Gene-centric analysis is commonly used to chart the structure, function, and activity of microbial communities in natural and engineered environments. A common approach is to create custom ad hoc reference marker gene sets, but these come with the typical disadvantages of inaccuracy and limited utility beyond assigning query sequences taxonomic labels. The Tree-based Sensitive and Accurate Phylogenetic Profiler (TreeSAPP) software package standardizes analysis of phylogenetic and functional marker genes and improves predictive performance using a classification algorithm that leverages information-rich reference packages consisting of a multiple sequence alignment, a profile hidden Markov model, taxonomic lineage information, and a phylogenetic tree.
University of British Columbia researchers provide a set of protocols that link the various analysis modules in TreeSAPP into a coherent process that both informs and directs the user experience. This workflow, initiated from a collection of candidate reference sequences, progresses through construction and refinement of a reference package to marker identification and normalized relative abundance calculations for homologous sequences in metagenomic and metatranscriptomic datasets. The alpha subunit of methyl-coenzyme M reductase (McrA) involved in biological methane cycling is presented as a use case given its dual role as a phylogenetic and functional marker gene driving an ecologically relevant process. These protocols fill several gaps in prior TreeSAPP documentation and provide best practices for reference package construction and refinement, including manual curation steps from trusted sources in support of reproducible gene-centric analysis.
TreeSAPP software package modules for creation and quality control of reference packages and for assignment and visualization of protein sequence profiles using phylogenetic trees.
Availability – TreeSAPP was developed in Python 3 as an open-source Python package and is available on GitHub at https://github.com/hallamlab/TreeSAPP.