PhylOTU: A High-Throughput Procedure Quantifies Microbial Community Diversity and Resolves Novel Taxa from Metagenomic Data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?Author Summary: Microorganisms comprise the majority of the biodiversity on the planet. Because the overwhelming majority of microbes are not readily cultured in the laboratory, researchers often rely on PCR-based investigations of genomic sequence to characterize microbial diversity. These analyses have dramatically expanded our understanding of biodiversity, but due to methodological biases PCR-based approaches may only reveal part of the microbial biosphere. Shotgun sequencing of environmental DNA, known as metagenomics, avoids the biases associated with targeted amplification of genomic sequence and can provide insight into the diversity hidden from traditional investigations. However, the fragmentary, non-overlapping nature of shotgun sequence data makes it intractable to analyze with existing tools. Here, we present PhylOTU, a novel computational method that enables accurate characterization of microbial diversity from metagenomic data. We process over 10 million metagenomic sequences obtained from the global open ocean to identify novel Bacterial taxa and reveal the presence of microorganisms overlooked by investigation of PCR-based sequences from the same samples. These results suggest that to fully characterize microbial biodiversity requires a novel bioinformatics toolbox for analysis of shotgun metagenomic data.