Abstract
Abundant bioinformatics resources are available for the study of complex microbial metagenomes, however their utility in viral metagenomics is limited. HoloVir is a robust and flexible data analysis pipeline that provides an optimized and validated workflow for taxonomic and functional characterization of viral metagenomes derived from invertebrate holobionts. Simulated viral metagenomes comprising varying levels of viral diversity and abundance were used to determine the optimal assembly and gene prediction strategy, and multiple sequence assembly methods and gene prediction tools were tested in order to optimize our analysis workflow. HoloVir performs pairwise comparisons of single read and predicted gene datasets against the viral RefSeq database to assign taxonomy and additional comparison to phage-specific and cellular markers is undertaken to support the taxonomic assignments and identify potential cellular contamination. Broad functional classification of the predicted genes is provided by assignment of COG microbial functional category classifications using EggNOG and higher resolution functional analysis is achieved by searching for enrichment of specific Swiss-Prot keywords within the viral metagenome. Application of HoloVir to viral metagenomes from the coral Pocillopora damicornis and the sponge Rhopaloeides odorabile demonstrated that HoloVir provides a valuable tool to characterize holobiont viral communities across species, environments, or experiments.
Highlights
Marine viruses are the most abundant biological entities in the oceans, often exceeding the number of bacteria 10-fold (Wommack and Colwell, 2000; Suttle, 2005, 2007) and having high local and global diversity (Brum et al, 2015)
In order to facilitate rapid in-house analysis of marine holobiont viral metagenome sequence data, we have developed and validated a flexible and robust script-based workflow that accepts overlapping paired-end Illumina data [after basic Quality Control (QC) and trimming]
While the largest contigs produced by Trinity and Ray Meta were comparable in length to CLC Genomics Workbench (177,419 and 179,062 bp respectively), Trinity assembled a total of 960,610 bp which is considerably larger than the original genome size and Ray Meta failed to assemble any contigs originating from the Podoviral Prochlorococcus phage P-SSP7, as well as covering only 76.7% of the original genomes
Summary
Marine viruses are the most abundant biological entities in the oceans, often exceeding the number of bacteria 10-fold (Wommack and Colwell, 2000; Suttle, 2005, 2007) and having high local and global diversity (Brum et al, 2015). Due to limitations in traditional methodologies used for studying complex viral populations, including a lack of suitable marker genes, and limited methods designed for viruses, our understanding about the specific roles viruses play in marine ecosystems has lagged behind our knowledge of the functional roles of cellular microorganisms Despite these limitations, research over the past decade has shown that viruses play a vital role in biogeochemical cycles as they modulate microbially-driven processes through mortality and subsequent release of organic matter and inorganic nutrients that become available for other microorganisms to consume (Suttle, 2005; Weitz and Wilhelm, 2012). Horizontal gene transfer and metabolic reprogramming by viral-encoded auxiliary metabolic genes is another important ecosystem role (Jiang and Paul, 1998; Hurwitz et al, 2015) with the recombination of viral and host genes during infection often triggering changes in host metabolism, immunity, distribution and evolution (Rohwer and Thurber, 2009) as well as shaping viral genomes (Lindell et al, 2007)
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