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Promoting SINgle cell GEnomics to explore the ecology and evolution of hidden microeuKaryotes

Periodic Reporting for period 2 - SINGEK (Promoting SINgle cell GEnomics to explore the ecology and evolution of hidden microeuKaryotes)

Reporting period: 2018-01-01 to 2020-05-31

Most of the extant diversity of eukaryotes remains poorly characterized, limiting society's understanding of, for example, the rise and diversification of parasite lineages, the evolutionary origin and variability of metabolic and cellular networks, and which microbes provide certain key functions in the global environment. Classical methodologies have provided up to now limited progress in our understanding of eukaryotic biology. The SINGEK project exploited the Single cell genomics (SCG), one of the most promising modern techniques given its capacity to provide genomic information of one cell at a time, a possibility unimaginable a few years ago. Applying SCG to microbial eukaryotes was particularly challenging when compared to SCG on prokaryotes, because of target genomes of greater complexity. The exploitation of SCG in this field was crucial for two main reasons: 1) to get de novo genomes of microbial species for which a pure culture was not available, and 2) to study the cell variability of genomic data and expressed genes within a microbial population.

Despite the challenges and practical barriers to explore SCG to its full potential, the main objective of the SINGEK project was to train a new generation of researchers in SCG of microbial eukaryotes and do so by addressing a set of fundamental and inter-related scientific questions on evolution and ecology.

Results and data generated during this project served to consolidate the SCG approach from back to end, improving the routines for single cell sorting, for whole genome amplification and sequencing, and for bioinformatic processing (including assembly, gene prediction and functional annotation). Multiple de novo genomes, representing hidden taxa in different positions of the eukaryotic tree of life, were obtained. These new genomes were used to improve the reconstruction of the eukaryotic radiation, to unveil parasitic or symbiotic interactions, to investigate the ecological potential of unknown species in base of the gene set detected, and to study the biogeography and gene expression of uncultured lineages across natural systems. Tools to study cell to cell variation in gene content and gene expression were also developed. The new datasets and tools generated are available in both public repositories and in-house web-searching platforms prepared by SINGEK members.
The main training and scientific milestones of the SINGEK project were accomplished , leading to the following achievements:

- PHDs
A total of 4 ESRs have already obtained their PhD degree and 9 ESRs are in the process of finishing it in the next following months.

24 peer-reviewed publications (and 6 more already submitted) co-authored by ESRs of the SINGEK network ( including a special issue entitled “Single Cell Ecology” published in the prestigious journal Philosophical Transactions of the Royal Society B by some consortium members. Also, 49 contributions to International Conferences and more than 30 manuscripts currently in preparation.

Workshops on: Collaborative learning, Core Comm. Skills and Time Management, Genomics, Online Sci. Comm., Research Ethics, Single Cell Sorting and Analysis, Genome Assembly, Advanced Scientific Writing, Genome Annotation, Thesis Writing and Presenting, and Career Development.

Website ( Intranet Site, Facebook page, Research Gate profile, Twitter account (@SINGEK_ITN), 4 videos ( Internal forum tool, Blog ( genome databases and web applications ( and project (
The core of the research programme of the SINGEK project covered all aspects of the SCG pipeline. The scientific activities and major research questions addressed by the ESRs were:
ESR4: Javier Florenza (PI: S. Bertilsson, Uppsala Univ.) - WP3
A sequencing-based approximation to phytoflagellate bacterivory at individual resolution

ESR12: Sara Castillo (PI: S. Pagliara, Univ. of Exeter) - WP3
Novel Microfluidics for the study of microeukaryotes

ESR6: Atefeh Lafzi (PI: I. Gut / H. Heyn, CNAG) - WP4
Benchmarking Single-Cell RNA sequencing protocols for the Human Cell Atlas Projects

ESR10: François Bucchini (PI: K. Vandepoele, VIB, Ghent Univ.) - WP4
Taxonomic and functional analysis of de novo transcriptomes of microeukaryotes

ESR15: Imer Muhovic (PI: R. Massana, ICM-CSIC) - WP4
Simple tools for Microbial Ecology

ESR1: Aurelie Labarre (PI: R. Massana , ICM-CSIC) - WP5
Understanding the ocean: Functioning of the hidden majority of uncultured bacterivorous

ESR2: Ina Deutschmann (PI: R. Logares, ICM-CSIC) - WP5
Disentangling microbial association networks in the ocean

ESR8: Laura Rubinat (PI: C. de Vargas, CNRS-Roscoff) - WP5
Towards the functional characterization of underwater forests

ESR11: Vanessa Smilanski (PI: T. Richards, Univ. of Exeter) - WP5
Phylogenetic analysis of an amphibian pathogen reveals its rapid geographic expansion

ESR14: Alexandra Beliavskaia (PI: A. Darby, Univ. of Liverpool) - WP5
Genomic analysis of protists infecting blood-feeding arthropods

ESR3: Konstantina Mitsi (PI: I. Ruiz-Trillo, IBE-CSIC) - WP6
Eukaryotic megasystematics through the lens of metabarcoding and metagenomics

ESR5: Max Emil Schön (PI: T. Ettema, Uppsala Univ.) - WP6
Advancing the knowledge of the deep root of eukaryotes

ESR7: Luis Javier Galindo (PI: P. López-García, CNRS-PSUD) - WP6
Deep eukaryotic phylogenomics: The Holomycota branch

ESR9: Luis Felipe de Almeida Benites (PI: G. Piganeau, CNRS-Banyuls) - WP6
Single Cell Ecogenomics of picoplanktonic algae

ESR13: Raphael Gollnisch (PI: K. Rengefors / D. Ahrén, Lund Univ.) - WP6
Single-cell population genomics of an invasive microeukaryote

The SINGEK project covered a number of aspects which will have a long-term impact on the ESRs’ careers: (1) a cross disciplinary environment, (2) a dedicated scientific training, (3) an extensive training and mentoring programme on complementary skills, (4) a primary tool to facilitate the progress of their individual research and (5) secondments. In overall, being part of the SINGEK Consortium enhanced ESR’s knowledge and skills beyond what would have been possible by individual organisations.

For the SINGEK host institutions, their ability to attract high qualified and talented ESRs provided a significant impact. The visibility of the activities undertaken by all SINGEK Consortium Members increased with the involvement of ESRs, which played a key role in the establishment of new interactions and collaborations.

As a benefit for the ERA, SINGEK brought together individual partners from different sectors with varied and diverse expertises under one umbrella to cover all aspects of an emerging technology with high scientific potential: SCG. So, for th The networking of SINGEK scientific teams and their associated ESRs worked together for the first time and placed the European community in a unique position in terms of the capacity to innovate SCG science, becoming pivotal for the future application of SCG to address ecological, evolutionary as well a diverse array of biotechnological or biomedicine questions.