GeneStructureTools 1.4.0
GeneStructureTools is a package for the manipulation and analysis of transcribed gene structures.
We have provided functions for importing Whippet and leafcutter alternative splicing data, and the analysis of these splicing events. Splicing events can also be defined manually if you are using a different splicing analysis tool to Whippet. For specific events - currently including exon skipping, intron retention, alternative splice site usage and alternative first/last exons - transcripts can be made in silico which use the two splicing modes - i.e. transcripts containing and transcripts skipping an exon. These transcripts do not have to be pre-annotated, and thus all potential isoforms can be compared for an event.
Current comparisons of transcripts include annotating and analysing ORF and UTR features (length, locations, difference/similarity between transcripts), and predicting nonsense mediated decay (NMD) potential.
We also have functions for re-annotation of .GTF features, such as annotating UTRs as 3’ or 5’, and assigning a broader biotype for genes and transcripts so more informative analysis can be performed between these classes.
Currently, very few available tools output splicing event type information (i.e. exon skipping, intron retention) within tested genes. GeneStructureTools currently has functions for processing data from:
Data Preparation
We have pre-prepared data from mouse embryonic stem cell (ESC) development (Gloss et. al, 2017, Accession Number GSE75028), at days 0 and 5, and run whippet on each replicate using the reccomended parameters and the Gencode vM14 annotation. You can download the whippet, leafcutter, and DEXSeq files here.
You will also need to download the Gencode GTF file from here.
For the purposes of this vignette, small subsets of these data are available in the package data (inst/extdata).
Data provided is typical output for leafcutter and Whippet. For details on what each file contains, please refer to their respective manuals ( leafcutter | Whippet ).
DEXSeq data is processed as reccomended by the (DEXSeq) manual. The script used to process raw output is here
To run a full analysis on Whippet output, you will need the raw .psi.gz (percent spliced in) and .jnc.gz (junction read counts) files for each sample. In addition, you will need to compare conditions using whippet-delta.jl
and have a resulting .diff.gz file.
Read in Whippet files from downloaded data
# Load packages
library(GeneStructureTools)
library(GenomicRanges)
library(stringr)
library(BSgenome.Mmusculus.UCSC.mm10)
library(Gviz)
library(rtracklayer)
# list of files in the whippet directory
whippet_file_directory <- "~/Downloads/GeneStructureTools_VignetteFiles/"
# read in files as a whippetDataSet
wds <- readWhippetDataSet(whippet_file_directory)
# create a sample table with sample id, condition and replicate
whippet_sampleTable <- data.frame(sample=c("A01","B01","A21","B21"),
condition=c("01","01","21","21"),
replicate=(c("A","B","A","B")))
# read in gtf annotation
gtf <- rtracklayer::import("~/Downloads/gencode.vM14.annotation.gtf.gz")
exons <- gtf[gtf$type=="exon"]
transcripts <- gtf[gtf$type=="transcript"]
# add first/last annotation (speeds up later steps)
if(!("first_last" %in% colnames(mcols(exons)))){
t <- as.data.frame(table(exons$transcript_id))
exons$first_last <- NA
exons$first_last[exons$exon_number == 1] <- "first"
exons$first_last[exons$exon_number ==
t$Freq[match(exons$transcript_id, t$Var1)]] <- "last"
}
# specify the BSGenome annotation
g <- BSgenome.Mmusculus.UCSC.mm10::BSgenome.Mmusculus.UCSC.mm10
Read in Whippet files from package data
# Load packages
suppressPackageStartupMessages({
library(GeneStructureTools)
library(GenomicRanges)
library(stringr)
library(BSgenome.Mmusculus.UCSC.mm10)
library(Gviz)
library(rtracklayer)
})
# list of files in the whippet directory
whippet_file_directory <- system.file("extdata","whippet/",
package = "GeneStructureTools")
# read in files as a whippetDataSet
wds <- readWhippetDataSet(whippet_file_directory)
# create a sample table with sample id, condition and replicate
whippet_sampleTable <- data.frame(sample=c("A01","B01","A21","B21"),
condition=c("01","01","21","21"),
replicate=(c("A","B","A","B")))
# read in gtf annotation
gtf <- rtracklayer::import(system.file("extdata","example_gtf.gtf",
package = "GeneStructureTools"))
exons <- gtf[gtf$type=="exon"]
transcripts <- gtf[gtf$type=="transcript"]
# add first/last annotation (speeds up later steps)
if(!("first_last" %in% colnames(mcols(exons)))){
t <- as.data.frame(table(exons$transcript_id))
exons$first_last <- NA
exons$first_last[exons$exon_number == 1] <- "first"
exons$first_last[exons$exon_number ==
t$Freq[match(exons$transcript_id, t$Var1)]] <- "last"
}
# specify the BSGenome annotation
g <- BSgenome.Mmusculus.UCSC.mm10::BSgenome.Mmusculus.UCSC.mm10
Only the file containg the leafcutter results for each intron, and the .gtf file used with leafcutter needs to be read in for results processing. The leafcutter results file is generated after running prepare_results.R on your data, then extracting out the intron data table.
