Figure 1. Location of the HMMV sampling site

Metagenome pyrosequencing of Håkon Mosby Mud Volcano sediments Othilde Elise Håvelsrud1, 2, 3, Thomas H.A. Haverkamp3,4, Tom Kristensen2, Anne Gunn Rike1and Kjetill S. Jakobsen3,4

1 Norwegian Geotechnical Institute, Oslo, Norway. 2 Department of Molecular Biosciences, University of Oslo, Oslo, Norway. 3 Microbial Evolution Research Group, University of Oslo, Oslo, Norway. 4 Centre for Evolutionary and Ecological Synthesis, University of Oslo, Oslo, Norway.

Methods A sediment sample (2-20cm bsf) obtained from the northern outer rim of the HMMV, was used for DNA extraction. Extracted DNA was sequenced following the standard GS FLX protocols using the 70x75 format PicoTiterPlateTM on a GS FLX instrument at Roche Penzburg. Analysis of the metagenome was done as shown in the flowchart (Fig. 2) using MG-RAST [2], KAAS [3] and MEGAN [4].

References [1] Niemann et al. (2006). Novel microbial communities of the Håkon Mosby mud volcano and their role as a methane sink. Nature 443: 854-858. [2] Meyer F, et al. (2008). The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9: Article No.: 386. [3] Moriya Y et al. (2007). KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35: W182-185. [4] Huson et al. (2009). Methods for comparative metagenomics. BMC Bioinformatics 10 Suppl 1: S12.

Contact information e-mail: thhaverk@bio.uio.no

webpage T. Haverkamp: http://www.cees.uio.no/about/staff/frida/407495.xml Figure 2. Flowchart to generate taxon specific

annotation of metagenomic reads

Results The HMMV metagenome has 392.628 reads, average length: 222 bp; average GC content 44 %. 68% of the HMMV metagenome reads were assigned to three dominating taxa: Proteobacteria, Archaea and Bacteriodetes (Fig. 3). KAAS annotated reads showed metabolic capacity differences between these groups for: Anaerobic oxidation of methane (Fig.4); Methane metabolism (Fig.5); Sulfur metabolism (Fig.6) and Nitrogen metabolism (Fig.7).

Results Conclusions The dominating taxa within the HMMV prokaryotic community belong to Proteobacteria, Bacteroidetes and Archaea.

The combination of taxonomic binning and subsequent annotation of the binned reads, as performed here is particularly powerful in revealing taxon- or group-specific metabolism.

Essential steps in anaerobic oxidation of methane are only found among archaeal reads.

The Proteobacteria have the capacity for aerobic oxidation of methane.

Only among proteobacterial reads a full pathway for sulfate reduction could be detected.

The Bacteriodetes at the HMMV are not involved in direct methane metabolism.

Introduction The Håkon Mosby Mud Volcano (HMMV) is an active methane driven cold seep at the bottom of the Barents Sea (Fig. 1). This cold seep harbors a highly diverse microbial community that uses the reverse methanogenesis pathway to anaerobically oxidize methane [1]. The energy released is used by the prokaryotic community. To understand the diversity and metabolic capacity of the prokaryotic community we created a metagenomic library using 454 GS FLX technology. We combined taxonomic binning of sequences with metabolic annotation to analyze the metabolic capacity of different taxa found in the HMMV microbial community.

Acknowledgements The VISTA program for financial support.�Jochen M. Knies (NGU, Trondheim, Norway) for providing samples. �The crew of RV Håkon Mosby (IMR, Bergen, Norway). Roche Penzburg for 454 sequencing. Lex Nederbragt for help with bioinformatic analyses (NSC, Oslo, Norway).

Figure 3. A) Taxonomic assignment of HMMV metagenome reads using MEGAN. B) Proteobacterial taxa. C) Archaeal taxa.

Figure 4. KEGG map folate biosynthesis, including the reverse methanogenesis pathway.

Figure 5. KEGG map of methane metabolism.

Figure 6. KEGG map of sulfur metabolism

Figure 7. KEGG map of nitrogen metabolism