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Target sequence design of padlock probes based on experimentally determined in situ synthesized cDNA fragments
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0001-7509-8071
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Padlock probes are widely used to target a short fragment of DNA. For example, in in situ sequencing (ISS), an image-based technology for highly multiplexed spatial gene expression analysis, cDNA target detection is mediated by padlock probes. Transcript counts from ISS generally has good correlation with next-generation sequencing read counts, but bias between different genes are also observed. Therefore, we developed a new method to isolate and sequence in situ synthesized cDNA and sought to use the read coverage information from it to guide padlock probe design. The results show limited correlation between cDNA library sequencing and ISS counts, but it can still help the probe design process by eliminating target sequences that are very unlikely to be detected. In addition, the method provides a way to systematically characterize in situ reverse transcription.

Keywords [en]
probe design, in situ sequencing
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-174737OAI: oai:DiVA.org:su-174737DiVA, id: diva2:1359446
Available from: 2019-10-09 Created: 2019-10-09 Last updated: 2019-10-10Bibliographically approved
In thesis
1. Towards comprehensive cellular atlases: High-throughput cell mapping by in situ sequencing
Open this publication in new window or tab >>Towards comprehensive cellular atlases: High-throughput cell mapping by in situ sequencing
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

With recent technological advancements in single-cell biology, many aspects of individual cells are characterized with unprecedented resolution and details. Cell types in human and model organisms are redefined, and multiple organ-wide atlases are proposed to integrate different types of data to provide a comprehensive view of biological systems at cellular resolution. Incorporating location information of cells in such atlases is crucial to understanding the structure and functions. Several spatially resolved transcriptomics technologies may serve this purpose, and in situ sequencing (ISS) is among the most powerful ones.

ISS detects the expression of tens to hundreds of genes in situ, i.e. inside preserved cells and tissues. ISS is a targeted approach, using probes designed to identify specific transcripts. Its key advantages, as compared to other spatially resolved gene expression analysis methods, are high throughput, cellular resolution and tissue compatibility, making it a tool ideally suited for spatial cell mapping. The work included in this thesis aims to develop tools and methods for this application.

In paper I, a network analysis tool was developed to analyze ISS and other spatially resolved data. The tool enables smooth visualization of large datasets and generates networks based on colocalization. It also includes functions to test statistical significance and resolve tissue heterogeneity.

In paper II, we studied spatio-temporal patterns of immune response in tuberculosis granuloma by targeting immune markers with ISS. Using the tool developed in paper I together with other methods, we established an immune response time course at the granuloma sites and found histologically different granulomas based on transcriptional information. The paper demonstrated that ISS can robustly detect transcripts in formalin-fixed paraffin-embedded tissues across biological samples and reveal biologically relevant structures.

In paper III, we developed probabilistic cell typing by in situ sequencing (pciSeq), a method to spatially map cell types defined by single-cell RNA-sequencing. pciSeq is an integrated pipeline that includes gene selection, image analysis, barcode calling and cell type calling. We mapped closely related interneuron cell types of the mouse hippocampal CA1 region in 14 coronal sections and validated the results against ground truth.

In paper IV, we investigated the quantification bias of ISS resulting from the probe target selection. We developed a method to sequence in situ synthesized cDNA and found that the read coverage of in situ cDNA library reflected ISS counts more closely than conventional RNA sequencing, making it possible, to some extent, to predict a probe’s performance and guide the probe design.

Taken together, the developments described in this thesis comprise several tools that make ISS suitable for building cellular atlases via large-scale spatial mapping.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 60
Keywords
Spatially resolved transcriptomics, in situ sequencing, cell type, spatial analysis
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-174755 (URN)978-91-7797-883-1 (ISBN)978-91-7797-884-8 (ISBN)
Public defence
2019-11-25, Air & Fire, SciLifeLab, Tomtebodavägen 23 A, Solna, 09:30 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Accepted. Paper 4: Manuscript.

Available from: 2019-10-30 Created: 2019-10-10 Last updated: 2019-10-22Bibliographically approved

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