Analysis of Benthic Meiofauna Communities Using 454 Pyrosequencing

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Erik Pilgrim U.S. Environmental Protection Agency Collaborator
Anthony Chariton CSIRO Collaborator
Robin Giblin-Davis University of Florida Collaborator
Dorota Porazinska University of Florida Collaborator
Simon Creer University of Bangor Collaborator
W. Kelley Thomas UNH - Department of Molecular, Cellular & Biomedical Sciences Principal Investigator
Holly Bik UNH - Department of Molecular, Cellular & Biomedical Sciences Co-Principal Investigator
Cheryl Whistler UNH - Department of Molecular, Cellular & Biomedical Sciences Collaborator
Vaughn Cooper UNH - Department of Molecular, Cellular & Biomedical Sciences Collaborator
Subhash Minocha UNH - Department of Biological Sciences Collaborator
Rakesh Minocha UNH - Department of Natural Resources & the Environment Collaborator
Michael Lesser UNH - Department of Biological Sciences Collaborator
Nancy Kinner UNH - Department of Civil Engineering Collaborator
Kay Ho U.S. Environmental Protection Agency Collaborator
Dina Proestou U.S. Environmental Protection Agency Collaborator
Caludia Gunsch Duke University Collaborator

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Intellectual Merit
Accurate measures of species richness and abundance are critical for studies of biodiversity. However, for most small metazoan organisms (e.g. nematodes), assessing environmental diversity is currently a laborious and time consuming process. New, high-throughput sequencing methods (e.g. GS FLX, Roche) offer vast potential for characterizing metazoan communities from environmental samples, but such ‘metagenomic’ studies face many computational challenges.
The proposed study aims to further develop analytical tools and protocols that can be used to accurately characterize metazoan communities (primarily nematodes) from marine environmental samples, using two diagnostic regions from the 18S ribosomal RNA gene. The recently developed OCTUPUS pipeline (Sung et al., in prep.) will be used to designate Operational Clustered Taxonomic Units (OCTUs) from GS FLX Titanium sequence reads (~400bps). Preliminary metagenomic datasets have already been obtained from eight deep-sea sites and two coastal locations (H. Bik, unpublished data), and are currently awaiting further analysis. Initial analysis recovered a diversity of eukaryotic taxa from this metagenomic dataset, with high species richness observed in all groups. Similar taxonomic compositions were also recovered from two independent analyses. 
Preliminary hypotheses need to be rigorously evaluated with comprehensive data analysis. The primary objectives of the proposed study include:
1) Test and refine computational pipelines for metagenomic analysis
2) Characterize deep-sea metazoan communities from three sites in the North Atlantic
3) Establish a database to support bioinformatic analysis of metagenomic data. 
One specific aim is to investigate how different sequence identity cutoffs (e.g. 90-99%) can generate different OCTUs sets that partition the data in biologically meaningful ways. This study will also tackle theoretical and computational challenges concerning abundance estimates and sequence chimeras. Another aim is to expand metagenomic analysis to incorporate additional genetic loci and use emerging methodology such as primer extension capture (Briggs et al. 2009). This investigation will represent the first comprehensive molecular assessment of deep-sea metazoan communities. Additional metagenomic datasets will be generated from existing sediment samples. 
This study will be used to address specific biological questions:
1) Do deep-sea sites share any OCTUs?
2) How do metazoan communities vary on both small and large scales (e.g. within a site and between different sampling areas, respectively)?
3) Can we correlate variations in metazoan communities with any physical or biological factors? 
Broader Impacts
Outputs from this study will benefit future metagenomic research, support industrial applications of high-throughput methodology, and contribute fundamental knowledge to the field of deep-sea biology. Rigorous methodological and analytical tests carried out during this investigation will facilitate the development of refined and empirically sound metagenomics protocols. Such protocols will allow for broad application of metagenomic methods, encouraging future biodiversity research in other eukaryotic phyla, as well as promoting industrial applications (e.g. for environmental surveys and monitoring). The establishment of a long-term database structure will be invaluable to these goals—such a resource will facilitate the discovery of significant and biologically meaningful associations in metagenomic data and associated ecological metadata. 
Analysis of deep-sea metazoan communities will fundamentally contribute to our understanding of this largely unknown habitat, elucidating species distributions and physical factors which may govern community composition. Finally, this study will incorporate outreach activities focused on biodiversity investigations, running training workshops for teachers and developing laboratory curricula for classroom use. 
Briggs, A. et al. (2009) Targeted retrieval and analysis of five neandertal mtDNA genomes. Science, 325, 252.
Sung, W., et al. (in preparation) OCTUPUS: A package for defining operationally clustered taxonomic units from parallel-tagged ultra sequencing. Beta version available from the Thomas lab on request.