Last updated: 2024-11-01
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Deleted: data/piceifrons/deg_counts/STAR_newparams/piceifrons/featurecounts/finalcounts/SPICE_S_Iso_SRR11815261_counts.txt
Deleted: data/piceifrons/deg_counts/STAR_newparams/piceifrons/featurecounts/finalcounts/SPICE_S_Iso_SRR11815273_counts.txt
Deleted: data/piceifrons/deg_counts/STAR_newparams/piceifrons/featurecounts/finalcounts/SPICE_S_Iso_SRR11815274_counts.txt
Deleted: data/venn_diagramm_down_head.png
Deleted: data/venn_diagramm_down_thorax.png
Deleted: data/venn_diagramm_up_head.png
Deleted: data/venn_diagramm_up_thorax.png
Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.
There are no past versions. Publish this analysis with
wflow_publish() to start tracking its development.
edgeR analysis using STAR inputWe followed partially the comprehensive tutorial for edgeR.
We start by loading all the required R packages.
#(install first from CRAN or Bioconductor)
library("knitr")
library("rmdformats")
library("tidyverse")
library("DT") # for making interactive search table
library("plotly") # for interactive plots
library("ggthemes") # for theme_calc
library("reshape2")
library("edgeR")
library("data.table")
library("apeglm")
library("ggpubr")
library("ggplot2")
library("ggrepel")
library("EnhancedVolcano")
library("SARTools")
library("pheatmap")
Here we will be using the html report function of edgeR
to export all the results, however, the option to do it step by step
will be also provided below.
We need to specify the path of our working directories, where are located our gene counts files and where the .html report will be created:
## PARAMETERS for running the script
homeDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics"
workDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics/data" # Working directory
# path to the directory containing raw counts files
rawDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics/data/gregaria/deg_counts/STAR_newparams/gregaria/featurecounts/finalcounts/"
## Details to create the html report
projectName <- "SPECIESXXX_HEAD" # name of the project
author <- "Maeva TECHER" # author of the statistical analysis/report
## Create all the needed directories for the html report
setwd(workDir)
Dirname <- paste("edgeR_", projectName, sep = "")
dir.create(Dirname)
setwd(Dirname)
workDir_DEseq2 <- getwd()
We then define the parameters that we will be running with
edgeR:
## PARAMETERS for running EdgeR
tresh_logfold <- 1 # Treshold for log2(foldchange) in final DE-files
tresh_padj <- 0.05 # Treshold for adjusted p-valued in final DE-files
alpha_edgeR <- 0.05 # threshold of statistical significance
pAdjustMethod_edgeR <- "BH" # p-value adjustment method: "BH" (default) or "BY"
featuresToRemove <- c(NULL) # names of the features to be removed, NULL if none or if using Idxstats
varInt <- "RearingCondition" # factor of interest
condRef <- "Isolated" # reference biological condition
batch <- NULL # blocking factor: NULL (default) or "batch" for example
cpmCutoff <- 1 # counts-per-million cut-off to filter low counts
gene.selection <- "pairwise" # selection of the features in MDSPlot
normalizationMethod <- "TMM" # normalization method: "TMM" (default), "RLE" (DESeq) or "upperquartile"
# Path and name of targetfile containing conditions and file names
targetFile <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics/data/list/HeadSPECIESXXX.txt"
colors <- c("#B31B21", "#1465AC", "green3", "orange")
We then import the metadata table and format it as such as there is no error downstream.
Here our targetFile is our metadata which should have a
column for each: sample ID, the file name (exactly the same as in your
folder, be careful with ” ” or “_”) and the conditions.
If everything has been loaded properly, the next chunk of code should not show any warning and we can simply proceed to the actual DEG analysis.
# checking parameters
setwd(workDir_edgeR)
checkParameters.edgeR(projectName=projectName,author=author,targetFile=targetFile,
rawDir=rawDir,featuresToRemove=featuresToRemove,varInt=varInt,
condRef=condRef,batch=batch,alpha=alpha_edgeR,pAdjustMethod=pAdjustMethod_edgeR,
cpmCutoff=cpmCutoff,gene.selection=gene.selection,
normalizationMethod=normalizationMethod,colors=colors)
Here if you just want a quick report without being able to tailor step by step, you can use the code below and obtain several graphs and details for your DEG analysis.
