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Published on: November 11, 2014
Gene expression profiling by massively parallel sequencing
Tatiana Teixeira Torres1, Muralidhar Metta, Birgit Ottenwälder
1Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, 1210 Vienna, Austria.
Genome Research
|November 23, 2007
Summary
Massively parallel sequencing using 454 technology shows promise for gene expression profiling. Optimizing cDNA fragment size can overcome limitations, enabling accurate gene expression measurements and cross-species comparisons.
Area of Science:
- Genomics
- Molecular Biology
Background:
- Massively parallel sequencing offers high throughput and accuracy for gene expression profiling.
- Limited data existed on the suitability of 454 sequencing for this application.
Purpose of the Study:
- To evaluate the potential of 454 sequencing technology for gene expression profiling.
- To assess the suitability of 454 sequencing for analyzing gene expression in Drosophila melanogaster.
Main Methods:
- Utilized 454 sequencing technology to analyze cDNA fragments.
- Investigated the impact of cDNA fragment length on sequencing reads.
- Employed nebulization to generate 3' cDNA fragments.
- Performed gene expression measurements using restriction analysis and nebulization.
- Mapped cDNA fragments to genomic DNA.
Main Results:
- Short (<80 bp) and long (>300-400 bp) cDNA fragments were under-represented in 454 reads.
- Sequencing 3' cDNA fragments via nebulization mitigated length bias.
- Gene expression measurements for 80-300 bp fragments showed high correlation with microarray data (0.83-0.91).
- 97% of cDNA fragments mapped unambiguously to genomic DNA, indicating high accuracy.
Conclusions:
- 454 sequencing technology has significant potential for gene expression profiling.
- High mapping accuracy facilitates cross-species expression profile comparisons.
- Nebulization of cDNA fragments is a viable strategy to overcome length bias in 454 sequencing.
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