First, find the location of the leafviz2table.R script:
#find location of the script
system.file("extdata","leafviz2table.R", package = "GeneStructureTools")
Then run it on your leafviz output .RData file. The first argument is the leafviz output RData file, and the second is the name of the table you wish to write the intron results to.
Rscript leafviz2table.R leafviz.RData per_intron_results.tab
We have an processed example file available in extdata/leafcutter with a small sample of significant events.
# read in gtf annotation
gtf <- rtracklayer::import(system.file("extdata","example_gtf.gtf", package = "GeneStructureTools"))
exons <- gtf[gtf$type=="exon"]
# specify the BSGenome annotation
g <- BSgenome.Mmusculus.UCSC.mm10::BSgenome.Mmusculus.UCSC.mm10
# list of files in the leafcutter directory
leafcutter_files <- list.files(system.file("extdata","leafcutter/", package = "GeneStructureTools"),full.names = TRUE)
intron_results <- read.delim(leafcutter_files[grep("per_intron_results.tab", leafcutter_files)], stringsAsFactors = FALSE)
# filter events for significance
wds <- filterWhippetEvents(
whippetDataSet = wds,
probability = 0.95, # min probability
psiDelta = 0.1, # min change in PSI
eventTypes = "all", # all event types
minCounts = 100, # mean of at least 100 counts in one condition
sampleTable = whippet_sampleTable)
# check for changes in gene/transcript structure
whippet_summary <- whippetTranscriptChangeSummary(wds,
exons = exons,
transcripts = transcripts,
BSgenome = g,
NMD = FALSE # ignore nonsense mediated decay
)
head(whippet_summary)
## gene node coord strand type psi_a
## 1 ENSMUSG00000032883.15 3 chr1:78663141-78663280 + CE 0.35304
## 2 ENSMUSG00000024038.16 6 chr17:31527310-31528401 + CE 0.13662
## 3 ENSMUSG00000018379.17 5 chr11:88049216-88049411 + RI 0.37136
## 4 ENSMUSG00000034064.14 3 chr16:38549434-38549636 - AA 0.38094
## 5 ENSMUSG00000035478.14 2 chr10:80399122-80399217 - AD 0.45017
## 6 ENSMUSG00000026421.14 2 chr1:135729147-135729274 + AF 0.84840
## psi_b psi_delta probability complexity entropy
## 1 0.13494 0.21810 1.000 K1 0.9619
## 2 0.27537 -0.13875 0.998 K1 0.9006
## 3 0.21276 0.15861 1.000 K1 0.9590
## 4 0.23379 0.14716 0.990 K1 0.9638
## 5 0.33980 0.11036 0.953 K1 0.9937
## 6 0.99487 -0.14647 1.000 K1 0.6421
## unique_name comparison condition_1
## 1 ENSMUSG00000032883.15_chr1:78663141-78663280_CE_3 01_v_21 01
## 2 ENSMUSG00000024038.16_chr17:31527310-31528401_CE_6 01_v_21 01
## 3 ENSMUSG00000018379.17_chr11:88049216-88049411_RI_5 01_v_21 01
## 4 ENSMUSG00000034064.14_chr16:38549434-38549636_AA_3 01_v_21 01
## 5 ENSMUSG00000035478.14_chr10:80399122-80399217_AD_2 01_v_21 01
## 6 ENSMUSG00000026421.14_chr1:135729147-135729274_AF_2 01_v_21 