## import the sample sheet that indicates Rearing Conditions and Tissue origins
setwd(workDir_edgeR)
# loading target file
target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
# loading counts
counts <- loadCountData(target=target, rawDir=rawDir, featuresToRemove=featuresToRemove)
# description plots
majSequences <- descriptionPlots(counts=counts, group=target[,varInt], col=colors)
# edgeR analysis
out.edgeR <- run.edgeR(counts=counts, target=target, varInt=varInt, condRef=condRef,
batch=batch, cpmCutoff=cpmCutoff, normalizationMethod=normalizationMethod,
pAdjustMethod=pAdjustMethod_edgeR)
# MDS + clustering
exploreCounts(object=out.edgeR$dge, group=target[,varInt], gene.selection=gene.selection, col=colors)
# summary of the analysis (boxplots, dispersions, export table, nDiffTotal, histograms, MA plot)
summaryResults <- summarizeResults.edgeR(out.edgeR, group=target[,varInt], counts=counts, alpha=alpha_edgeR, col=colors)
# generating HTML report
writeReport.edgeR(target=target, counts=counts, out.edgeR=out.edgeR, summaryResults=summaryResults,
majSequences=majSequences, workDir=workDir_edgeR, projectName=projectName, author=author,
targetFile=targetFile, rawDir=rawDir, featuresToRemove=featuresToRemove, varInt=varInt,
condRef=condRef, batch=batch, alpha=alpha_edgeR, pAdjustMethod=pAdjustMethod_edgeR, colors=colors,
gene.selection=gene.selection, normalizationMethod=normalizationMethod, cpmCutoff =cpmCutoff)
Here we only need to specify the path of our raw counts and metadata files.
## PARAMETERS for running the script
homeDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics"
workDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics/data" # Working directory
# path to the directory containing raw counts files
rawDir <- "/Users/alphamanae/Documents/GitHub/locust-comparative-genomics/data/gregaria/deg_counts/STAR_newparams/gregaria/featurecounts/finalcounts/"
We then define the parameters that we will be running with
edgeR:
## PARAMETERS for running DEseq2
tresh_logfold <- 1 # Treshold for log2(foldchange) in final DE-files
tresh_padj <- 0.05 # Treshold for adjusted p-valued in final DE-files
alpha_DEseq2 <- 0.05 # threshold of statistical significance
pAdjustMethod_DEseq2 <- "BH" # p-value adjustment method: "BH" (default) or "BY"
featuresToRemove <- c(NULL) # names of the features to be removed, NULL if none or if using Idxstats
varInt <- "RearingCondition" # factor of interest
condRef <- "Crowded" # reference biological condition
batch <- NULL # blocking factor: NULL (default) or "batch" for example
fitType <- "parametric" # mean-variance relationship: "parametric" (default) or "local"
cooksCutoff <- TRUE # TRUE/FALSE to perform the outliers detection (default is TRUE)
independentFiltering <- TRUE # TRUE/FALSE to perform independent filtering (default is TRUE)
typeTrans <- "rlog" # transformation for PCA/clustering: "VST" or "rlog"
locfunc <- "median"
We then import the metadata table and load the count files.
# loading target file
target <- loadTargetFile(targetFile=targetFile, varInt=varInt, condRef=condRef, batch=batch)
# loading counts
counts <- loadCountData(target=target, rawDir=rawDir, featuresToRemove=featuresToRemove)
## Create DGEList data class
musashi <- DGEList(counts = counts, group = target[,varInt])
musashi
## Filter out the genes with low counts
keep <- filterByExpr(y = musashi)
musashi <- musashi[keep, , keep.lib.sizes=FALSE]
musashi
## Normalization and effective library sizes
musashi <- calcNormFactors(object = musashi)
musashi
## Dispersion estimation
musashi <- estimateDisp(y = musashi)
## Testing for differential gene expression
kojiro <- exactTest(object = musashi)
