Microarrays are a widely used tool for conducting high-throughput analyses in many areas of biological research. Microarray designs have become increasingly probe-rich, enabling the targeting of specific features, such as individual exons or single nucleotide polymorphisms (SNPs). These arrays have the potential to achieve quantitative high-throughput estimates of transcript abundances, but currently these estimates are affected by probe-specific biases such as hybridization to background or off-target transcripts. This thesis describes new methodologies developed for the "low-level" analysis of gene expression microarrays. By "low-level" we mean the parts of the data analysis that deal with background correction and the computation of quantitative indexes of transcript abundances.
We develop methods to improve low-level analysis of a new probe-rich microarray, Affymetrix exon arrays. Our methods include a probe-specific background correction in which the probe sequence is used to predict hybridization to non-specific transcripts and a probe selection strategy, which selects a subset of probes with highly correlated intensities across multiple samples to summarize gene expression. To study cross-hybridization, we map probes to a set of annotated mRNA transcripts, allowing a small number of mismatches or insertion-deletions between the two sequences. Based on a systematic study of the degree to which probes with a given match type to a transcript are affected by cross-hybridization, we developed a strategy to correct for cross-hybridization biases of gene-level expression estimates.
Our methods are shown to be effective for generating gene-level expression estimates for the analysis of a panel of diverse tissue samples. We compare exon array expression estimates with independent sources of biological data including Solexa ultra-high-throughput sequencing, serial analysis of gene expression (SAGE) and cross-species comparisons to validate our results.
With the transition of array technology from probe-poor to probe-rich designs, new arrays offer the possibility to study biological questions not assessable by current (probe-poor) arrays that have only a few probes targeted to localized regions of the transcript. Appropriate consideration of probe low-level effects will be useful for the detection of alternative splicing on Affymetrix exon arrays and for future microarray analysis.
|School Location:||United States -- California|
|Source:||DAI-B 69/10, Dissertation Abstracts International|
|Keywords:||Cross-hybridization, Exon-targeting microarrays, Gene expression, Microarray probes|
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