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Gene expression analysis by signature pyrosequencing
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 289, no 1-2, 31-39 p.Article in journal (Refereed) Published
Abstract [en]

 We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.

Place, publisher, year, edition, pages
2002. Vol. 289, no 1-2, 31-39 p.
Keyword [en]
3 '-tagged cDNA library, virtual chip, atherosclerosis, DNA sequencing
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5169DOI: 10.1016/S0378-1119(02)00548-6ISI: 000177867000005OAI: oai:DiVA.org:kth-5169DiVA: diva2:7975
Note
QC 20100915Available from: 2004-10-08 Created: 2004-10-08 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Computational approaches for in-depth analysis of cDNA sequence tags
Open this publication in new window or tab >>Computational approaches for in-depth analysis of cDNA sequence tags
2004 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Major recent improvements in biotechnology have led to an accelerated production of DNA sequences. The completion of the human genome sequence, along with the genomes of more than two hundred other species, has marked the arrival of the genome era. The ultimate goal is to understand the structure and function of genomes and their genes. This thesis has focused on the computational analysis of complementary DNA (cDNA) sequences. These are copies of mRNA transcripts that correspond to the coding regions of genomes.

Studying the expression patterns of genes is essential for understanding gene function. Many gene expression profiling techniques generate short sequence tags that derive from transcripts. A pilot study was performed to assess the feasibility of using the pyrosequencing platform for gene expression analysis. The sequences generated by pyrosequencing in most cases (≈ 85%) were long enough (> 18 nucleotides) to uniquely identify the corresponding transcripts through database searches. Aspects of transcript identification by short sequence tags were further investigated in a number of public databases, revealing that a tag length 16-17 nucleotides was sufficient for unique identifi- cation.

Longer transcript representations are obtained from expressed sequence tag (EST) sequencing. Method development for the analysis and maintenance of large EST data sets has been performed on data from poplar, which is a tree of commercial interest to the forest biotechnology industry. In 2003 a large ESTsequencing project reached > 100 000 reads, providing a unique resource for tree biology research. ESTs have been grouped into clusters and singletons that represent potential genes. Preliminary analyses have estimated gene content in Populus to be very similar to that of model organism Arabidopsis thaliana.

EST data collections provide a rich source for mining polymorphisms. A software application was developed and applied to EST data from two Populus species, and candidate single nucleotide polymorphisms (SNPs) were recorded. A study of genetic variation between the species revealed a striking similarity, with orthologous pairs being > 98% identical on the protein level.

Keywords: cDNA, EST, gene expression, SNP, SAGE, polymorphism, assembly, clustering, DNA sequencing, pyrosequencing, mRNA transcript, orthology, tree biotechnology, restriction enzyme

Place, publisher, year, edition, pages
Bioteknologi, 2004
Keyword
Biotechnology, cDNA, EST, gene expression, SNP, SAGE, polymorphism, Bioteknik
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-23 (URN)91-7283-837-X (ISBN)
Public defence
2004-10-08, E1, KTH, Lindstedsvägen 3, Stockholm, 13:00
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Available from: 2004-10-08 Created: 2004-10-08 Last updated: 2012-03-21Bibliographically approved

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Uhlén, Mathias

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