Differential Transcript Expression of 42 vs 44 colorectal cancer samples in R
Salomón Marquez
2026-02-03
1 Load dataset
Let’s ensure that the previously mentioned file are in our working directory.
# Specify a working directory
directory <- "/Volumes/DataWinBackup/00_MASTER UOC BIOINFORMATICA - TEMPORAL/TFM-UOC/DTU44CRC"
setwd(directory)
list.files() [1] "all_parameters_isoforms.csv"
[2] "DTU ANALYSIS CLUSTER"
[3] "DTU_2crc.RData"
[4] "Dtu_44crc_cache"
[5] "Dtu_44crc_cluster.Rmd"
[6] "Dtu_44crc.html"
[7] "DTU_44crc.RData"
[8] "Dtu_44crc.Rmd"
[9] "dtu-read-rds-cluster_cache"
[10] "dtu-read-rds-cluster.html"
[11] "dtu-read-rds-cluster.Rmd"
[12] "dtu-read-rds-isoforms_42_44 copy.Rmd"
[13] "dtu-read-rds-isoforms_42_44_cache"
[14] "dtu-read-rds-isoforms_42_44_files"
[15] "dtu-read-rds-isoforms_42_44-copy_cache"
[16] "dtu-read-rds-isoforms_42_44-copy.Rmd"
[17] "dtu-read-rds-isoforms_42_44.html"
[18] "dtu-read-rds-isoforms_42_44.Rmd"
[19] "dtu-read-rds-isoforms.Rmd"
[20] "extractConsequenceEnrichmentComparison.csv"
[21] "extractConsequenceSummary.csv"
[22] "extractSplicingEnrichmentComparison.csv"
[23] "extractSplicingGenomeWide.csv"
[24] "extractSplicingSummary.csv"
[25] "figures"
[26] "gencode.v49.primary_assembly.annotation.filtered.gtf.gz"
[27] "gene.SummarizedExperiment.metadata.rds"
[28] "genome.transcripts.fa.gz"
[29] "genome.transcripts.gencode.fa.gz"
[30] "Homo_sapiens.GRCh38.115.filtered.gtf.gz"
[31] "mySwitchList_consequences_42_44.rds"
[32] "mySwitchList_saturn_consequences.rds"
[33] "salmon.merged.transcript_counts_gencode.tsv"
[34] "salmon.merged.transcript_counts.tsv"
[35] "salmon.merged.transcript_tpm_gencode.tsv"
[36] "salmon.merged.transcript_tpm.tsv"
[37] "salmon.merged.transcript.SummarizedExperiment.rds"
[38] "salmonQuant_inspection.rds"
[39] "tables"
[40] "top_switch_genes.csv"
[41] "top_switch_isoforms.csv"
[42] "tx2gene.tsv" # Load file
mySwitchList <- readRDS("mySwitchList_consequences_42_44.rds")
# [1] 'aSwitchList' 'counts' 'directory' 'myDesign' 'tpm'# View salmonQuant
names(mySwitchList) [1] "isoformFeatures" "exons"
[3] "conditions" "designMatrix"
[5] "sourceId" "isoformCountMatrix"
[7] "isoformRepExpression" "runInfo"
[9] "orfAnalysis" "isoformRepIF"
[11] "ntSequence" "isoformSwitchAnalysis"
[13] "aaSequence" "AlternativeSplicingAnalysis"
[15] "intronRetentionAnalysis" "switchConsequence" head(mySwitchList, 3) iso_ref gene_ref isoform_id gene_id
110561 isoComp_00001664 geneComp_00000111 ENST00000337334.7 ABHD12B
condition_1 condition_2 gene_name gene_biotype iso_biotype
110561 normal_eocrc tumor_eocrc ABHD12B protein_coding protein_coding
gene_overall_mean gene_value_1 gene_value_2 gene_stderr_1 gene_stderr_2
110561 2.970463 2.135247 3.788574 0.4050145 1.35647
gene_log2_fold_change gene_q_value iso_overall_mean iso_value_1
110561 0.8243139 NA 0.3661316 0.3411125
iso_value_2 iso_stderr_1 iso_stderr_2 iso_log2_fold_change iso_q_value
110561 0.3847805 0.07567231 0.2025121 0.1691176 NA
