Results from the univariate regressions performed using can be combined in a post-processing step to perform multivariate hypothesis testing. In this example, we fit on transcript-level counts and then perform multivariate hypothesis testing by combining transcripts at the gene-level. This is done with the function.

Import transcript-level counts

Read in transcript counts from the package.

library(readr)
library(tximport)
library(tximportData)

# specify directory
path <- system.file("extdata", package = "tximportData")

# read sample meta-data
samples <- read.table(file.path(path, "samples.txt"), header = TRUE)
samples.ext <- read.table(file.path(path, "samples_extended.txt"), header = TRUE, sep = "\t")

# read assignment of transcripts to genes
# remove genes on the PAR, since these are present twice
tx2gene <- read_csv(file.path(path, "tx2gene.gencode.v27.csv"))
tx2gene <- tx2gene[grep("PAR_Y", tx2gene$GENEID, invert = TRUE), ]

# read transcript-level quatifictions
files <- file.path(path, "salmon", samples$run, "quant.sf.gz")
txi <- tximport(files, type = "salmon", txOut = TRUE)

# Create metadata simulating two conditions
sampleTable <- data.frame(condition = factor(rep(c("A", "B"), each = 3)))
rownames(sampleTable) <- paste0("Sample", 1:6)

Standard dream analysis

Perform standard analysis at the transcript-level

library(variancePartition)
library(edgeR)

# Prepare transcript-level reads
dge <- DGEList(txi$counts)
design <- model.matrix(~condition, data = sampleTable)
isexpr <- filterByExpr(dge, design)
dge <- dge[isexpr, ]
dge <- calcNormFactors(dge)

# Estimate precision weights
vobj <- voomWithDreamWeights(dge, ~condition, sampleTable)

# Fit regression model one transcript at a time
fit <- dream(vobj, ~condition, sampleTable)
fit <- eBayes(fit)

Multivariate analysis

Combine the transcript-level results at the gene-level. The mapping between transcript and gene is stored in as a list.

# Prepare transcript to gene mapping
# keep only transcripts present in vobj
# then convert to list with key GENEID and values TXNAMEs
keep <- tx2gene$TXNAME %in% rownames(vobj)
tx2gene.lst <- unstack(tx2gene[keep, ])

# Run multivariate test on entries in each feature set
# Default method is "FE.empirical", but use "FE" here to reduce runtime
res <- mvTest(fit, vobj, tx2gene.lst, coef = "conditionB", method = "FE")

# truncate gene names since they have version numbers
# ENST00000498289.5 -> ENST00000498289
res$ID.short <- gsub("\\..+", "", res$ID)

Gene set analysis

Perform gene set analysis using on the gene-level test statistics.

# must have zenith > v1.0.2
library(zenith)
library(GSEABase)

gs <- get_MSigDB("C1", to = "ENSEMBL")

df_gsa <- zenithPR_gsa(res$stat, res$ID.short, gs, inter.gene.cor = .05)

head(df_gsa)
##                NGenes Correlation      delta        se     p.less   p.greater     PValue Direction
## M7078_chr2p16      30        0.05  1.4208384 0.5610910 0.99432899 0.005671015 0.01134203        Up
## M14982_chr7p13     26        0.05  1.1335492 0.5777005 0.97512013 0.024879873 0.04975975        Up
## M7314_chr4p14      25        0.05 -1.1344103 0.5825608 0.02575932 0.974240679 0.05151864      Down
## M5824_chr11p13     30        0.05 -1.0120371 0.5612285 0.03568377 0.964316230 0.07136754      Down
## M3783_chr2q37      73        0.05  0.8367603 0.4929617 0.95518099 0.044819012 0.08963802        Up
## M10517_chr4q24     21        0.05 -1.0062435 0.6060832 0.04844305 0.951556955 0.09688609      Down
##                      FDR
## M7078_chr2p16  0.9992274
## M14982_chr7p13 0.9992274
## M7314_chr4p14  0.9992274
## M5824_chr11p13 0.9992274
## M3783_chr2q37  0.9992274
## M10517_chr4q24 0.9992274

