The construction and analysis of marker gene libraries

Marker genes for viruses are typically amplified from aquatic samples to determine whether specific viruses are present in the sample, or to examine the diversity of a group of related viruses. In this chapter, we will provide an overview of common methods used to amplify, clone, sequence, and analyze virus marker genes, and will focus our discussion on viruses infecting algae, bacteria, and heterotrophic flagellates. Within this chapter, we endeavor to highlight critical aspects and components of these methods. To this end, instead of providing a detailed experimental protocol for each of the steps involved in examining virus marker gene libraries, we have provided a few key considerations, recommendations, and options for each step. We conclude this chapter with a brief discussion of research on a major capsid protein (g20) of cyanomyoviruses using this work as a case study for polymerase chain reaction primer design and development. By building on the experience of numerous labs, this chapter should not only be useful to the new virus ecologist, but also serve as a valuable resource to established research groups.