| 1. |
- Klevebring, Daniel, 1981-
(författare)
-
On Transcriptome Sequencing
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.
|
|
| 2. |
- Johansson, Tomas, et al.
(författare)
-
Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue
- 2004
-
Ingår i: Molecular Plant-Microbe Interactions. - 0894-0282 .- 1943-7706. ; 17:2, s. 202-215
-
Tidskriftsartikel (refereegranskat)abstract
- In order to obtain information on genes specifically expressed in the ectomycorrhizal symbiosis, 3,555 expressed sequence tags (ESTs) were analyzed from a cDNA library constructed from ectomycorrhiza formed between the basidiomycete Paxillus involutus and birch (Betula pendula). cDNA libraries from saprophytically growing fungus (3,964 ESTs) and from axenic plants (2,532 ESTs) were analyzed in parallel. By clustering all the EST obtained, a nonredundant set of 2,284 unique transcripts of either fungal or plant origin were identified. The expression pattern of these genes was analyzed using cDNA microarrays. The analyses showed that the plant and fungus responded to the symbiosis by altering the expression levels of a number of enzymes involved in carbon metabolism. Several plant transcripts with sequence similarities to genes encoding enzymes in the tricarboxylic cycle and electron transport chain were down regulated as compared with the levels in free-living roots. In the fungal partner, a number of genes encoding enzymes in the lipid and secondary metabolism were down regulated in mycorrhiza as compared with the saprophytically growing mycelium. A substantial number of the ESTs analyzed displayed significant sequence similarities to proteins involved in biotic stress responses, but only a few of them showed differential expression in the mycorrhizal tissue, including plant and fungal metallothioneins and a plant defensin homologue. Several of the genes that were differentially expressed in the mycorrhizal root tissue displayed sequence similarity to genes that are known to regulate growth and development of plant roots and fungal hyphae, including transcription factors and Rho-like GTPases.
|
|
| 3. |
- Zhu, Y., et al.
(författare)
-
Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis
- 2017
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 45:5, s. 2629-2643
-
Tidskriftsartikel (refereegranskat)abstract
- Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.
|
|
| 4. |
- Asp, Michaela, et al.
(författare)
-
Spatially Resolved Transcriptomes : Next Generation Toolsfor Tissue Exploration
- 2020
-
Ingår i: Bioessays. - : Wiley. - 0265-9247 .- 1521-1878. ; 42:10, s. 1900221-
-
Tidskriftsartikel (refereegranskat)abstract
- Recent advances in spatially resolved transcriptomics have greatly expandedthe knowledge of complex multicellular biological systems. The field hasquickly expanded in recent years, and several new technologies have beendeveloped that all aim to combine gene expression data with spatialinformation. The vast array of methodologies displays fundamentaldierences in their approach to obtain this information, and thus,demonstrate method-specific advantages and shortcomings. While the field ismoving forward at a rapid pace, there are still multiple challenges presentedto be addressed, including sensitivity, labor extensiveness, tissue-typedependence, and limited capacity to obtain detailed single-cell information.No single method can currently address all these key parameters. In thisreview, available spatial transcriptomics methods are described and theirapplications as well as their strengths and weaknesses are discussed. Futuredevelopments are explored and where the field is heading to is deliberatedupon.
|
|
| 5. |
- Hasmats, Johanna, et al.
(författare)
-
Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1, s. e84785-
-
Tidskriftsartikel (refereegranskat)abstract
- Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.
|
|
| 6. |
- Hildebrandt, Franziska, 1994-, et al.
(författare)
-
Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver
- 2021
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology. Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along transcriptional gradients and suggest the presence of previously uncharacterized structures within liver tissue.
|
|
| 7. |
- Lundeberg, Joakim
(författare)
-
Spatially resolved gene expression
- 2022
-
Ingår i: European Journal of Human Genetics. - : SPRINGERNATURE. - 1018-4813 .- 1476-5438. ; 30:SUPPL 1, s. 7-7
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
|
|
| 8. |
- Savolainen, Peter, et al.
