ena-STUDY-INSTITUTE OF EVOLUTIONARY BIOLOGY-10-11-2020-17:01:51:858-316
Generating SAGs of microeukaryotes to better understand the functions and the ecology of uncultured eukaryotes in marine systems
Biodiversity studies of marine microbial eukaryotes have revealed a large diversity of new uncultured lineages, especially among heterotrophic flagellates. Single-cell genomics offer the opportunity to have access to the genomic information of these uncultured and ecologically relevant taxa. Therefore, it will provide a better understanding of the function and roles of microeukaryotic diversity in marine ecosystems. The technique consists in 6 main steps: collection of cells from the environment, sample preparation, cryopreservation, cell isolation (through different techniques, most commonly used is by Fluorescence Activated Cell Sorting, FACS), cell lysis, whole genome amplification (MDA), DNA library preparation and sequencing. In previous studies, often cell collection and cell sorting are temporally decoupled, thus cells must be cryopreserved and this may affect the yield of whole genome amplification and the genome recovery, which it is already low for microeukaryotes. Thus, establishing protocols that can dimish such limitations, are key to a better performance of the technique. Here, we attempted to generate a new collection of single-cell amplified genomes from microeukaryotes isolated from Blanes Bay (Barcelona, Spain), for a better understanding of the function, the ecology and also the evolution of the uncultured microeukaryotes of marine systems. We accomplished it by coupling in the same day the cell collection with the cell sorting to avoid possible problems occurring during cryoprerservation. Finally, we took advantage of the cell sorting lasers to isolate and separate different eco-groups of interest: Photosynthetic organisms (pico sized 2-3 um), Nanosized heterotrophic flagellates (nano sized >3um) and Picosized heterotrophic flagellates (2-3um).
Biodiversity studies of marine microbial eukaryotes have revealed a large diversity of new uncultured lineages, especially among heterotrophic flagellates. Single-cell genomics offer the opportunity to have access to the genomic information of these uncultured and ecologically relevant taxa. Therefore, it will provide a better understanding of the function and roles of microeukaryotic diversity in marine ecosystems. The technique consists in 6 main steps: collection of cells from the environment, sample preparation, cryopreservation, cell isolation (through different techniques, most commonly used is by Fluorescence Activated Cell Sorting, FACS), cell lysis, whole genome amplification (MDA), DNA library preparation and sequencing. In previous studies, often cell collection and cell sorting are temporally decoupled, thus cells must be cryopreserved and this may affect the yield of whole genome amplification and the genome recovery, which it is already low for microeukaryotes. Thus, establishing protocols that can dimish such limitations, are key to a better performance of the technique. Here, we attempted to generate a new collection of single-cell amplified genomes from microeukaryotes isolated from Blanes Bay (Barcelona, Spain), for a better understanding of the function, the ecology and also the evolution of the uncultured microeukaryotes of marine systems. We accomplished it by coupling in the same day the cell collection with the cell sorting to avoid possible problems occurring during cryoprerservation. Finally, we took advantage of the cell sorting lasers to isolate and separate different eco-groups of interest: Photosynthetic organisms (pico sized 2-3 um), Nanosized heterotrophic flagellates (nano sized >3um) and Picosized heterotrophic flagellates (2-3um).