Identifying differential exon splicing using linear models and correlation coefficients.

BACKGROUND: With the availability of the Affymetrix exon arrays a number of tools have been developed to enable the analysis. These however can be expensive or have several pre-installation requirements. This led us to develop an analysis workflow for analysing differential splicing using freely available software packages that are already being widely used for gene expression analysis. The workflow uses the packages in the standard installation of R and Bioconductor (BiocLite) to identify differential splicing. We use the splice index method with the LIMMA framework. The main drawback with this approach is that it relies on accurate estimates of gene expression from the probe-level data. Methods such as RMA and PLIER may misestimate when a large proportion of exons are spliced. We therefore present the novel concept of a gene correlation coefficient calculated using only the probeset expression pattern within a gene. We show that genes with lower correlation coefficients are likely to be differentially spliced. RESULTS: The LIMMA approach was used to identify several tissue-specific transcripts and splicing events that are supported by previous experimental studies. Filtering the data is necessary, particularly removing exons and genes that are not expressed in all samples and cross-hybridising probesets, in order to reduce the false positive rate. The LIMMA approach ranked genes containing single or few differentially spliced exons much higher than genes containing several differentially spliced exons. On the other hand we found the gene correlation coefficient approach better for identifying genes with a large number of differentially spliced exons. CONCLUSION: We show that LIMMA can be used to identify differential exon splicing from Affymetrix exon array data. Though further work would be necessary to develop the use of correlation coefficients into a complete analysis approach, the preliminary results demonstrate their usefulness for identifying differentially spliced genes. The two approaches work complementary as they can potentially identify different subsets of genes (single/few spliced exons vs. large transcript structure differences).

Epigenetic Signatures of Cigarette Smoking.

BACKGROUND: DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders. METHODS AND RESULTS: To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15 907 blood-derived DNA samples from participants in 16 cohorts (including 2433 current, 6518 former, and 6956 never smokers). Comparing current versus never smokers, 2623 cytosine-phosphate-guanine sites (CpGs), annotated to 1405 genes, were statistically significantly differentially methylated at Bonferroni threshold of P<1×10-7 (18 760 CpGs at false discovery rate <0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases, and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant P<1×10-7 (2623 CpGs at false discovery rate <0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs. CONCLUSIONS: Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biological effects of smoking and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke.

Complex disease genetics: present and future translational applications.

A report on the British Atherosclerosis Society autumn meeting 'Genetics of Complex Diseases', Cambridge, UK, 17-18 September 2009.