VIROME: a standard operating procedure for analysis of viral metagenome sequences

One consistent finding among studies using shotgun metagenomics to analyze whole viral communities is that most viral sequences show no significant homology to known sequences. Thus, bioinformatic analyses based on sequence collections such as GenBank nr, which are largely comprised of sequences from known organisms, tend to ignore a majority of sequences within most shotgun viral metagenome libraries. Here we describe a bioinformatic pipeline, the Viral Informatics Resource for Metagenome Exploration (VIROME), that emphasizes the classification of viral metagenome sequences (predicted open-reading frames) based on homology search results against both known and environmental sequences. Functional and taxonomic information is derived from five annotated sequence databases which are linked to the UniRef 100 database. Environmental classifications are obtained from hits against a custom database, MetaGenomes On-Line, which contains 49 million predicted environmental peptides. Each predicted viral metagenomic ORF run through the VIROME pipeline is placed into one of seven ORF classes, thus, every sequence receives a meaningful annotation. Additionally, the pipeline includes quality control measures to remove contaminating and poor quality sequence and assesses the potential amount of cellular DNA contamination in a viral metagenome library by screening for rRNA genes. Access to the VIROME pipeline and analysis results are provided through a web-application interface that is dynamically linked to a relational back-end database. The VIROME web-application interface is designed to allow users flexibility in retrieving sequences (reads, ORFs, predicted peptides) and search results for focused secondary analyses.