(författare)
-
mtDNA tandem repeats in domestic dogs and wolves : Mutation mechanism studied by analysis of the sequence of imperfect repeats
- 2000
-
Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 17:4, s. 474-488
-
Tidskriftsartikel (refereegranskat)abstract
- The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations.
|
|
| 9. |
- Ståhl, Patrik, Dr., et al.
(författare)
-
Visualization and analysis of gene expression in tissue sections by spatial transcriptomics
- 2016
-
Ingår i: Science. - : AMER ASSOC ADVANCEMENT SCIENCE. - 0036-8075 .- 1095-9203. ; 353:6294, s. 78-82
-
Tidskriftsartikel (refereegranskat)abstract
- Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.
|
|
| 10. |
- Larsson, Ludvig
(författare)
-
Mapping Transcriptomes in Tissues
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the past few decades, the advent of pioneering biotechnological methods has allowed scientists to analyze the molecular components of multicellular organisms with remarkable precision. The field of transcriptomics has witnessed a rapid development of technologies for gene expression profiling of biological samples. These gene expression profiles offer crucial insights into biological processes that underlie organism development, homeostasis, and disease-causing dysregulation. Modern transcriptomics technologies can profile samples at various degrees of precision and resolution, and when combined, they contribute to a comprehensive understanding of the complex molecular mechanisms that shape entire organisms. Some of these molecular mechanisms occur at the microscopic scale, controlled by communication between nearby cells. Other mechanisms depend on coordinated efforts between large networks of cells organized into tissues and organs. Cells, tissues and organs represent hierarchical levels of structural organization, and each level plays a vital role in the proper functioning of the organism. Gene expression profiling technologies yield comprehensive data that can be harnessed to explore and characterize biological phenomena within and across these structural levels. The central theme of this thesis revolves around the use of experimental technologies and computational methods in the field of transcriptomics to enhance our understanding of multicellular life. Particular attention is directed at a technology known as Visium, which has held an important position in the field in recent years. The research articles included in this thesis demonstrate the applications of Visium and related technologies in biological research.In article I, we present a computational toolbox for processing, analyzing, and visualizing Visium data, assembled into an open-source package written in the R programming language. The package facilitates the characterization of gene expression profiles in tissue sections and seamlessly integrates expression data with corresponding histological images. This computational framework was used extensively for the data analyses presented in articles II, III and IV and the articles listed in the extended list of publications.In article II, we report one of the first spatiotemporal, transcriptomics atlases of the developing human heart. The atlas encompasses three developmental time points during the first trimester, and is constructed from gene expression data from isolated cells and intact tissue sections. Joint analysis of this data enabled characterization of the transcriptomic profiles and the cellular composition of anatomical domains within the heart, illuminating biological processes that underlie cardiac morphogenesis in humans.Article III constitutes a study of the transcriptomic landscape of the murine colon generated using spatially resolved transcriptomics. By folding the organ into a roll, we successfully obtained tissue sections covering the entire colon, enabling organ-wide transcriptomic profiling. Sections were acquired from a healthy colon and a colon recovering from damage due to treatment with a tissue-damaging substance. Data-driven analysis of the healthy colon unveiled a previously undiscovered molecular regionalization from the proximal to distal parts. In the recovering colon, we observed dramatic alterations in the distal tissues, while the proximal parts remained more similar to the healthy colon. In the injured distal colon, we mapped multiple gene expression programs associated with distinct biological responses to tissue injury.In article IV, we introduce an experimental protocol that makes the Visium method compatible with fresh frozen tissue samples with low RNA quality. The protocol was tested on human prostate cancer, lung, colon, small intestine and pediatric brain tumor tissue samples, as well as mouse brain and cartilage tissue samples. Together, these tissue samples represented a wide selection of specimens with varying composition and RNA quality. Through comparative analyses, we demonstrated that the proposed experimental protocol surpassed the standard Visium protocol in performance for samples with low to moderate RNA integrity.Finally, in article V, we present an updated R package for Visium data analysis. This R package builds upon the work presented in article I, but offers a more versatile and efficient computational framework. The package features web-based tools for interactive data exploration, image processing methods and methods to map cell types in tissue sections. Additionally, it includes several spatially aware analysis methods that incorporate information about distances between measurements to investigate biological phenomena that exhibit spatial patterns.
|
|
