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

The amplified cDNA was used as a template for GS FLX analysis (454 Life Sciences). A 70675 PicoTiterPlate device (gasket for 16 regions) was divided into 2 regions for each of 8 samples. The obtained data were then subjected to a data analysis pipeline. Data analysis was performed on each read sequence by computational tools, as constructed previously [20] with some modifications. The analysis steps were: (i) remove tag sequences; (ii) execute a BLASTN search by Hi-per BLAST (Fujitsu); (iii) identify the scientific name for each read based on the NCBI taxonomy database; (iv) extract viral reads and perform mapping to reference data by SSEARCH. This analysis pipeline was constructed by utilizing BioRuby [39] , BioPerl [40] , and MySQL. After classification, particular human and bacterial reads were further analyzed as follows. Human genome mapping was performed by MEGABLAST search against the Human Genome, Homo_sapiens.NCBI36.49, using a threshold of 1E-40. Bacterial rRNA typing was performed by BLASTN search against the comprehensive rRNA database ''silva'' release 94 [41] using a threshold of 80% match per read.