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

In the severe acute respiratory syndrome (SARS)-CoV outbreak in 2003, it was demonstrated that a combination of stool, pooled nasal, and throat swab specimens gave the highest yield for SARS-CoV detection by RT-PCR [32] . Thus, not only respiratory specimens but also gastric and digestive specimens are important for the diagnosis of emerging infectious viruses, including those with airborne transmission. In this study, we isolated whole RNA and detected viral genes from nasal and stool samples with the 454 high-throughput sequencing system. Flu sequences were present in 20-460 of the 21,858-30,958 reads in each nasopharyngeal aspirate (Table 1) , and the cover rates ranged from 8.1-58.3% (Table 2) , which was sufficient for subtype identification in all three specimens. Furthermore, the near-complete norovirus genome sequence was obtained in two fecal specimens (#N2 and #N3), and more than 75% of the genome was covered in the other two specimens (#N4 and #N5) ( Table 3) . Recently, we subjected stool sample-extracted DNAs to 454 pyrosequencing, and found that nearly 20% of the reads had best hits that matched currently-reported bacterial DNA sequences [20] . These previous results, together with our findings here, indicate that two protocols, namely direct DNA extraction for bacteria and cell/bacterial removal by centrifugation followed by RNA/DNA extraction for virus, could be used to comprehensively identify pathogenic microbes in clinical samples.