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tx2ale: detect alternative last exons (ALE) from transcript data

Source: R/jambio-tx.R
tx2ale.Rd

detect alternative last exons (ALE) from transcript data

Usage

tx2ale(
  gtf = NULL,
  txdb = NULL,
  threeUtrGRL = NULL,
  detectedTx = NULL,
  tx2geneDF = NULL,
  iMatrix = NULL,
  aleMethod = c("first", "range"),
  verbose = FALSE,
  ...
)

Arguments

gtf

character path to a GTF file, used to import gene models using Bioconductor txdbmaker::makeTxDbFromGFF() to create a transcriptDb object. If supplied, and if tx2geneDF is NULL, then tx2geneDF will be created using makeTx2geneFromGtf().

txdb

a TxDb object as defined by Bioconductor package GenomicFeatures. Note that when reloading an R session, these objects usually become defunct, since they refer internally to a sqlite database stored in a temporary file. Therefore, when saving and reloading an R session, it is recommended to use the relevant functions AnnotationDbi::saveDb and AnnotationDbi::loadDb.

threeUtrGRL

GRangesList object of 3'UTR exons by transcript, whose names are available in the tx2geneDF data.frame with colname transcript_id.

detectedTx

optional character vector of transcripts detected, whose values match the transcript names in gtf, txdb, threeUtrGRL, and/or tx2geneDF as relevant to the supplied input data.

tx2geneDF

optional data.frame with colnames gene_id and transcript_id which are used to relate transcripts to genes. If either gtf or txdb are supplied, this data.frame can be determined from that data.

iMatrix

optional numeric matrix of transcript rows, and sample columns, containing expression abundance data. When supplied, a matrix will be created iMatrixByALE where the transcript abundance values have been aggregated per ALE as defined.

aleMethod

character value describing the method used to define ALE regions.

verbose

logical whether to print verbose output during processing.

Value

aleGRL GRangesList object containing the ALE ranges after processing. Compare to transcript models to see if 3'UTR regions have been properly handled.

multiALEgenes vector of gene_name values which contain multiple ALEs after processing.

tx2ale vector containing ALE_name values, named by transcript_id, used to aggregate per-transcript data into per-ALE data.

iMatrixByALE optional data matrix created only if iMatrix was supplied as input. The data matrix rows are ALE_name values after aggregating transcripts into ALEs. Note that only multi-ALE genes are included, all single-ALE genes are excluded.

Details

This function is intended to encapsulate logic used to define alternative last exons (ALE) based upon gene-transcript models. Specifically, it uses either the 5' end of all 3'UTR regions aleMethod="first", or the full 3'UTR range aleMethod="range", optionally using only detected transcripts if given by detectedTx. Any transcripts overlapping the criteria above are merged together by gene, to define a unique set of ALEs for each gene.

Note that when using aleMethod="first", 3'UTRs will be combined if the 5'end of the 3'UTR is identical, which means the length of the 3'UTR is not considered in terms of defining an ALE. The goal is to determine whether the ALE is or is not maintained during transcript processing.

Note that when using aleMethod="range", 3'UTRs are converted first to a range, which converts multi-exon 3'UTRs to the full range covered by the 3'UTR exons, and not specific exon ranges contained. It is mainly intended to focus on the specific scenario where alternative 3'UTRs for a give gene do not overlap in any way. In reality, several sources of GTF gene models showed unusual transcript isoforms that contained premature stop codons, thus annotating the remaining exons all as "3'UTR" and therefore causing all subsequent 3'UTR regions to be combined into one large spanning range. For this reason, we recommend using aleMethod="first" by default.

We noted substantially improved results when supplying detected transcripts via the parameter detectedTx, which greatly reduces the full set of possible ALE regions to use only those regions relevant and observed in the given RNA-seq dataset. In other words, there are a large number of possible ALE regions which have no supporting measured data in a particular RNA-seq experiment. It is possible that generating a superset of ALE regions may not be practically problematic in downstream analysis steps, however the impact may be seen predominantly during fase discovery rate (FDR) adjustment for multiple testing, especially if over half the annotated ALE regions have zero reported measurements. If they have no supporting measurements, they are effectively not being tested. As GTF files become increasingly annotated to cover every possible scenario, this issue is likely to become more impactful over time.

Input data can be in one of three forms:

  • gtf a GTF file such as those GTF files provided by Gencode

  • txdb a TxDb R object as produced by the Bioconductor package GenomicFeatures, suitable for use in calling GenomicFeatures::threeUTRsByTranscript()

  • threeUtrGRL a GenomicRanges::GRangesList object which is a list of 3'UTR exons by transcript. This input also requires tx2geneDF which is used to relate transcripts to genes.

Two important outputs are tx2ale which converts transcripts to ALE names, and if a matrix of transcript expression is supplied with iMatrix, then iMatrixByALE is a matrix of expression aggregated at the ALE level, suitable for differential testing in limma::diffSplice() or DEXSeq.

This function depends upon custom functions: annotateGRLfromGRL(), annotateGRfromGR(), and assignGRLexonNames().

See also

Other ALE and codon functions: ale2violin(), codonUsage2df(), dna2codon(), getFirstStrandedFromGRL(), jamCai()