This proposal describes the acquisition of an Illumina GAIIx nucleic acid sequencer and related instrumentation to enhance microbial ecology and genomics research programs at the Marine Biological Laboratory and the broader Woods Hole scientific community. The instrumentation will dramatically reduce the cost of microbial profiling, genome sequencing and RNA-Seq profiles while leveraging existing experience with advanced DNA sequencing platforms. The requested equipment would provide a daily throughput capacity of ~5 billion nt/day, sufficient for the ~1755 billion nucleotides (nt) of sequencing needs identified by major and minor users of the equipment under existing NSF and NIH funded programs; these estimates do not include projected use by MBL high school, undergraduate, and graduate level courses as described below.
Intellectual Merit: The massively parallel DNA sequencer and a supporting DNA sample preparation instrument requested for shared use in this MRI proposal will serve NSF-funded and other investigators at the Marine Biological Laboratory in Woods Hole and collaborators at the Woods Hole Oceanographic Institution inclusive of Massachusetts Institute of Technology scientists who participate in the Woods Hole Center for Oceans and Human Health funded by the NSF. The instruments to be purchased include an Illumina GAIIx massively parallel DNA sequencing machine plus an accessory Covaris S-2 DNA acoustic shearer. The equipment will accommodate new DNA sequencing requirements for at least eight funded projects falling within two major themes: microbial community profiling and genomics/transcriptomics. Microbial ecology projects include profiling of human-impacted fresh water supplies, long-term ecological research sites, controlled mesocosms, and the human gut; genomics projects include studies of genome evolution that focus on mechanisms and consequences of deleterious evolution in bacterial symbionts, the role of microRNAs in asexual rotifers, and genomic variation in natural communities, including the phyllosphere and the subseafloor. This equipment will dramatically increase our massively parallel sequencing capacity while at the same time reducing sequencing costs by many fold. Acquisition of an Illumina GAIIx will additionally enable investigators to increase the scope and depth of their ongoing research and consider sequence-based approaches to new questions in ecology and evolutionary biology. The equipment will operate within the W. M. Keck Ecological and Evolutionary Genetics Facility within the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution (BPC) at the Marine Biological Laboratory (MBL), a shared-use resource for investigators in the Woods Hole scientific community operational since 1997.
Broader Impacts: The new instrumentation will provide exciting opportunities for students and researchers at all levels. The PIs currently mentor postdoctoral scholars and Brown-MBL graduate students who will immediately benefit from the addition of a GAIIx to our sequencing facility. Additionally, MBL laboratories will host undergraduates in the MBL?s NSF Research Experience for Undergraduates and Semester in Environmental Sciences programs. These students will benefit from the development of a new curriculum module, From Seawater to Sequences, which will incorporate use of the GAIIx data. This curriculum module will also serve upper-level high school students through our collaboration with the Zephyr Education Foundation, a local non-profit marine education group in Woods Hole. In addition, through the integration of the GAIIx sequencing facility into MBL summer courses, we will extend the training of a diverse population of graduate and postdoctoral students next generation sequencing methods that will have national and international impact. Finally, visiting and summer faculty who might usually outsource their sequencing will have access to the instrument while in residence, learning first hand how sequence datasets are generated and processed, what protocols are possible, and perhaps equally importantly, the limitations and sources of error in next-generation sequencing. This combination of new curriculum development geared towards high school and undergraduate students with rigorous training for summer and visiting faculty, graduate students, and postdocs will allow for a strong integration of the GAIIx with both new and established training opportunities at the MBL.
Advances in massively parallel DNA sequencing have revolutionized nearly every corner of biology including studies of the smallest life forms – microbes – to genome studies of multicellular plants and animals. This project funded the acquisition of an Illumina DNA sequencing platform that accelerated the pace of discovery for many projects in the Woods Hole Scientific Community. Some involved evolutionary studies of gene regulatory networks that shape development in relatively simple invertebrates while others elucidated developmental pathways spanning the interval from unfertilized Xenopus laevis (a powerful model organism for studies of development) eggs to swimming embryos. Microbial studies focused on defining shifts in microbial diversity under different environmental conditions and / or their impact on biodiversity and health of host organisms including humans. For example, one investigation focused on a single-cell photosynthetic organism from the Antarctic that shifts its lifestyle in response to the concentration of iron in the water column. Through studies of gene expression made possible by the Illumina sequencing platform it was possible to identify the molecular mechanisms that regulate alga blooms along the Antarctica coastline during the austral summer. Using marker gene analyses on the Illumina platform, studies of microbial communities associated with sewage release identified nearly 100 molecular targets that provide a complex signature for human fecal pollution that can be differentiated from non-human fecal contamination. This is of significant health importance because traditional indicators of sewage pollution rely upon the growth of E. coli that can colonize many different animal species. Genome and gene expression studies in the rotifer Adineta vaga (a near-microscopic pseudocoelomate animal with only 957 cells) under stress of desiccation demonstrated extensive gene conversion following desiccation, consistent with the synthesis-depended strand-annealing model of DNA repair. Because this facility impacted the research of more than a dozen independently funded projects from the NSF and NIH, this report cannot summarize the impact of each research initiative or its major discoveries. Yet the collective results clearly show this instrument took each of the projects to new levels of accomplishment. In addition to a broad range of scientific advances, this instrument contributed to the training of nearly 100 graduate students and post docs who have enrolled in the MBL-Brown graduate program or who have attended one of the MBL summer courses in microbial diversity or embryology. Finally, data generated on this instrument provided the basis for publication of 10 manuscripts, submission of 11 others (currently in review or in preparation) and the awarding of 15 new grants plus pending reviews for 6 other applications.
