Project description:In this study we applied MASC-seq (massive and parallel microarray sequencing, https://doi.org/10.1038/ncomms13182), a scRNA-seq method that facilitates sequencing of thousands of cells in parallel, and that couples microscope images with the single cell transcriptome data. For this method, fixed cells are spread over a microarray with 100 μm-sized spots of DNA capture probes with spot-specific indices. The cells are first imaged using a scanning microscope and then permeabilized, releasing their RNA out of the cells and bind to the probes on the array. cDNA is synthesized, harvested and sequenced, and, using the spot-specific barcode-sequences, cDNA sequences stemming from a specific spot (i.e., cell) can be linked to the microscope image of the corresponding cell. However, until now, the MASC-seq method has only been applied to mammalian cells. The aim of this study was to test and adapt the MASC-seq method for application on unicellular eukaryotic plankton. We applied and optimized the method on three cultured plankton representatives, abundant in communities of aquatic environments, Phaeodactylum tricornutum (a diatom, silica and polysaccharide cell walls 23), Heterocapsa sp. (a dinoflagellate, cellulose thecal plates 24) and Tetrahymena thermophila (a ciliate, lipid membrane 25) which all have different size and diverse cell surface structures common to plankton. We optimized several steps in the protocol to make it more suitable for planktonic cells and compared the results from MASC-seq generated single cell transcriptomes to bulk RNA sequencing.
Project description:We used single-cell sequencing data and imaging to investigate Eukaryotic plankton from environmental marine samples collected from Coogee, NSW, Australia.
Project description:Protist community composition and seasonal dynamics are of major importance for the production of higher trophic levels, such as zooplankton and fish. Our aim was to reveal how the protist community in the Skagerrak changes through the seasons by combining high-throughput sequencing and microscopy of plankton collected monthly over two years. The V4 region of the 18S rRNA gene was amplified by eukaryote universal primers from the total RNA/cDNA. We found a strong seasonal variation in protist composition and proportional abundances, and a difference between two depths within the euphotic zone. Highest protist richness was found in late summer-early autumn, and lowest in winter. Temperature was the abiotic factor explaining most of the variation in diversity. Dinoflagellates was the most abundant and diverse group followed by ciliates and diatoms. We found about 70 new taxa recorded for the first time in the Skagerrak. The seasonal pattern in relative read abundance of major phytoplankton groups was well in accordance with microscopical biovolumes. This is the first metabarcoding study of the protist plankton community of all taxonomic groups and through seasons in the Skagerrak, which may serve as a baseline for future surveys to reveal effects of climate and environmental changes.