Session info

## R version 4.3.1 (2023-06-16)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.3 LTS
## 
## Matrix products: default
## BLAS:   /home/biocbuild/bbs-3.18-bioc/R/lib/libRblas.so 
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=en_GB             
##  [4] LC_COLLATE=C               LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                  LC_ADDRESS=C              
## [10] LC_TELEPHONE=C             LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## time zone: America/New_York
## tzcode source: system (glibc)
## 
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
##  [1] org.Hs.eg.db_3.18.0      msigdbr_7.5.1            GSEABase_1.64.0         
##  [4] graph_1.80.0             annotate_1.80.0          XML_3.99-0.14           
##  [7] AnnotationDbi_1.64.0     IRanges_2.36.0           S4Vectors_0.40.0        
## [10] Biobase_2.62.0           BiocGenerics_0.48.0      zenith_1.4.0            
## [13] tximportData_1.29.0      tximport_1.30.0          readr_2.1.4             
## [16] edgeR_4.0.0              pander_0.6.5             variancePartition_1.32.0
## [19] BiocParallel_1.36.0      limma_3.58.0             ggplot2_3.4.4           
## [22] knitr_1.44              
## 
## loaded via a namespace (and not attached):
##   [1] jsonlite_1.8.7              magrittr_2.0.3              farver_2.1.1               
##   [4] nloptr_2.0.3                rmarkdown_2.25              zlibbioc_1.48.0            
##   [7] vctrs_0.6.4                 memoise_2.0.1               minqa_1.2.6                
##  [10] RCurl_1.98-1.12             progress_1.2.2              S4Arrays_1.2.0             
##  [13] htmltools_0.5.6.1           curl_5.1.0                  broom_1.0.5                
##  [16] SparseArray_1.2.0           sass_0.4.7                  KernSmooth_2.23-22         
##  [19] bslib_0.5.1                 pbkrtest_0.5.2              plyr_1.8.9                 
##  [22] cachem_1.0.8                lifecycle_1.0.3             iterators_1.0.14           
##  [25] pkgconfig_2.0.3             Matrix_1.6-1.1              R6_2.5.1                   
##  [28] fastmap_1.1.1               GenomeInfoDbData_1.2.11     rbibutils_2.2.15           
##  [31] MatrixGenerics_1.14.0       digest_0.6.33               numDeriv_2016.8-1.1        
##  [34] colorspace_2.1-0            GenomicRanges_1.54.0        RSQLite_2.3.1              
##  [37] filelock_1.0.2              labeling_0.4.3              RcppZiggurat_0.1.6         
##  [40] fansi_1.0.5                 abind_1.4-5                 httr_1.4.7                 
##  [43] compiler_4.3.1              bit64_4.0.5                 aod_1.3.2                  
##  [46] withr_2.5.1                 backports_1.4.1             DBI_1.1.3                  
##  [49] gplots_3.1.3                MASS_7.3-60                 DelayedArray_0.28.0        
##  [52] corpcor_1.6.10              gtools_3.9.4                caTools_1.18.2             
##  [55] tools_4.3.1                 remaCor_0.0.16              glue_1.6.2                 
##  [58] nlme_3.1-163                grid_4.3.1                  reshape2_1.4.4             
##  [61] generics_0.1.3              snow_0.4-4                  gtable_0.3.4               
##  [64] tzdb_0.4.0                  tidyr_1.3.0                 hms_1.1.3                  
##  [67] utf8_1.2.4                  XVector_0.42.0              pillar_1.9.0               
##  [70] stringr_1.5.0               babelgene_22.9              vroom_1.6.4                
##  [73] splines_4.3.1               dplyr_1.1.3                 BiocFileCache_2.10.0       
##  [76] lattice_0.22-5              bit_4.0.5                   tidyselect_1.2.0           
##  [79] locfit_1.5-9.8              Biostrings_2.70.0           SummarizedExperiment_1.32.0
##  [82] RhpcBLASctl_0.23-42         xfun_0.40                   statmod_1.5.0              
##  [85] matrixStats_1.0.0           KEGGgraph_1.62.0            stringi_1.7.12             
##  [88] yaml_2.3.7                  boot_1.3-28.1               evaluate_0.22              
##  [91] codetools_0.2-19            archive_1.1.6               tibble_3.2.1               
##  [94] Rgraphviz_2.46.0            cli_3.6.1                   xtable_1.8-4               
##  [97] Rdpack_2.5                  munsell_0.5.0               jquerylib_0.1.4            
## [100] Rcpp_1.0.11                 GenomeInfoDb_1.38.0         EnvStats_2.8.1             
## [103] dbplyr_2.3.4                png_0.1-8                   Rfast_2.0.8                
## [106] parallel_4.3.1              blob_1.2.4                  prettyunits_1.2.0          
## [109] bitops_1.0-7                lme4_1.1-34                 mvtnorm_1.2-3              
## [112] lmerTest_3.1-3              scales_1.2.1                purrr_1.0.2                
## [115] crayon_1.5.2                fANCOVA_0.6-1               rlang_1.1.1                
## [118] EnrichmentBrowser_2.32.0    KEGGREST_1.42.0

<>

References