Microbial and Viral Ecology Analysis for Metagenomic Data

The explosion in known microbial diversity in the last two decades has made it abundantly clear that microbes in the environment do not exist in isolation; they are members of communities. Accordingly, omics approaches such as metagenomics have revealed that interactions between diverse groups of community members such as archaea, bacteria, and viruses (bacteriophage) are common and have significant impacts on entire microbiomes. Thus, to have a well-developed understanding of microbes as they naturally exist in the environment, biological entities of all kinds must be studied together. While numerous protocols for metagenome analysis exist, comprehensive published protocols for the simultaneous analysis of viruses and prokaryotes together are scarce. Further, as bioinformatic methods for microbiology rapidly advance, existing metagenomic tools and pipelines require frequent reevaluation. This ensures the adherence of best practices for microbiome and metagenomic data analysis. Here, we offer an expansive approach for the joint analysis of bulk sequence data from a mixed microbial community (metagenomes) and viral-sized fraction communities (viromes). This chapter serves as a beginner's-level guide for researchers with limited bioinformatics expertise who wish to engage in multi-scale metagenome and virome analyses. We cover steps from initial study design to sequence read processing, metagenome assembly, quality control, virus identification, microbial and viral genome binning, taxonomic characterization, species-level clustering, and host-virus predictions. We also provide the bioinformatic scripts used in our workflow for reuse in one's own computational methods. Lastly, we discuss additional approaches a researcher can take after processing data with this workflow.