 (average 23,994) reads, as summarized in Table 3 . In contrast with influenza virus, almost the whole genome was covered in #N2 (7,302 reads) and #N3 (15,260 reads) samples, with average cover depths of 141.5 and 258.7, respectively (Table 3 ). More than 75% of the genome was covered in #N4 (484 reads) and #N5 (611 reads) samples (Table 3) . A BLAST search of each sequence strongly indicated that these four patients were infected with a similar genotype, GII.4 (Table S2) , consistent with previous diagnostic results [26] . In contrast, only 7 reads were detected in sample #N1 (Table 3) , which was under-detectable with single round of PCR ( Figure 1C ), suggesting that the diagnostic method using high-throughput pyrosequencing is more sensitive than conventional PCR analysis.