## Extract the table with adjusted p-values (FDR = padj for DEseq2)
top_degs = topTags(object = kojiro, n = "Inf")
top_degs
## Obtain the summary
summary(decideTests(object = kojiro, lfc = 1))
## Export the table
#write.csv(as.data.frame(top_degs), file="condition_infected_vs_control_dge.csv")
sakaki <- as.data.frame(top_degs)
keyvals <-ifelse(
sakaki$logFC >= 1 & sakaki$FDR <= 0.05, '#B31B21',
ifelse(sakaki$logFC <= -1 & sakaki$FDR <= 0.05, '#1465AC', 'darkgray'))
keyvals[is.na(keyvals)] <-'darkgray'
names(keyvals)[keyvals == "#B31B21"] <-'Upregulated'
names(keyvals)[keyvals == "#1465AC"] <-'Downregulated'
names(keyvals)[keyvals == 'darkgray'] <-'NS'
EnhancedVolcano(sakaki,
lab = rownames(sakaki),
x = 'logFC',
y = 'FDR',
pCutoff = 0.05,
FCcutoff = 1,
pointSize = 3,
labSize = 4,
colAlpha = 4/5,
colCustom = keyvals,
drawConnectors = TRUE)
plotMD(object = kojiro, p.value = 0.05)
DESeq2 and edgeR resultsTo do… by hand for now
sessionInfo()
R version 4.4.1 (2024-06-14)
Platform: aarch64-apple-darwin20
Running under: macOS Sonoma 14.7
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: Asia/Tokyo
tzcode source: internal
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] pheatmap_1.0.12 SARTools_1.8.1
[3] kableExtra_1.4.0 ashr_2.2-63
[5] DESeq2_1.44.0 SummarizedExperiment_1.34.0
[7] Biobase_2.64.0 MatrixGenerics_1.16.0
[9] matrixStats_1.4.1 GenomicRanges_1.56.2
[11] GenomeInfoDb_1.40.1 IRanges_2.38.1
[13] S4Vectors_0.42.1 BiocGenerics_0.50.0
[15] EnhancedVolcano_1.22.0 ggrepel_0.9.6
[17] ggpubr_0.6.0 apeglm_1.26.1
[19] data.table_1.16.2 edgeR_4.2.2
[21] limma_3.60.6 reshape2_1.4.4
[23] ggthemes_5.1.0 plotly_4.10.4
[25] DT_0.33 lubridate_1.9.3
[27] forcats_1.0.0 stringr_1.5.1
[29] dplyr_1.1.4 purrr_1.0.2
[31] readr_2.1.5 tidyr_1.3.1
[33] tibble_3.2.1 ggplot2_3.5.1
[35] tidyverse_2.0.0 rmdformats_1.0.4
[37] knitr_1.48
loaded via a namespace (and not attached):
[1] RColorBrewer_1.1-3 ggdendro_0.2.0 rstudioapi_0.17.1
[4] jsonlite_1.8.9 magrittr_2.0.3 rmarkdown_2.28
[7] fs_1.6.5 zlibbioc_1.50.0 vctrs_0.6.5
[10] memoise_2.0.1 SQUAREM_2021.1 rstatix_0.7.2
[13] mixsqp_0.3-54 htmltools_0.5.8.1 S4Arrays_1.4.1
[16] truncnorm_1.0-9 broom_1.0.7 SparseArray_1.4.8
[19] Formula_1.2-5 sass_0.4.9 bslib_0.8.0
[22] htmlwidgets_1.6.4 plyr_1.8.9 cachem_1.1.0
[25] lifecycle_1.0.4 pkgconfig_2.0.3 Matrix_1.7-1
[28] R6_2.5.1 fastmap_1.2.0 GenomeInfoDbData_1.2.12
[31] digest_0.6.37 numDeriv_2016.8-1.1 colorspace_2.1-1
[34] GGally_2.2.1 AnnotationDbi_1.66.0 rprojroot_2.0.4
[37] irlba_2.3.5.1 RSQLite_2.3.7 invgamma_1.1
[40] fansi_1.0.6 timechange_0.3.0 httr_1.4.7
[43] abind_1.4-8 compiler_4.4.1 bit64_4.5.2
[46] withr_3.0.2 backports_1.5.0 BiocParallel_1.38.0
[49] DBI_1.2.3 carData_3.0-5 ggstats_0.7.0
[52] ggsignif_0.6.4 MASS_7.3-61 DelayedArray_0.30.1
[55] tools_4.4.1 httpuv_1.6.15 glue_1.8.0
[58] promises_1.3.0 grid_4.4.1 generics_0.1.3
[61] gtable_0.3.6 tzdb_0.4.0 hms_1.1.3
[64] xml2_1.3.6 car_3.1-3 utf8_1.2.4
[67] XVector_0.44.0 pillar_1.9.0 emdbook_1.3.13
[70] genefilter_1.86.0 later_1.3.2 splines_4.4.1
[73] lattice_0.22-6 survival_3.7-0 bit_4.5.0
[76] annotate_1.82.0 tidyselect_1.2.1 locfit_1.5-9.10
[79] Biostrings_2.72.1 git2r_0.35.0 gridExtra_2.3
[82] bookdown_0.41 svglite_2.1.3 xfun_0.49
[85] statmod_1.5.0 stringi_1.8.4 UCSC.utils_1.0.0
[88] workflowr_1.7.1 lazyeval_0.2.2 yaml_2.3.10
[91] evaluate_1.0.1 codetools_0.2-20 bbmle_1.0.25.1
[94] cli_3.6.3 xtable_1.8-4 systemfonts_1.1.0
[97] munsell_0.5.1 jquerylib_0.1.4 Rcpp_1.0.13
[100] png_0.1-8 coda_0.19-4.1 XML_3.99-0.17
[103] bdsmatrix_1.3-7 parallel_4.4.1 blob_1.2.4
[106] viridisLite_0.4.2 mvtnorm_1.3-1 scales_1.3.0
[109] crayon_1.5.3 rlang_1.1.4 KEGGREST_1.44.1