Re-purposing software for functional characterization of the microbiome

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Background: Widespread bioinformatic resource development generates a constantly evolving and abundant landscape of workflows and software. For analysis of the microbiome, workflows typically begin with taxonomic classification of the microorganisms that are present in a given environment. Additional investigation is then required to uncover the functionality of the microbial community, in order to characterize its currently or potentially active biological processes. Such functional analysis of metagenomic data can be computationally demanding for high-throughput sequencing experiments. Instead, we can directly compare sequencing reads to a functionally annotated database. However, since reads frequently match multiple sequences equally well, analyses benefit from a hierarchical annotation tree, e.g. for taxonomic classification where reads are assigned to the lowest taxonomic unit. Results: To facilitate functional microbiome analysis, we re-purpose well-known taxonomic classification tools to allow us to perform direct functional sequencing read classification with the added benefit of a functional hierarchy. To enable this, we develop and present a tree-shaped functional hierarchy representing the molecular function subset of the Gene Ontology annotation structure. We use this functional hierarchy to replace the standard phylogenetic taxonomy used by the classification tools and assign query sequences accurately to the lowest possible molecular function in the tree. We demonstrate this with simulated and experimental datasets, where we reveal new biological insights. Conclusions: We demonstrate that improved functional classification of metagenomic sequencing reads is possible by re-purposing a range of taxonomic classification tools that are already well-established, in conjunction with either protein or nucleotide reference databases. We leverage the advances in speed, accuracy and efficiency that have been made for taxonomic classification and translate these benefits for the rapid functional classification of microbiomes. While we focus on a specific set of commonly used methods, the functional annotation approach has broad applicability across other sequence classification tools. We hope that re-purposing becomes a routine consideration during bioinformatic resource development. [MediaObject not available: see fulltext.].