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

Newly-developed ''next-generation'' sequencing technologies, such as 454 (Roche), Solexa (Illumina), and SOLiD (ABI), allow researchers, in an unbiased manner, to obtain millions of sequences in a single round of operation [5] . Among these sequencing technologies, 454 currently offers by far the longest read length, ,250 bp on the Genome Sequencer (GS) FLX platform [6] . Sequencing error levels are low (,1%) and arise primarily from homopolymer runs [7] , but tend to be resolved in cases where there is sufficient coverage depth to allow the assembly of overlapping reads [8] . Many studies have used 454 pyrosequencing for the analysis of PCR amplicons, bacterial artificial chromosomes, genomic, mitochondrial, plastid DNA, and expression profiling [9, 10, 11, 12, 13, 14] . 454 is also a powerful tool for pathogen discovery [15] , and was used with the GS platform to identify a new arenavirus transmitted through solid-organ transplantation [16] and a new polyomavirus in samples of Merkel cell skin carcinoma [17] . The 454 sequencing technique was also used to implicate Israeli acute paralysis virus as a significant marker for colony collapse disorder in honey bees [18] . Another group reported the whole genome analysis of Gallid herpesvirus, and showed that .99.0% coverage was obtained by assembling the raw sequence data to an overall average coverage depth of 13 [19] .