IF_overall IF1 IF2 dIF isoform_switch_q_value
110561 0.1631526 0.1817588 0.072125 -0.1096338 0.9997642
gene_switch_q_value PTC IR switchConsequencesGene
110561 0.4261566 FALSE 0 NA
[ reached 'max' / getOption("max.print") -- omitted 2 rows ]# Summary
mySwitchList$isoformFeatures iso_ref gene_ref isoform_id gene_id
110561 isoComp_00001664 geneComp_00000111 ENST00000337334.7 ABHD12B
condition_1 condition_2 gene_name gene_biotype iso_biotype
110561 normal_eocrc tumor_eocrc ABHD12B protein_coding protein_coding
gene_overall_mean gene_value_1 gene_value_2 gene_stderr_1 gene_stderr_2
110561 2.970463 2.135247 3.788574 0.4050145 1.35647
gene_log2_fold_change gene_q_value iso_overall_mean iso_value_1
110561 0.8243139 NA 0.3661316 0.3411125
iso_value_2 iso_stderr_1 iso_stderr_2 iso_log2_fold_change iso_q_value
110561 0.3847805 0.07567231 0.2025121 0.1691176 NA
IF_overall IF1 IF2 dIF isoform_switch_q_value
110561 0.1631526 0.1817588 0.072125 -0.1096338 0.9997642
gene_switch_q_value PTC IR switchConsequencesGene
110561 0.4261566 FALSE 0 NA
[ reached 'max' / getOption("max.print") -- omitted 1842 rows ]2 Review mySwithList object
# Summary
extractSwitchSummary(mySwitchList, filterForConsequences = T) Comparison nrIsoforms nrSwitches nrGenes
1 normal_eocrc vs tumor_eocrc 14 10 10
2 normal_locrc vs tumor_locrc 132 122 103
3 Combined 140 128 109# Summary
extractSwitchSummary(mySwitchList, filterForConsequences = TRUE, alpha = 0.05, dIFcutoff = 0.05) Comparison nrIsoforms nrSwitches nrGenes
1 normal_eocrc vs tumor_eocrc 14 14 10
2 normal_locrc vs tumor_locrc 140 189 103
3 Combined 148 198 109
# Extract top switching genes (by q-value) from each comparison
top_switch_genes <- extractTopSwitches(mySwitchList, filterForConsequences = TRUE,
extractGenes = TRUE, alpha = 0.05, dIFcutoff = 0.05, n = 109, inEachComparison = FALSE,
sortByQvals = TRUE)
keep <- c("gene_id", "condition_1", "condition_2", "gene_switch_q_value", "Rank")
top_switch_genes <- top_switch_genes[keep]
top_switch_genes gene_id condition_1 condition_2 gene_switch_q_value Rank
1 TPM2 normal_eocrc tumor_eocrc 0.0001116680 1
2 SORBS2 normal_locrc tumor_locrc 0.0001544585 2
3 TPM2 normal_locrc tumor_locrc 0.0001544585 3
4 FSIP1 normal_locrc tumor_locrc 0.0001622092 4
5 OSBPL1A normal_locrc tumor_locrc 0.0002382206 5
6 MAP3K20 normal_locrc tumor_locrc 0.0003502090 6
7 CD44 normal_locrc tumor_locrc 0.0004922797 7
8 CXCL12 normal_locrc tumor_locrc 0.0006191435 8
9 NLN normal_locrc tumor_locrc 0.0006191435 9
10 HNF4A normal_locrc tumor_locrc 0.0009308128 10
[ reached 'max' / getOption("max.print") -- omitted 99 rows ]# Save results
write.csv(top_switch_genes, "top_switch_genes.csv")# Extract top switching genes (by q-value) from each comparison
top_switch_isoforms <- extractTopSwitches(mySwitchList, filterForConsequences = TRUE,
extractGenes = FALSE, alpha = 0.05, dIFcutoff = 0.05, n = 148, inEachComparison = FALSE,
sortByQvals = TRUE)
