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Single-cell transcriptomics expands sampled protist diversity and provides insights into niche adaptation
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
(English)Manuscript (preprint) (Other academic)
National Category
Evolutionary Biology
Identifiers
URN: urn:nbn:se:uu:diva-392616OAI: oai:DiVA.org:uu-392616DiVA, id: diva2:1349179
Note

The majority of eukaryotic diversity is dominated by microbial species, also referred to as protists. Among this diversity are several taxonomic groups where access to genomic data is sparse. In an effort to provide gene content information about such species and potentially identify new lineages of protists, we sequenced 124 single-cell transcriptomes covering eight of the major clades in the eukaryotic tree. Among those, we generated transcriptome data for free-living prokinetoplastids and osmotrophic euglenids, two groups of protists for which very limited sequence data has been available to date. We also have significantly expanded the known genomic diversity within Metamonada and Katablepharidae. Additionally, we report several new algae and ciliate species that are only distantly related to lineages that have been previously found. The data generated here has both enabled us to confidently place our newly identified, free-living protist lineages in the eukaryotic tree, and to get an insight into their biology and different niche adaptation strategies.

Available from: 2019-09-06 Created: 2019-09-06 Last updated: 2019-09-10
In thesis
1. Novel insights into protist diversity and niche adaptation using single cell transcriptomics
Open this publication in new window or tab >>Novel insights into protist diversity and niche adaptation using single cell transcriptomics
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protists are a polyphyletic group of microbes that represents the vast majority of eukaryotic diversity. Despite this, most sequencing efforts targeting eukaryotes have been focused on animals, fungi and plants. The sequencing bias towards multicellular organisms can partially be explained by the difficulty in cultivating protists, which is needed in traditional sequencing workflows. In this thesis, single-cell RNA sequencing has been used to generate transcriptome data from environmental protists, without being dependent on establishing a culture. These transcriptome data have been used to discover novel protist diversity, as well as exploring the cell biology of two ciliates.

In the first chapter, transcriptomes of cell fragments were generated for the ciliate Stentor. This ciliate is well-known for its ability to repair drastic cellular wounds, and the transcriptomes uncovered genes involved in processes such as cell cycle, signaling and microtubule-based movement to be activated during Stentor regeneration.

Spirostomum semivirescens is another ciliate, whose transcriptome was generated using single-cell RNA sequencing. The transcriptome data suggest that S. semivirescens is using rhodoquinol-dependent fumarate reduction for respiration in environments with low levels of oxygen.

Single-cell RNA sequencing was further used to target cells smaller than Stentor and Spirostomum. By generating 124 transcriptomes of environmental protists, a high number of novel lineages could be identified. The generated transcriptome data included free-living prokinetoplastids, non-photosynthetic euglenids, metamonads and katablepharids.

A few modifications to the single-cell RNA sequencing protocol Smart-seq2 were necessary to generate the 124 transcriptomes of small protists cells. The impact of these modifications to Smart-seq2 was benchmarked using Giardia intestinalis. The generated single-cell transcriptomes revealed that addition of freeze-thaw cycles to Smart-seq2 improved transcript recovery. Finally, we propose a protocol that allows identification of failed cDNA reactions, based only on measuring DNA concentration, without compromising on transcript recovery. Reducing the dependency on quality control will be important if single-cell RNA sequencing would be done in a high-throughput workflow.

In conclusion, single-cell RNA sequencing can be a powerful tool for studying protist diversity and biology. In particular, it has the potential to efficiently uncover protist diversity, provided that a robust and efficient method to isolate single cells from the environment is established.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 52
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1853
Keywords
Protists, microbial eukaryotes, cultivation-independent methods, single-cell RNA sequencing, phylogenomics
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-392618 (URN)978-91-513-0747-3 (ISBN)
Public defence
2019-10-25, B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-10-01 Created: 2019-09-07 Last updated: 2019-10-15

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