01
## condition_2 coord_match condition_1_counts condition_2_counts node
## 1 21 1 89.5 213.5 3
## 2 21 2 706.0 456.5 6
## 3 21 3 569.5 662.0 5
## 4 21 4 103.5 109.0 3
## 5 21 5 98.5 107.5 2
## 6 21 6 51.5 196.5 2
## coord strand type psi_a psi_b psi_delta probability
## 1 chr1:78663141-78663280 + CE 0.35304 0.13494 0.21810 1.000
## 2 chr17:31527310-31528401 + CE 0.13662 0.27537 -0.13875 0.998
## 3 chr11:88049216-88049411 + RI 0.37136 0.21276 0.15861 1.000
## 4 chr16:38549434-38549636 - AA 0.38094 0.23379 0.14716 0.990
## 5 chr10:80399122-80399217 - AD 0.45017 0.33980 0.11036 0.953
## 6 chr1:135729147-135729274 + AF 0.84840 0.99487 -0.14647 1.000
## complexity entropy unique_name
## 1 K1 0.9619 ENSMUSG00000032883.15_chr1:78663141-78663280_CE_3
## 2 K1 0.9006 ENSMUSG00000024038.16_chr17:31527310-31528401_CE_6
## 3 K1 0.9590 ENSMUSG00000018379.17_chr11:88049216-88049411_RI_5
## 4 K1 0.9638 ENSMUSG00000034064.14_chr16:38549434-38549636_AA_3
## 5 K1 0.9937 ENSMUSG00000035478.14_chr10:80399122-80399217_AD_2
## 6 K1 0.6421 ENSMUSG00000026421.14_chr1:135729147-135729274_AF_2
## comparison condition_1 condition_2 coord_match condition_1_counts
## 1 01_v_21 01 21 1 89.5
## 2 01_v_21 01 21 2 706.0
## 3 01_v_21 01 21 3 569.5
## 4 01_v_21 01 21 4 103.5
## 5 01_v_21 01 21 5 98.5
## 6 01_v_21 01 21 6 51.5
## condition_2_counts id orf_length_bygroup_x
## 1 213.5 chr1:78663141-78663280 720
## 2 456.5 chr17:31527310-31528401 104
## 3 662.0 chr11:88049216-88049411 201
## 4 109.0 chr16:38549434-38549636 410
## 5 107.5 chr10:80399122-80399217 313
## 6 196.5 chr1:135729147-135729274 166
## orf_length_bygroup_y utr3_length_bygroup_x utr3_length_bygroup_y
## 1 720 1328 1328
## 2 468 74 74
## 3 253 763 411
## 4 317 1485 1488
## 5 281 185 185
## 6 166 1898 709
## utr5_length_bygroup_x utr5_length_bygroup_y filtered percent_orf_shared
## 1 460 320 FALSE 1.0000000
## 2 57 57 FALSE 0.2222222
## 3 37 37 FALSE 0.0000000
## 4 2 520 FALSE 0.7731707
## 5 192 192 FALSE 0.8977636
## 6 1135 2596 FALSE 1.0000000
## max_percent_orf_shared orf_percent_kept_x orf_percent_kept_y
## 1 1.0000000 1.0000000 1.0000000
## 2 0.2222222 1.0000000 0.2222222
## 3 0.7944664 0.0000000 0.0000000
## 4 0.7731707 0.7731707 1.0000000
## 5 0.8977636 0.8977636 1.0000000
## 6 1.0000000 1.0000000 1.0000000
###leafcutter
leafcutter_summary <- leafcutterTranscriptChangeSummary(intron_results,
exons = exons,
BSgenome = g,
NMD = FALSE,
showProgressBar = FALSE)
head(leafcutter_summary[!duplicated(leafcutter_summary$cluster),])
