extractGenomeWideSplicingAnalysis: Genome wide Analysis of alternative splicing

Description Usage Arguments Details Value Author(s) References See Also Examples

Description

This function enables a genome wide analysis of changes in isoform usage of isoforms with a common annotation.

Specifically this function extract isoforms of interest and for each splicing type (such as exon skipping) the global distribution of IF (measuring isoform usage) are plotted for each subset of features in that category (e.g with exons skipping vs without exon skipping). This enables a global analysis of isoforms with a common annotation.

The isoforms of interest can either be defined by isoforms form gene differentially expressed, isoform that are differential expressed or isoforms from genes with isoform switching - as controlled by featureToExtract. Please note that the extractSplicingEnrichment function probably more relevant than using featureToExtract='isoformUsage' since it directly uses the paired information from switches.

This function offers both visualization of the result as well as analysis via summary statistics of the comparisons.

Usage

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extractSplicingGenomeWide(
    switchAnalyzeRlist,
    featureToExtract = 'isoformUsage',
    splicingToAnalyze = 'all',
    alpha=0.05,
    dIFcutoff = 0.1,
    log2FCcutoff = 1,
    violinPlot=TRUE,
    alphas=c(0.05, 0.001),
    localTheme=theme_bw(),
    plot=TRUE,
    returnResult=TRUE
)

Arguments

switchAnalyzeRlist

A switchAnalyzeRlist object containing the result of an isoform switch analysis (such as the one provided by isoformSwitchTestDEXSeq()) as well as additional annotation data for the isoforms.

featureToExtract

This argument, given as a string, defines the set isoforms which should be analyzed. The available options are:

  • 'isoformUsage' (Default): Analyze a subset of isoforms defined by change in isoform usage (controlled by dIFcutoff) and the significance of the change in isoform expression (controlled by alpha). Please note that the extractSplicingEnrichment function probably more relevant than using featureToExtract='isoformUsage' since it directly uses the paired information from switches.

  • 'isoformExp' :Analyze a subset of isoforms defined by change in isoform expression (controlled by log2FCcutoff) and the significance of the change in isoform expression (controlled by alpha)

  • 'geneExp' :Analyze all isoforms from a subset of genes defined by by change in gene expression (controlled by log2FCcutoff) and the significance of the change in gene expression (controlled by alpha)

  • 'all' : Analyze all isoforms stored in the switchAnalyzeRlist (note that this is highly depending on the parameter reduceToSwitchingGenes in isoformSwitchTestDEXSeq - which should be set to FALSE (default is TRUE) if the 'all' option should be used here).

splicingToAnalyze

A string indicating which consequences should be considered. See details for description. Default is all.

alpha

The cutoff which the FDR correct p-values (q-values) must be smaller than for calling significant switches. Default is 0.05.

dIFcutoff

The cutoff which the changes in (absolute) isoform usage must be larger than before an isoform is considered switching. This cutoff can remove cases where isoforms with (very) low dIF values are deemed significant and thereby included in the downstream analysis. This cutoff is analogous to having a cutoff on log2 fold change in a normal differential expression analysis of genes to ensure the genes have a certain effect size. Default is 0.1 (10%).

log2FCcutoff

The cutoff which the changes in (absolute) isoform or gene expression must be larger than before an isoform is considered for inclusion.

violinPlot

A logical indicating whether to make a violin plots (if TRUE) or boxplots (if FALSE). Violin plots will always have added 3 black dots, one of each of the 25th, 50th (median) and 75th percentile of the data. Default is TRUE.

alphas

A numeric vector of length two giving the significance levels represented in plots. The numbers indicate the q-value cutoff for significant (*) and highly significant (***) respectively. Default 0.05 and 0.001 which should be interpret as q<0.05 and q<0.001 respectively). If q-values are higher than this they will be annotated as 'ns' (not significant).

localTheme

General ggplo2 theme with which the plot is made, see ?ggplot2::theme for more info. Default is theme_bw().

plot

A logic indicting whether the analysis should be plotted. If TRUE and returnResult = FALSE the ggplot2 object will be returned instead. Default is TRUE.

returnResult

A logical indicating whether to return a data.frame with summary statistics of the comparisons (if TRUE) or not (if FALSE). If FALSE (and plot=TRUE) the ggplot2 object will be returned instead. Default is TRUE.

Details

The classification of alternative splicing is always compared to the hypothetical pre-mRNA constructed by concatenating all exons from isoforms of the same gene.

The alternative splicing types, which can be passed to splicingToAnalyze must be a combination of:

The significance test is performed with R's build in wilcox.test() (aka 'Mann-Whitney-U') with default parameters and resulting p-values are corrected via p.adjust() using FDR (Benjamini-Hochberg).

Value

If plot=TRUE: A plot of the distribution of IF values as a function of the annotation and condition compared. If returnResult=TRUE: A data.frame with the summary statistics from the comparison of the two conditions with a Wilcox.test.

Author(s)

Kristoffer Vitting-Seerup

References

Vitting-Seerup et al. The Landscape of Isoform Switches in Human Cancers. Mol. Cancer Res. (2017).

See Also

isoformSwitchTestDEXSeq
isoformSwitchTestDRIMSeq
analyzeAlternativeSplicing
extractSplicingSummary
extractSplicingEnrichment
extractSplicingEnrichmentComparison

Examples

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### Load example data
data("exampleSwitchListAnalyzed")

extractSplicingGenomeWide( exampleSwitchListAnalyzed )

kvittingseerup/IsoformSwitchAnalyzeR documentation built on May 12, 2021, 12:25 p.m.