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An efficient single-cell transcriptomics workflow to assess protist diversity and lifestyle
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
MS, Mississippi State, Mississippi State University, Department of Biological Sciences.
MS, Mississippi State, Mississippi State University, Department of Biological Sciences.
MS, Mississippi State, Mississippi State University, Department of Biological Sciences.
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(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: urn:nbn:se:uu:diva-392617OAI: oai:DiVA.org:uu-392617DiVA, id: diva2:1349182
Available from: 2019-09-06 Created: 2019-09-06 Last updated: 2019-09-16
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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