## cluster status loglr df p p.adjust genes
## 5 chr16:clu_1396 Success 20.16004 2 1.756329e-09 2.720554e-06 Eif4a2
## 1 chr10:clu_1204 Success 18.48627 2 9.365149e-09 7.253308e-06 Bclaf1
## 6 chr15:clu_1488 Success 23.03735 6 2.860878e-08 1.107875e-05 Tarbp2
## 2 chr17:clu_1281 Success 20.57316 4 2.506720e-08 1.107875e-05 Rnps1
## 8 chr5:clu_225 Success 16.41082 2 7.462287e-08 2.311817e-05 Hnrnpd
## 7 chr14:clu_1512 Success 15.00163 2 3.054042e-07 7.884518e-05 Ktn1
## FDR intron logef T01
## 5 2.720554e-06 chr16:23111896:23112351:clu_1396 0.30823371 0.2657849
## 1 7.253308e-06 chr10:20333572:20334499:clu_1204 -0.56647927 0.3521539
## 6 1.107875e-05 chr15:102518602:102519122:clu_1488 -0.47200141 0.2294254
## 2 1.107875e-05 chr17:24414734:24415041:clu_1281 -0.07302291 0.1145809
## 8 2.311817e-05 chr5:99967387:99976534:clu_225 0.55565460 0.3165242
## 7 7.884518e-05 chr14:47724970:47725929:clu_1512 -0.46578048 0.3338764
## T02 deltapsi chr start end clusterID verdict
## 5 0.4410376 0.1752527121 chr16 23111896 23112351 clu_1396 annotated
## 1 0.2269207 -0.1252331984 chr10 20333572 20334499 clu_1204 annotated
## 6 0.2597079 0.0302825713 chr15 102518602 102519122 clu_1488 annotated
## 2 0.1140728 -0.0005080613 chr17 24414734 24415041 clu_1281 annotated
## 8 0.3905444 0.0740202209 chr5 99967387 99976534 clu_225 annotated
## 7 0.1656536 -0.1682228169 chr14 47724970 47725929 clu_1512 annotated
## gene ensemblID transcripts constitutive.score
## 5 Eif4a2 ENSMUSG00000022884.14 + 1
## 1 Bclaf1 ENSMUSG00000037608.16 + 1
## 6 Tarbp2 ENSMUSG00000023051.10 + 1
## 2 Rnps1 ENSMUSG00000034681.16 + 1
## 8 Hnrnpd ENSMUSG00000000568.15 - 1
## 7 Ktn1 ENSMUSG00000021843.16 + 1
## orf_length_bygroup_x orf_length_bygroup_y utr3_length_bygroup_x
## 5 408 363 615
## 1 919 870 2442
## 6 132 274 1265
## 2 305 282 710
## 8 336 361 366
## 7 1327 1303 469
## utr3_length_bygroup_y utr5_length_bygroup_x utr5_length_bygroup_y filtered
## 5 857 34 34 FALSE
## 1 2442 236 236 FALSE
## 6 334 224 92 FALSE
## 2 710 293 59 FALSE
## 8 366 294 294 FALSE
## 7 469 32 32 FALSE
## percent_orf_shared max_percent_orf_shared orf_percent_kept_x
## 5 0.7671569 0.8897059 0.7671569
## 1 0.9466812 0.9466812 0.9466812
## 6 0.0000000 0.4817518 0.0000000
## 2 0.9245902 0.9245902 0.9245902
## 8 0.9307479 0.9307479 1.0000000
## 7 0.9819141 0.9819141 0.9819141
## orf_percent_kept_y
## 5 0.8622590
## 1 1.0000000
## 6 0.0000000
## 2 1.0000000
## 8 0.9307479
## 7 1.0000000
whippetTranscriptChangeSummary() combines several functions for analysing changes in gene structures. While this has been made to simplify analysis from whippet data, individual functions can be used on other data sources or manually annotated gene structures. It may also be helpful to run each individual step if you would like to manually investigate changes to genes.
Exon skipping, or cassette exon usage, occurs when a single exon is spliced out of the mature transcript.