keep <- c("isoform_id", "gene_id", "condition_1", "condition_2", "dIF", "isoform_switch_q_value",
"Rank")
top_switch_isoforms <- top_switch_isoforms[keep]
top_switch_isoforms isoform_id gene_id condition_1 condition_2 dIF
1 ENST00000378292.9 TPM2 normal_eocrc tumor_eocrc -0.133
2 ENST00000698539.1 SORBS2 normal_locrc tumor_locrc -0.233
3 ENST00000378292.9 TPM2 normal_locrc tumor_locrc -0.173
4 ENST00000642527.1 FSIP1 normal_locrc tumor_locrc 0.112
5 ENST00000319481.8 OSBPL1A normal_locrc tumor_locrc 0.294
6 ENST00000338983.7 MAP3K20 normal_locrc tumor_locrc -0.273
7 ENST00000263398.11 CD44 normal_locrc tumor_locrc -0.311
isoform_switch_q_value Rank
1 0.0001116680 1
2 0.0001544585 2
3 0.0001544585 3
4 0.0001622092 4
5 0.0002382206 5
6 0.0003502090 6
7 0.0004922797 7
[ reached 'max' / getOption("max.print") -- omitted 141 rows ]# Save results
write.csv(top_switch_isoforms, "top_switch_isoforms.csv")3 Analysis of Individual Isoform Switching
3.1 Top 3 EOCRC switch genes
3.2 Top 3 LOCRC switch genes
4 Genes with overlapped isoforms
5 Genome-wide Summaries
5.1 Consequence Enrichment Analysis
{r, extract_consequence_enrichment, echo=FALSE extractConsequenceEnrichment( mySwitchList, consequencesToAnalyze='all', countGenes = FALSE, analysisOppositeConsequence = TRUE, localTheme = theme_bw(base_size = 14), returnResult = TRUE )
5.2 Splicing Enrichment Analysis
condition_1 condition_2 AStype nUp nDown
1 normal_eocrc tumor_eocrc A3 gain (paired with A3 loss) 1 2
2 normal_eocrc tumor_eocrc A5 gain (paired with A5 loss) 1 3
3 normal_eocrc tumor_eocrc ATSS gain (paired with ATSS loss) 2 4
4 normal_eocrc tumor_eocrc ATTS gain (paired with ATTS loss) 5 2
propUp propUpCiLo propUpCiHi propUpPval propUpQval Significant
1 0.3333333 0.008403759 0.9057007 1.000000 1 FALSE
2 0.2500000 0.006309463 0.8058796 0.625000 1 FALSE
3 0.3333333 0.043271868 0.7772219 0.687500 1 FALSE
4 0.7142857 0.290420864 0.9633074 0.453125 1 FALSE
Comparison
1 normal_eocrc\nvs\ntumor_eocrc
2 normal_eocrc\nvs\ntumor_eocrc
3 normal_eocrc\nvs\ntumor_eocrc
4 normal_eocrc\nvs\ntumor_eocrc
[ reached 'max' / getOption("max.print") -- omitted 11 rows ]
5.3 Overview Plots
6 Analysis of overlapped isoforms
# Extract gene data
overlapGenes <- c("TPM2", "LMNB2", "MAP3K20", "HOXB-AS3")
four_genes_data <- mySwitchList$isoformFeatures %>%
dplyr::filter(gene_name %in% overlapGenes, isoform_switch_q_value < 0.05, abs(dIF) >
0.05) %>%
dplyr::select(isoform_id, gene_name, iso_biotype, condition_1, condition_2, dIF,
isoform_switch_q_value, gene_switch_q_value, gene_log2_fold_change) %>%
dplyr::arrange(isoform_switch_q_value) %>%
dplyr::mutate(direction = ifelse(dIF > 0, "Up in tumor", "Down in tumor"))
# View results
print(four_genes_data) isoform_id gene_name iso_biotype condition_1 condition_2
1 ENST00000378292.9 TPM2 protein_coding normal_eocrc tumor_eocrc
2 ENST00000378292.9 TPM2 protein_coding normal_locrc tumor_locrc
3 ENST00000338983.7 MAP3K20 protein_coding normal_locrc tumor_locrc
