Award: OCE-1148118

Direct application of high-throughput DNA sequencing (HTS) technology to the analysis of environmental DNA has provided many of the most transformative scientific discoveries in microbiology within the past five years. At the core of this renaissance has been the ability to accurately describe the species composition of microbial communities within a broad range of environments at unprecedented resolution and depth. However, environmental microbiologists have only begun to tap into the scientific promise of HTS for understanding the influence of microbial communities on larger ecosystems. Part of the limitation to reaching the full scientific potential of HTS in microbiological research has come from the sequencing technology itself. In particular, HTS technologies are limited by short DNA sequence read lengths and low sample throughput. The dominant HTS platforms in widespread use are simply not designed for the demands of environmental microbiology research. In April, 2011 Pacific Biosciences Corp. released an instrument, the PacBio RS, based on a new technological approach to high-throughput DNA sequencing — Single Molecule Real Time (SMRT) sequencing. The novel engineering of the PacBio RS addresses several limitations of current high-throughput DNA sequencers, namely sample throughput, assay cost, and sequence read length. The project investigated the application of PacBio sequencing to three common microbial ecology research problems: whole genome sequencing; community profiling by PCR amplicon sequencing; and shotgun metagenomic sequencing. In each of these investigations, PacBio benefited scientific output over more established sequencing technologies. The team successfully showed that PacBio was superior to short read technologies for sequencing bacterial genomes, especially genomes that had proven intractable to closure with short read technologies. This work was published in the Journal of Genome Announcements (DeBruyn, et al. 2014) The long DNA sequence reads resulting from PacBio were critical to uncovering gene associations shaping the biology of unknown marine viruses. In particular, the team found that the gene encoding Ribonucleotide Reductase could broadly predict the identity of bacteria infected by an unknown marine bacteriophage (virus of a bacteria) as well as the physiological conditions necessary for the virus to replicate. This work was reported in the Proceedings of the National Academy of Sciences (Sakowski, et al. 2014). In carefully controlled experiments the team was able to identify systemic bias in a commonly used sample preparation technique, bias that was shown to substantially alter scientific conclusions surrounding the composition of a microbial community based on metagenome DNA sequence data. This work was published in the journal Microbiome (Marine, et al., 2014). The research activities of the project provided high-quality training experiences for undergraduates and graduate students. In all, three undergraduates and four graduate students were intimately involved in the project and provided much of the scientific output. Nearly all of the student participants were authors on publications resulting from the project. Contributions such as these can be critical in their preparation for scientific careers. Publications resulting from this project thus far: DeBruyn, J.M., Radosevich, M., Wommack, K.E., Polson, S.W., Hauser, L.J., Fawaz, M.N., Korlach, J., and Tsai, Y.-C. (2014). Genome Sequence and Methylome of Soil Bacterium Gemmatirosa kalamazoonensis KBS708T, a Member of the Rarely Cultivated Gemmatimonadetes Phylum. Genome announcements 2. Marine, R., McCarren, C., Vorrasane, V., Nasko, D., Crowgey, E., Polson, S.W., and Wommack, K.E. (2014). Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome. Microbiome 2, 3. Sakowski, E.G., Munsell, E.V., Hyatt, M., Kr...