Viral detection by high-throughput sequencing

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1236; p. 125
• Main Authors: Motooka, Daisuke, Nakamura, Shota, Hagiwara, Katsuro, Nakaya, Takaaki
• Format: Journal Article
• Language: English
• Published: United States 01.01.2015
• Subjects: Animals; Bacteriophages - genetics; Bacteriophages - isolation & purification; Cattle; DNA - isolation & purification; DNA, Complementary; Feces - virology; Female; Gene Library; Genome, Viral; High-Throughput Nucleotide Sequencing - instrumentation; High-Throughput Nucleotide Sequencing - methods; Insect Viruses - genetics; Insect Viruses - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA - isolation & purification; Viruses - genetics; Viruses - isolation & purification
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-1743-3_11

We applied a high-throughput sequencing platform, Ion PGM, for viral detection in fecal samples from adult cows collected in Hokkaido, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.25 ml of fecal specimens (N = 8), and more than 5 μg of cDNA was synthesized. Unbiased high-throughput sequencing using the 318 v2 semiconductor chip of these eight samples yielded 57-580 K (average: 270 K, after data analysis) reads in a single run. As a result, viral genome sequences were detected in each specimen. In addition to bacteriophage, mammal- and insect-derived viruses, partial genome sequences of plant, algal, and protozoal viruses were detected. Thus, this metagenomic analysis of fecal specimens could be useful to comprehensively understand viral populations of the intestine and food sources in animals.