4 ENST00000329305.6 TPM2 protein_coding normal_locrc tumor_locrc
5 ENST00000329305.6 TPM2 protein_coding normal_eocrc tumor_eocrc
dIF isoform_switch_q_value gene_switch_q_value gene_log2_fold_change
1 -0.1325048 0.0001116680 0.0001116680 -2.476681
2 -0.1732318 0.0001544585 0.0001544585 -2.536791
3 -0.2727864 0.0003502090 0.0003502090 1.306201
4 0.1148136 0.0018008163 0.0001544585 -2.536791
5 0.1009333 0.0025111748 0.0001116680 -2.476681
direction
1 Down in tumor
2 Down in tumor
3 Down in tumor
4 Up in tumor
5 Up in tumor
[ reached 'max' / getOption("max.print") -- omitted 7 rows ]# Save results Format decimals of top_genes dataframe
format_numeric <- function(x) {
if (is.numeric(x)) {
formatted <- ifelse(abs(x) < 0.001 & !is.na(x), formatC(x, format = "e",
digits = 2), as.character(round(x, 4)))
return(formatted)
} else {
return(x)
}
}
# Apply format_numeric function
four_genes_data_formatted <- four_genes_data %>%
mutate(across(where(is.numeric), format_numeric))
four_genes_data_formatted isoform_id gene_name iso_biotype condition_1 condition_2 dIF
1 ENST00000378292.9 TPM2 protein_coding normal_eocrc tumor_eocrc -0.1325
2 ENST00000378292.9 TPM2 protein_coding normal_locrc tumor_locrc -0.1732
3 ENST00000338983.7 MAP3K20 protein_coding normal_locrc tumor_locrc -0.2728
4 ENST00000329305.6 TPM2 protein_coding normal_locrc tumor_locrc 0.1148
5 ENST00000329305.6 TPM2 protein_coding normal_eocrc tumor_eocrc 0.1009
isoform_switch_q_value gene_switch_q_value gene_log2_fold_change
1 1.12e-04 1.12e-04 -2.4767
2 1.54e-04 1.54e-04 -2.5368
3 3.50e-04 3.50e-04 1.3062
4 0.0018 1.54e-04 -2.5368
5 0.0025 1.12e-04 -2.4767
direction
1 Down in tumor
2 Down in tumor
3 Down in tumor
4 Up in tumor
5 Up in tumor
[ reached 'max' / getOption("max.print") -- omitted 7 rows ]# Export table to .tex format
table_tex <- kable(four_genes_data_formatted, format = "latex", booktabs = TRUE,
linesep = "")
writeLines(table_tex, "tables/6_shared_isoforms.tex")# library(dplyr) overlapGenes <- c('TPM2', 'LMNB2', 'MAP3K20', 'HOXB-AS3') #
# Extraer todos los parámetros con los nombres correctos all_parameters <-
# mySwitchList$isoformFeatures %>% dplyr::filter(gene_name %in% overlapGenes,
# isoform_switch_q_value < 0.05, abs(dIF) > 0.05) %>% dplyr::select( #
# =========== IDENTIFICADORES =========== isoform_id, gene_id, gene_name,
# gene_biotype, iso_biotype, # =========== CONDICIONES =========== condition_1,
# condition_2, # =========== 1. GENE EXPRESSION =========== # Expresión génica
# total gene_exp_cond1 = gene_value_1, # Normal gene_exp_cond2 = gene_value_2,
# # Tumor gene_overall_mean, # Promedio general gene_stderr_1, # Error estándar
# cond1 gene_stderr_2, # Error estándar cond2 gene_log2_fold_change, # Log2FC
# del gen gene_q_value, # q-value del gen # =========== 2. ISOFORM EXPRESSION
# =========== # Expresión por isoforma iso_exp_cond1 = iso_value_1, # Normal
# iso_exp_cond2 = iso_value_2, # Tumor iso_overall_mean, # Promedio