A statistical toolbox for metagenomics: assessing functional diversity in microbial communities

Patrick D Schloss,Jo Handelsman

doi:10.1186/1471-2105-9-34

Abstract

BackgroundThe 99% of bacteria in the environment that are recalcitrant to culturing have spurred the development of metagenomics, a culture-independent approach to sample and characterize microbial genomes. Massive datasets of metagenomic sequences have been accumulated, but analysis of these sequences has focused primarily on the descriptive comparison of the relative abundance of proteins that belong to specific functional categories. More robust statistical methods are needed to make inferences from metagenomic data. In this study, we developed and applied a suite of tools to describe and compare the richness, membership, and structure of microbial communities using peptide fragment sequences extracted from metagenomic sequence data.ResultsApplication of these tools to acid mine drainage, soil, and whale fall metagenomic sequence collections revealed groups of peptide fragments with a relatively high abundance and no known function. When combined with analysis of 16S rRNA gene fragments from the same communities these tools enabled us to demonstrate that although there was no overlap in the types of 16S rRNA gene sequence observed, there was a core collection of operational protein families that was shared among the three environments.ConclusionThe results of comparisons between the three habitats were surprising considering the relatively low overlap of membership and the distinctively different characteristics of the three habitats. These tools will facilitate the use of metagenomics to pursue statistically sound genome-based ecological analyses.

Highlights

The 99% of bacteria in the environment that are recalcitrant to culturing have spurred the development of metagenomics, a culture-independent approach to sample and characterize microbial genomes
To make comparisons among communities, we propose grouping ORFs into operational protein families (OPFs) which are analogous to operational taxonomic units (OTUs) derived from 16S rRNA gene sequences
By analogy to the analysis of 16S rRNA gene sequences of uncultured bacteria where OTUs are developed based on a distance matrix, we propose using BLAST score ratios (BSRs) values to define OPFs

Summary

Introduction

The 99% of bacteria in the environment that are recalcitrant to culturing have spurred the development of metagenomics, a culture-independent approach to sample and characterize microbial genomes. Massive datasets of metagenomic sequences have been accumulated, but analysis of these sequences has focused primarily on the descriptive comparison of the relative abundance of proteins that belong to specific functional categories. The analysis used to describe these communities has primarily focused on the descriptive characterization and comparison of the relative abundance of proteins that belong to specific functional categories. Labbased cultures that are sequenced do evolve, the differences between lab stocks is minimal compared to the changes faced by natural communities over short periods of time. This makes it difficult to reanalyze a community once a genome sequence has been obtained to improve annotations and understand gene expression

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