Starting Month: M1
Ending Month: M15
This will help to identify the depths (time-points) that will be prioritized in following WPs as inclusion in culture collections, and in the multi omics approach that will be deployed. We are going to adopt culture-independent surveys (such as sequencing ITS and 16S rRNA amplicons) of large numbers of microbiomes in high resolution intervals (every 3-5 cm).
Task 1.1 – Sample collection, barcoding and metadata collection M1-M4.
Task Leader: Assoc. Prof. Ierotheos Zacharias, University of Patras
Task 1.2 – Phylogenetic characterization M3-M15.
Task Leader: Assoc. Prof. George Tsiamis, University of Patras
Three cores from each of the stations A8 and A9 will be used for analysis (Figure 3.1). All collection equipment and supplies such as dredges, corers, spoons, scoops and compositing trays that may come into contact with the sample will be cleaned prior to use as follows: (a) wash with phosphate-free liquinox soap, (b) tap water rinse, (c) ASTM water (distilled water) rinse, (d) methanol rinse, (e) hexane rinse, (f) allow to air dry, and (g) cleaned, decontaminated, and dried equipment would be wrapped in aluminium foil or sealed in reclosable plastic bags. If field decontamination is necessary, all methanol and hexane rinses will be collected in appropriate containers for proper disposal at a later time. Cores from the Etoliko lagoon will be processed aseptically in the lab and samples will be collected every 3-5cm for amplicon sequence analysis, and selectively for metagenome, single cell genome and metatranscriptome analysis. Samples for Single Cell analysis will be kept in betaine as it is described previously 3. Samples for the culture-dependent approach will be processed immediately (see also WP4). All metadata, and future generated data will be stored in an SQL in a Web-based platform that will be developed.
16S rRNA and ITS2 amplicon sequencing will be used to phylogenetically characterize the cores in high resolution intervals using an Illumina HiSeq platform. In more detail, DNA will be extracted using the MoBio PowerSoil kit according to the manufacturer instructions. The V3-V4 region of the 16S rRNA gene will be amplified using universal primers 341F/805R 59 and the ITS2 region will be amplified with ITS2F-ITS4R primers. In addition to the specific priming regions, the above-mentioned primers will contain the Illumina adapters and unique indexing sequences to allow for multiplexing. Purified amplicons will be normalized to proper concentration by pooling equal amounts from each sample into a new microtube and will be sequenced using the Illumina HiSeq platform. Filtering and removal of short and poor quality sequences, as well as demultiplexing will be carried out with usearch v11.60. Quality filtered sequences will be clustered into OTUs according to sequence similarity using a 97% similarity threshold against the respective reference databases (Greengenes for prokaryotes and UNITE for eukaryotes) 61,62. The remaining sequences will be clustered into de novo OTUs with usearch3. Taxonomic identification and comparisons will be performed at the phylum and genus levels and alpha diversity values will be obtained using various diversity indices (observed species, Chao estimate, abundance-based coverage estimator [ACE], Shannon and Simpson diversity indices). The nucleotide sequence dataset will be deposited in the National Center for Biotechnology Information (NCBI) Short Read Archive (SRA). Based on the data analysis putative depths that contain microbial dark matter in a high proportion (more than 50%) will be selected for metagenomic, single cell and metatranscriptomic analysis. A minimum set of 5-8 depths will be selected.
Task 1.3 – Visual identity & promotional materials M1-M15.
Task Leader: Assoc. Prof. George Tsiamis, University of Patras
Milestones Sample collection (M4); 1.2 Identification of key depth for multi-omic analysis (M15)
Deliverables
Visual identity: a professional designer will be contracted to develop the project identity that will be applied to the website and communication material. A visual toolkit (templates, guidelines) will be provided to ensure overall consistency (M3). Project leaflet and poster: an informative leaflet and a poster will be produced by M6 to provide information about the project objectives. These will be made available as an electronic document and in printed version. Project website: a project website (M3 online) will be created and maintained, which will enable effective communication within the project and with external stakeholders, the press and the wider public. The project website will be a key dissemination and communication instrument, with up-to-date, easy and quick access to information on the project and the grant programme. Social media: Twitter (sending function) and LinkedIn (discussion feeds function), that will be integrated into the project website. The project will also have a Facebook page, to ensure wider dissemination to different age groups and target audiences. Project newsletter: a UniqELT e-newsletter will by produced (M18) to inform all potential stakeholders on the project progress. It will be published on the project website by an online integration to the news blog, accessible through the project website, to inform about the project latest events and relevant news items.
1.1 Core samples from Etoliko lagoon (T1.1; M4) - 1.2 Phylogenetic analysis based on 16S rRNA and ITS2 amplicon sequencing data (T1.2; M15) – 1.3 Project website (T1.3; M6) – 1.4 Project leaflet and poster (T1.3; M6) – 1.5 Project e-newsletter – release 1 (T1.3; M18)