Microbial Diversity Across an Oxygen Gradient Using Large-scale Phylogenetic-based Analysis of Marine Metagenomes

Insufficient reference sequence data for annotation of unknown environmental sequences and metagenomes has driven efforts to find alternative annotation methods that mitigate biases from missing information. The use of phylogenetic-placement algorithms shows promise as a robust sequence annotation technique that deals with missing reference information by allowing for annotation of sequences at internal nodes of a phylogenetic tree. However, using these methods for community level surveys of the thousands of genes found in metagenomes requires powerful computational systems and sophisticated software workflows. The main goal of this thesis is to outline a phylogenetic analysis pipeline built to process environmental metagenomic samples using the pplacer software suite, and a pilot study performed with this software pipeline to investigate community-level patterns in gene diversity for a marine oxygen minimum zone (OMZ) off the coast of Chile, South America. Reference sequence data was used to create a custom database and custom reference packages for 9,204 functional housekeeping genes, along with small sub-unit ribosomal genes (SSU) by Domain. A comparative analysis of metagenomic samples from the OMZ using our pipeline shows that while functional and SSU genes show similar spatial patterns of diversity across the oxygen gradient, higher overall diversity was identified via the functional genes. Ecologically relevant functional genes showed higher levels of diversity than either the total from all functional genes or SSU ribosomal genes, underlining the importance of diversity in ecosystem functions.