analyzeAlternativSplicing: Analyse alternative splicing (including intron retention(s))

In kvittingseerup/IsoformSwitchAnalyzeR: Identify, Annotate and Visualize Isoform Switches with Functional Consequences from both short- and long-read RNA-seq data

Analyse alternative splicing (including intron retention(s))

Description

These function utilize the analysis of alternative splicing previously implemented in the now deprecated spliceR package which compares each isoform in a gene to the hypothetical pre-RNA generated by combining all the exons within a gene and classify the changes in alternative splicing. Not this version also only considerd expressed (aka analyzed in the switchAnalyzeRlist).

Usage

analyzeAlternativeSplicing(
    switchAnalyzeRlist,
    onlySwitchingGenes=TRUE,
    alpha=0.05,
    dIFcutoff = 0.1,
    showProgress=TRUE,
    quiet=FALSE
)

analyzeIntronRetention(
    switchAnalyzeRlist,
    onlySwitchingGenes = TRUE,
    alpha = 0.05,
    dIFcutoff = 0.1,
    showProgress = TRUE,
    quiet = FALSE
)

Arguments

switchAnalyzeRlist	A switchAnalyzeRlist object.
onlySwitchingGenes	A logic indicating whether to only analyze genes with isoform switches (as indicated by the alpha and dIFcutoff parameters). Default is FALSE.
alpha	The Cutoff used on the FDR correct p-values (q-values) for calling significance. Default is 0.05.
dIFcutoff	Cutoff used for minimum changes in (absolute) isoform usage before an isoform is considered eligible for switch testing. This cutoff can remove cases where isoforms with extremely low IF values are deemed significant and thereby included in the downstream analysis. This cutoff is analogous to having a (log2) fold change in a normal differential expression analysis of genes to ensure the DE genes have a certain effect size. Default is 0.1 (10%).
showProgress	A logic indicating whether to make a progress bar (if TRUE) or not (if FALSE). Default is TRUE.
quiet	A logic indicating whether to avoid printing progress messages. Default is FALSE

Details

The analyzeIntronRetention() is just a convenient wrapper for the analyzeIntronRetention() function to ensure backward compatibility.

Alternative splicing (including alternative transcription start sites (ATSS) and alternative transcription termination sites (ATTS)) are classified for each isoform comparing that isoform to the hypothetical pre-RNA generated by combining all the exons (after exclusion of retained introns) within a gene. Retained introns is defined as when one "exon" of one isoform overlaps two separate exons in other isoform.

Since the comparison is to the hypothetical pre-RNA the interpretation of an event is as follows:

• ES: Exon Skipping. Compared to the hypothetical pre-RNA a single exon was skipped in the isoform analyzed (for every ES event annotated).

• MEE: Mutually exclusive exon. Special case were two isoforms form the same gene contains two mutually exclusive exons and which are not found in any of the other isoforms from that gene.

• MES: Multiple Exon Skipping. Compared to the hypothetical pre-RNA multiple consecutive exon was skipped in the isoform analyzed (for every MES event annotated).

• IR: Intron Retention. Compared to the hypothetical pre-RNA an intron was retained in the isoform analyzed.

• A5: Alternative 5'end donor site. Compared to the hypothetical pre-RNA an alternative 5'end donor site was used. Since it is compared to the pre-RNA, the donor site used is per definition more upstream than the the pre-RNA (the upstream exon is shorter).

• A3: Alternative 3'end acceptor site. Compared to the hypothetical pre-RNA an alternative 3'end acceptor site was used. Since it is compared to the pre-RNA, the donor site used is per definition more downstream than the the pre-RNA (the downstream exon is shorter).

• ATSS: Alternative Transcription Start Sites. Compared to the hypothetical pre-RNA an alternative transcription start sites was used. Since it is compared to the pre-RNA, the ATSS site used is per definition more downstream than the the pre-RNA .

• ATTS: Alternative Transcription Termination Sites. Compared to the hypothetical pre-RNA an alternative transcription Termination sites was used. Since it is compared to the pre-RNA, the ATTS site used is per definition more upstream than the the pre-RNA.

Value

A switchAnalyzeRlist where the column IR indicating the number of Intron Retentions found in each transcript have been added to the isoform_features entry. NA is used if the transcript was not analyzed. Furthermore a data.frame (called 'AlternativeSplicingAnalysis'), where for each isoform_id containing the number of alternative splicing events found as well as the genomic coordinates of the affected region(s), is added to the switchAnalyzeRlist. In this data.frame genomic coordinates for each splice event are separated by ";" except for cases where there are multiple MES, then each set of coordinates belonging to a MES is separated by ',' (and then the coordinates belong to a specific MES is separated by ';').

Author(s)

Kristoffer Vitting-Seerup

References

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

• Vitting-Seerup et al. IsoformSwitchAnalyzeR: Analysis of changes in genome-wide patterns of alternative splicing and its functional consequences. bioRxiv (2018).

See Also

extractSplicingSummary
extractSplicingEnrichment
extractSplicingEnrichmentComparison
extractSplicingGenomeWide

Examples

### Load data
data("exampleSwitchListIntermediary")

### Perform analysis
exampleSwitchListAnalyzed <- analyzeAlternativeSplicing(exampleSwitchListIntermediary, quiet=TRUE)

### Inspect result
head(exampleSwitchListAnalyzed$AlternativeSplicingAnalysis) # the first 6 does not have any intron retentions (IR)
table(exampleSwitchListAnalyzed$AlternativeSplicingAnalysis$IR) # there appear to be 7 transcripts that have an intron retention

kvittingseerup/IsoformSwitchAnalyzeR documentation built on Jan. 14, 2024, 11:30 p.m.