Award: OCE-1148017

Overview. Next-generation DNA sequencers introduced over the past seven years have given environmental researchers unprecedented access to DNA sequence data from unknown microorganisms. By and large, the technological advancements in DNA sequencing have come through providing increasingly cheaper, yet shorter read length sequences. This emphasis on cheap sequencing of short DNA fragments has been driven primarily by the demand for human genome re-sequencing, but it poses significant limitations when applied to the analysis of complex microbial communities from environmental samples. PacBio RS, a third-generation genomic sequencing technology, offers a departure from this trend, with read lengths averaging 2,500 bp, and these sequence reads are obtained from single template DNA molecules. In theory, this should significantly improve the value of genomic sequences obtained from environmental microbial communities. The central objective of this project was to determine whether the perceived advantages of PacBio translate in practice when applied to æreal worldÆ research scenarios in marine microbial ecology, including: a) examination of microbial diversity using polymerase chain reaction amplicon sequencing; b) bacterial genomic sequencing, including single-cell genomic analysis; and c) analysis of the genetic composition of microbial communities using shotgun metagenomics. Intellectual merit. At Bigelow Laboratory, our work has been focused on PacBio sequencing applications in microbial single cell genomics, a cutting-edge research technology pioneered by Bigelow scientists. By creating benchmark data sets from individual cells of previously sequenced strains of marine cyanobacteria Prochlorococcus, we were able to efficiently evaluate various genomic sequencing and assembly strategies. We tested PacBio sequencing using various DNA insert sizes and library preparation techniques, alone and in combination with Illumina sequencing (currently predominant short read sequencing technology). We determined that the success of genome recovery from individual cells can be predicted from the speed of single cell whole genome amplification, which will significantly improve data quality and reduce costs in this rapidly growing research area. We then applied the PacBio-only, Illumina-only and PacBio-Illumina hybrid sequencing approaches on SAGs of ten uncultured marine bacteria and archaea in the first single cell genomics-based analysis of virus-host interactions in their natural environment. In this pilot study we have identified a number of novel virus-host systems, which span three virus classes: Myoviridae, Siphoviridae and Podoviridae. We found these infections in diverse members of surface ocean bacterioplankton, including Verrucomicrobia, Marinimicrobia (SAR406), Proteobacteria, and Bacteroidetes. In difference to prior cultivation-independent research methods, this new approach offers a combination of several important benefits: a) ability to identify viruses as well as their hosts; b) recovery of near-complete genomes; and c) discrimination between lytic and lysogenic interaction. Thus, our results so far demonstrate that PacBio technology offers significant new opportunities to marine microbiologists. The paucity of suitable bioinformatics tools currently constitutes the main bottleneck to the broader application of PacBio sequencing technology in marine microbial ecology, and our current work is focused specifically to address this challenge. Broader impacts. This project has provided significant training and professional development experiences for two undergraduate students, three graduate students, two postdoctoral scientists and a bioinformatician. They have been involved in various aspects of laboratory work, computational analyses, result presentations and manuscript preparation. In the process, they obtained hands-on experience in new research areas and technologies, as well as project management and mentorship roles. B...