2 A tutorial for using TreeSAPP

2.1 Introduction and goals

In this series of tutorials, students will analyze a gene family by creating a TreeSAPP reference package (refpkg). Students will work through a typical workflow of TreeSAPP both with an example gene (XmoA) to familiarize yourself with the tools, and then you will repeat the steps with a gene assigned to your group for which no reference package exists. You will document your efforts for the new reference package in Problem Set 5.

2.2 TreeSAPP workflow

TreeSAPP is a Python package for gene-centric analysis. It uses custom protein sequence databases called reference packages (RefPkg)

• Acquiring reference sequence data.
• Creating a Reference Package For TreeSAPP.
• Classifying unknown sequences with TreeSAPP and updating a reference package.
• Calculating relative abundance of meta’omic data.
• Analysing the TreeSAPP classifications in R.

2.3 Genes for creating reference packages

Reference packages can be built for nearly any protein-encoding gene but to demonstrate the process of gene-centric analysis we will create and use a reference package for XmoA.

2.3.1 XmoA

The protein family we will be focusing on is that of the copper-containing membrane-bound monooxygenases (5). This family contains particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO) and well be building a reference package for the alpha subunits of these enzymes called XmoA. All students will work through this example individually.

2.4 Tools

2.4.1 Shell

Please use this short Shell cheat sheet for commonly used commands and review previous tutorials on Canvas.

2.4.2 TreeSAPP

Tree-based Sensitive and Accurate Phylogenetic Profiler (TreeSAPP) (3) can be found on GitHub including an excellent wiki with additional information on each of the treesapp subcommands.

2.4.3 iTOL

Interactive Tree Of Life (iTOL) (4) is a browser-based tool that allows you to visualize data generated in TreeSAPP as a phylogenetic tree with additional annotations.

2.5 Data

Other than The Saanich Inlet data set already located on the server, you may download data from different databases:

• FunGene, the functional gene pipeline and repository
• EggNOG, evolutionary genealogy of genes: Non-supervised Orthologous Groups