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Exome RNA sequencing reveals rare and novel alternative transcripts
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 1, p. e6-Article in journal (Refereed) Published
Abstract [en]

RNA sequencing has become an important method to perform hypothesis-free characterization of global gene expression. One of the limitations of RNA sequencing is that most sequence reads represent highly expressed transcripts, whereas low level transcripts are challenging to detect. To combine the benefits of traditional expression arrays with the advantages of RNA sequencing, we have used whole exome enrichment prior to sequencing of total RNA. We show that whole exome capture can be successfully applied to cDNA to study the transcriptional landscape in human tissues. By introducing the exome enrichment step, we are able to identify transcripts present at very low levels, which are below the level of detection in conventional RNA sequencing. Although the enrichment increases the ability to detect presence of transcripts, it also lowers the accuracy of quantification of expression levels. Our results yield a large number of novel exons and splice isoforms, suggesting that conventional RNA sequencing methods only detect a small fraction of the full transcript diversity. We propose that whole exome enrichment of RNA is a suitable strategy for genome-wide discovery of novel transcripts, alternative splice variants and fusion genes.

Place, publisher, year, edition, pages
2013. Vol. 41, no 1, p. e6-
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-180350DOI: 10.1093/nar/gks816ISI: 000312889900006PubMedID: 22941640OAI: oai:DiVA.org:uu-180350DiVA, id: diva2:549521
Available from: 2012-09-04 Created: 2012-09-04 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Genome-wide Characterization of RNA Expression and Processing
Open this publication in new window or tab >>Genome-wide Characterization of RNA Expression and Processing
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out.

In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis.

The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 939
Keywords
RNA sequencing, RNA splicing, RNA processing, Gene expression
National Category
Medical Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-209390 (URN)978-91-554-8784-3 (ISBN)
Public defence
2013-11-29, Rudbeck Salen, Dag Hammarskjölds väg 20,, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-10-18 Last updated: 2018-01-11
2. Sequence based analysis of neurodevelopmental disorders
Open this publication in new window or tab >>Sequence based analysis of neurodevelopmental disorders
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis the main focus is the use of methods and applications of next generation sequencing in order to study three of the most common neurodevelopmental disorders: intellectual disability, epilepsy and schizophrenia. A large fraction of the genes in our genome produce several distinct transcript isoforms through the process of splicing and there is an increasing amount of evidence pinpointing mutations affecting splicing as a mechanism of disease.  In Paper I we used exome capture of RNA in combination with sequencing in order to enrich for coding sequences. We show that this approach enables us to detect lowly expressed transcript and splice events that would have been missed in regular RNA sequencing using the same coverage.  In Paper II we selectively depleted the different transcripts of Quaking (QKI), a gene previously associated to schizophrenia. Using RNA sequencing we show that the effects of depletion differ between transcripts and that the QKI gene is a potential regulator of the Glial Fibrillary Acidic Protein (GFAP), a gene implicated in several diseases in the central nervous system.

De-novo mutations are frequently reported to be causative in neurodevelopmental disorders with a strong genetic component, such as epilepsy and intellectual disability. In Paper III we used exome sequencing in family trios where the child was diagnosed with both intellectual disability and epilepsy, focusing on finding de-novo mutations. We identified several previously unknown disease causing mutations in genes previously known to cause disease and used previously published interaction and mutation data to prioritize novel candidate genes. The most interesting result from this study are the implication of the HECW2 gene as a candidate gene in intellectual disability and epilepsy. In Paper IV we used RNA sequencing of post mortem brain tissue in a large cohort of schizophrenics and controls.  In this study we could show that the immune system and more specifically the complement system was dysregulated in a large fraction of patients. Further, using co-expression network we also found some evidence suggesting genes involved in axon development and maintenance.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1231
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-287407 (URN)978-91-554-9597-8 (ISBN)
Public defence
2016-06-14, C2:305, BMC, Husargatan 3, Uppsala, 09:30 (English)
Opponent
Supervisors
Available from: 2016-05-20 Created: 2016-04-24 Last updated: 2018-01-10

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Halvardson, JonatanZaghlool, AmmarFeuk, Lars

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