general
# iso_stderr_1, # Error estándar cond1 iso_stderr_2, # Error estándar cond2
# iso_log2_fold_change, # Log2FC del isoforma iso_q_value, # q-value del
# isoforma # =========== 3. ISOFORM USAGE =========== # Uso/fracción de
# isoforma IF_overall, # Fracción general IF_cond1 = IF1, # Fracción en
# condición 1 (0-1) IF_cond2 = IF2, # Fracción en condición 2 (0-1) dIF, #
# Cambio en fracción: IF2 - IF1 # =========== SIGNIFICANCIA SWITCHING
# =========== isoform_switch_q_value, gene_switch_q_value, # ===========
# CARACTERÍSTICAS ADICIONALES =========== PTC, # Premature Termination Codon
# IR, # Intron Retention switchConsequencesGene # Consecuencias del switching )
# %>% # =========== CÁLCULOS DERIVADOS =========== dplyr::mutate( # Fold
# changes lineales (a partir de log2FC) gene_fold_change =
# 2^gene_log2_fold_change, iso_fold_change = 2^iso_log2_fold_change, #
# Significancia de expresión génica gene_expr_sig = gene_q_value < 0.05, #
# Significancia de expresión de isoforma iso_expr_sig = iso_q_value < 0.05, #
# Significancia de switching switching_sig = isoform_switch_q_value < 0.05 &
# abs(dIF) > 0.05, # Dirección del switching switching_direction = case_when(
# dIF > 0.1 & isoform_switch_q_value < 0.05 ~ 'Significant Increase', dIF <
# -0.1 & isoform_switch_q_value < 0.05 ~ 'Significant Decrease',
# isoform_switch_q_value < 0.05 ~ 'Significant Small Change', TRUE ~
# 'Non-significant' ), # Clasificación por contribución
# isoform_contribution_cond1 = (iso_exp_cond1 / gene_exp_cond1) * 100,
# isoform_contribution_cond2 = (iso_exp_cond2 / gene_exp_cond2) * 100, # Cambio
# en contribución contribution_change = isoform_contribution_cond2 -
# isoform_contribution_cond1, # Dominancia dominance = case_when( IF_cond1 >
# 0.7 & IF_cond2 > 0.7 ~ 'Major in Both', IF_cond1 > 0.7 ~ 'Major in Normal',
# IF_cond2 > 0.7 ~ 'Major in Tumor', IF_cond1 > 0.5 | IF_cond2 > 0.5 ~
# 'Intermediate', TRUE ~ 'Minor' ) ) %>% # =========== ORDENAR ===========
# dplyr::arrange(gene_name, isoform_switch_q_value, desc(abs(dIF))) # Ver las
# primeras filas print(all_parameters, n = 15) write.csv(all_parameters,
# 'all_parameters_isoforms.csv', row.names = FALSE)7 R Session Information
─ Session info ───────────────────────────────────────────────────────────────
setting value
version R version 4.5.1 (2025-06-13)
os macOS Ventura 13.7.3
system x86_64, darwin20
ui X11
language (EN)
collate en_US.UTF-8
ctype en_US.UTF-8
tz Europe/Madrid
date 2026-02-03
pandoc 3.2 @ /Applications/RStudio.app/Contents/Resources/app/quarto/bin/tools/x86_64/ (via rmarkdown)
quarto 1.5.57 @ /Applications/RStudio.app/Contents/Resources/app/quarto/bin/quarto
─ Packages ───────────────────────────────────────────────────────────────────
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[1] /Library/Frameworks/R.framework/Versions/4.5-x86_64/Resources/library
* ── Packages attached to the search path.
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