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Novel insights into protist diversity and niche adaptation using single cell transcriptomics
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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 [en]
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: urn:nbn:se:uu:diva-392618ISBN: 978-91-513-0747-3 (print)OAI: oai:DiVA.org:uu-392618DiVA, id: diva2:1349263
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
List of papers
1. RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration
Open this publication in new window or tab >>RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration
2018 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 28, no 8, p. 1281-1288.e3Article in journal (Refereed) Published
Abstract [en]

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-354956 (URN)10.1016/j.cub.2018.02.055 (DOI)000430694900049 ()29628369 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Research Council, 621-2009-4813EU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research , SSF-FFL5
Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2019-09-07Bibliographically approved
2. Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records
Open this publication in new window or tab >>Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records
2018 (English)In: Protist, ISSN 1434-4610, E-ISSN 1618-0941, Vol. 169, no 6, p. 875-886Article in journal (Refereed) Published
Abstract [en]

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600 km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1 mm in size, could have a global biogeographical distribution.

Keywords
Protist, stop codon, RNA-seq, anaerobic respiration, symbiotic algae, Heterotrich
National Category
Ecology
Identifiers
urn:nbn:se:uu:diva-372698 (URN)10.1016/j.protis.2018.08.001 (DOI)000452432800005 ()30447617 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council, 310039-PUZZLE_CELLSwedish Foundation for Strategic Research Swedish Research Council, 2015-04959
Note

Hunter N. Hines and Henning Onsbring contributed equally to this work.

Available from: 2019-01-09 Created: 2019-01-09 Last updated: 2019-09-07Bibliographically approved
3. Single-cell transcriptomics expands sampled protist diversity and provides insights into niche adaptation
Open this publication in new window or tab >>Single-cell transcriptomics expands sampled protist diversity and provides insights into niche adaptation
(English)Manuscript (preprint) (Other academic)
National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-392616 (URN)
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
4. An efficient single-cell transcriptomics workflow to assess protist diversity and lifestyle
Open this publication in new window or tab >>An efficient single-cell transcriptomics workflow to assess protist diversity and lifestyle
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Most diversity in the eukaryotic tree of life are represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, it is challenging to establish protist cultures, which further influence what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists are often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed. In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol optimized for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinaliswas used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we tried reducing the reaction volume and purifying with alternative beads to test different cost-reducing changes of Smart-seq2. Neither did improve the procedure, and cutting the volumes by half actually led to significantly fewer genes detected. We also added a 5’ biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency.

National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-392617 (URN)
Available from: 2019-09-06 Created: 2019-09-06 Last updated: 2019-09-16

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