1.a. Find skipped exon events
# filter out skipped exon events (coded as "CE")
# we will be looking at Ndufv3 (ENSMUSG00000024038.16)
wds.ce <- filterWhippetEvents(wds, psiDelta=0,probability=0,
event="CE", idList="ENSMUSG00000024038.16")
diffSplicingResults(wds.ce)
## gene node coord strand type psi_a
## 2 ENSMUSG00000024038.16 6 chr17:31527310-31528401 + CE 0.13662
## psi_b psi_delta probability complexity entropy
## 2 0.27537 -0.13875 0.998 K1 0.9006
## unique_name comparison condition_1
## 2 ENSMUSG00000024038.16_chr17:31527310-31528401_CE_6 01_v_21 01
## condition_2 coord_match condition_1_counts condition_2_counts
## 2 21 2 706 456.5
# psi_a = 0.137, psi_b = 0.275
# percentage of transcripts skipping exon 3 decreases from timepoint 1 to 21
# whippet outputs the skipped exon coordinates
coordinates(wds.ce)
## GRanges object with 1 range and 1 metadata column:
## seqnames ranges strand | id
## <Rle> <IRanges> <Rle> | <character>
## [1] chr17 31527310-31528401 + | chr17:31527310-31528401
## -------
## seqinfo: 5 sequences from an unspecified genome; no seqlengths
2. Find transcripts which overlap the skipped exon, and create normal & skipped exon isoforms
# find exons in the gtf that overlap the skipped exon event
exons.ce <- findExonContainingTranscripts(wds.ce,
exons = exons,
transcripts = transcripts)
# make skipped and included exon transcripts
# removes the skipped exon from all transcripts which contain it
skippedExonTranscripts <- skipExonInTranscript(skippedExons = exons.ce,
exons=exons,
match="skip",
whippetDataSet = wds.ce)
## make Gvis models
# set up for visualisation
gtr <- GenomeAxisTrack()
# all transcripts for the gene
geneModel.all <- GeneRegionTrack(makeGeneModel(exons[exons$gene_id ==
skippedExonTranscripts$gene_id[1]]),
name="Reference Gene",
showId=TRUE,
transcriptAnnotation = "transcript")
# reference transcript
geneModelNormal <- GeneRegionTrack(makeGeneModel(
skippedExonTranscripts[skippedExonTranscripts$set=="included_exon"]),
name="Reference Isoform",
showId=TRUE, fill="#4D7ABE",
transcriptAnnotation = "transcript")
# for the skipped exon transcript
geneModelSkipped <- GeneRegionTrack(makeGeneModel(
skippedExonTranscripts[skippedExonTranscripts$set=="skipped_exon"]),
name="Alternative Isoform",
showId=TRUE, fill="#94AFD8",
transcriptAnnotation = "transcript")
plotTracks(list(gtr,geneModel.all,geneModelNormal, geneModelSkipped),
extend.left = 1000, extend.right = 1000)
# Only the transcript isoform containing the skipped exon (exon 3)
# is used for analysis, and a 'novel' isoform is created by exon skipping
Intron Retention occurs when an intron is not spliced out of the mature transcript.
1. Create normal and retained isoform structures from whippet coordinates
# filter out retained events (coded as "RI")
# we will be looking at Srsf1 (ENSMUSG00000018379.17)
wds.ri <- filterWhippetEvents(wds, psiDelta=0,probability=0,
event="RI", idList="ENSMUSG00000018379.17")
diffSplicingResults(wds.ri)
## gene node coord strand type psi_a
## 3 ENSMUSG00000018379.17 5 chr11:88049216-88049411 + RI 0.37136
## psi_b psi_delta probability complexity entropy
## 3 0.21276 0.15861 1 K1 0.959
## unique_name comparison condition_1
## 3 ENSMUSG00000018379.17_chr11:88049216-88049411_RI_5 01_v_21 01
## condition_2 coord_match condition_1_counts condition_2_counts
## 3 21 3 569.5 662
2. Find transcripts which overlap the intron, and create normal & retained intron isoforms
# find exons pairs in the gtf that bound the retained intron event
exons.ri <- findIntronContainingTranscripts(wds.ri,
exons)
# make retained and non-retained transcripts
# adds the intron into all transcripts which overlap it
retainedIntronTranscripts <- addIntronInTranscript(exons.ri,
exons = exons,
whippetDataSet = wds.ri,
glueExons = TRUE)
## make Gviz models
# all transcripts for the gene
geneModel.all <- GeneRegionTrack(makeGeneModel(exons[exons$gene_id == retainedIntronTranscripts$gene_id[1]]),
name="Reference Gene",
showId=TRUE,
transcriptAnnotation = "transcript")
# reference transcript
geneModelNormal <- GeneRegionTrack(makeGeneModel(
retainedIntronTranscripts[retainedIntronTranscripts$set=="spliced_intron"]),
name="Reference Isoform",
showId=TRUE, fill="#4D7ABE",
transcriptAnnotation = "transcript")
# for the retained intron transcript
geneModelRetained <- GeneRegionTrack(makeGeneModel(
retainedIntronTranscripts[retainedIntronTranscripts$set=="retained_intron"]),
name="Alternative Isoform",
showId=TRUE, fill="#94AFD8",
transcriptAnnotation = "transcript")
# Only the transcript isoforms with exons at the boundries of the retained intron are used for analysis, and 'novel' isoforms are created by intron retention
plotTracks(list(gtr,geneModel.all,geneModelNormal, geneModelRetained),
extend.left = 1000, extend.right = 1000)
Creation of alternative donor/acceptor isoforms currently relies on junction read counts supplied by whippet.