Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes

Abstract

Next-generation DNA sequencing (NGS) technologies have made generating genomic sequence for organisms of interest affordable and commonplace. However, NGS platforms and analysis software are generally tuned to be used on large and complex genomes or metagenomic samples. Determining the complete genome sequence of a single bacteriophage requires a somewhat different perspective, workflow, and sensitivity to the nature of phages. Because phage genomes consist of mostly coding regions (see Pope/Jacobs-Sera chapter), a very high standard should be adopted when completing these genomes so that the subsequent steps of annotation and analysis are not sabotaged by sequencing errors. While read quality and assembly algorithms have continued to improve, achieving this standard still requires a significant amount of human oversight and expertise. This chapter describes our workflow for sequencing, assembling, and finishing phage genomes to a high standard by the NGS platforms Illumina, Ion Torrent, and 454.