Flow Cytometry and Direct Sequencing of Viruses.

ABSTRACT

In order to describe a novel uncultured viral species, it is essential to obtain the DNA sequence of their full genomes. The ability to distinguish the viral genome from the genome of its bacterial host is the major challenge of the modern viromics. The major obstacles for mining of viral genomes in metagenomic assemblies is bacterial contamination in viromes and low DNA input for sequencing.These obstacles can be overcome by flow cytometry that allows collecting free viral particles from environmental samples. In addition, fluorescence activated cell sorting reduces the bacterial contamination. By using optimized sequencing protocols, the ultra-low input DNA samples can be sequenced directly, without the need for whole genome amplification. This chapter provides details for staining of environmental viruses, flow cytometry, and direct sequencing of ultra-low input DNA samples on Illumina platform.