Results DESeq2 per species
Maeva Techer
2024-11-01
Last updated: 2024-11-01
Checks: 5 2
Knit directory:
locust-comparative-genomics/
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- Load the libraries
We start by loading all the required R packages.
#(install first from CRAN or Bioconductor)
library("DESeq2")
library("tximport")
library("txdbmaker")
library("knitr")
library("rmdformats")
library("tidyverse")
library("data.table")
library("DT") # for making interactive search table
library("plotly") # for interactive plots
library("ggthemes") # for theme_calc
library("reshape2")
library("ComplexHeatmap")
library("RColorBrewer")
library("circlize")
library("apeglm")
library("ggpubr")
library("ggplot2")
library("ggrepel")
library("EnhancedVolcano")
library("SARTools")
library("pheatmap")
library("clusterProfiler")
library("sva")
library("cowplot")
library("ashr")
library("vsn")
library("ggdist")
library("ggConvexHull")
# Path for all species
workDir <- "/Users/maevatecher/Library/Mobile Documents/com~apple~CloudDocs/Documents/GitHub/locust-comparative-genomics/data"
setwd(workDir)
## 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 <- "Isolated" # 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"STRATEGY 1: One genome S. gregaria
Differential gene expression analysis
gregaria
rawDir <- file.path(workDir, "03-gregaria-DESeq2")
# Path and name of targetfile containing conditions and file names
species <- "gregaria"
targetFile <- file.path(workDir, "list", paste0("Head", "_", species, ".txt"))
sampletable <- fread(targetFile)
rownames(sampletable) <- sampletable$SampleName
sampletable$RearingCondition <- as.factor(sampletable$RearingCondition)
sampletable$Tissue <- as.factor(sampletable$Tissue)
## Import count files
satoshi <- DESeqDataSetFromHTSeqCount(sampleTable = sampletable,
directory = rawDir,
design = ~ RearingCondition )
#satoshi
smallestGroupSize <- 3
keep <- rowSums(counts(satoshi) >= 5) >= smallestGroupSize
satoshi <- satoshi[keep,]
#nrow(satoshi)
satoshi$RearingCondition <- relevel(satoshi$RearingCondition, ref = "Isolated")
# Fit the statistical model
shigeru <- DESeq(satoshi)
#cbind(resultsNames(shigeru))
res_shigeru <- results(shigeru)
sum(res_shigeru$padj < tresh_padj, na.rm = TRUE)[1] 5697A total of 5,697 genes out of the pre-filtered 16,305 features were showing significant differences in expression levels. The summary below showed how many were upregulated and downregulated in crowded compared to isolated.
brock <- results(shigeru, name = "RearingCondition_Crowded_vs_Isolated", alpha = alpha_DEseq2)
summary(brock)
out of 16305 with nonzero total read count
adjusted p-value < 0.05
LFC > 0 (up) : 2709, 17%
LFC < 0 (down) : 2988, 18%
outliers [1] : 99, 0.61%
low counts [2] : 0, 0%
(mean count < 1)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?resultsmcols(brock)$description[1] "mean of normalized counts for all samples"
[2] "log2 fold change (MLE): RearingCondition Crowded vs Isolated"
[3] "standard error: RearingCondition Crowded vs Isolated"
[4] "Wald statistic: RearingCondition Crowded vs Isolated"
[5] "Wald test p-value: RearingCondition Crowded vs Isolated"
[6] "BH adjusted p-values" head(brock)log2 fold change (MLE): RearingCondition Crowded vs Isolated
Wald test p-value: RearingCondition Crowded vs Isolated
DataFrame with 6 rows and 6 columns
baseMean log2FoldChange lfcSE stat pvalue
<numeric> <numeric> <numeric> <numeric> <numeric>
LOC126318536 277.0310 0.0378182 0.0721917 0.5238587 6.00377e-01
LOC126318656 63.3737 0.0909845 0.1567636 0.5803928 5.61650e-01
LOC126318743 99.5814 0.0188983 0.7549026 0.0250341 9.80028e-01
LOC126319294 198.1378 -0.3206079 0.1175191 -2.7281337 6.36938e-03
LOC126319460 594.5958 0.4286836 0.1080481 3.9675272 7.26222e-05
LOC126320026 87.7157 0.2909248 0.1535866 1.8942072 5.81975e-02
padj
<numeric>
LOC126318536 0.715630080
LOC126318656 0.682941257
LOC126318743 0.987400090
LOC126319294 0.022444476
LOC126319460 0.000549704
LOC126320026 0.127642289Plots
# Try with the data transformation
shigeru_vst <- vst(shigeru)
shigeru_rlog <- rlog(shigeru)
shigeru_ntd <- normTransform(shigeru)
itadori <- meanSdPlot(assay(shigeru_ntd))
itadori2 <- itadori$gg + ggtitle("Transformation with ntd")
itadori2
megumi <- meanSdPlot(assay(shigeru_vst))
megumi2 <- megumi$gg + ggtitle("Transformation with vst")
megumi2
nobara <- meanSdPlot(assay(shigeru_rlog))
nobara2 <-nobara$gg + ggtitle("Transformation with rlog")
nobara2
# Create the pca on the defined groups
pcaData1 <- plotPCA(object = shigeru_rlog, intgroup = c("RearingCondition"),returnData=TRUE)
percentVar <- round(100 * attr(pcaData1, "percentVar"))
pcaData1$RearingCondition<-factor(pcaData1$RearingCondition,levels=c("Crowded","Isolated"), labels=c("Crowded S. gregaria","Isolated S. gregaria"))
#levels(pcaData1$RearingCondition)
p1 <- ggplot(pcaData1, aes(PC1, PC2, color= RearingCondition)) +
geom_point(size=6) +
xlab(paste0("PC1: ", percentVar[1], "% variance")) +
ylab(paste0("PC2: ", percentVar[2], "% variance")) +
scale_color_manual(values = c("blue", "red")) +
#coord_fixed() +
theme_bw() +
theme(legend.title = element_blank()) +
theme(legend.text = element_text(face="bold", size=16)) +
theme(axis.text = element_text(size=14)) +
theme(axis.title = element_text(size=14))
p1 + geom_convexhull(aes(fill = RearingCondition, color = RearingCondition), alpha = 0.5) +
scale_fill_manual(values = c("blue", "red"))+
ggtitle("PCA on S. gregaria Head tissues", subtitle = "rlog transformation") 
pcaData2 <- plotPCA(object = shigeru_vst, intgroup = c("RearingCondition"),returnData=TRUE)
percentVar <- round(100 * attr(pcaData2, "percentVar"))
pcaData2$RearingCondition<-factor(pcaData2$RearingCondition,levels=c("Crowded","Isolated"), labels=c("Crowded S. gregaria","Isolated S. gregaria"))
#levels(pcaData2$RearingCondition)
p2 <-ggplot(pcaData2, aes(PC1, PC2, color= RearingCondition)) +
geom_point(size=6) +
xlab(paste0("PC1: ", percentVar[1], "% variance")) +
ylab(paste0("PC2: ", percentVar[2], "% variance")) +
scale_color_manual(values = c("blue", "red")) +
#coord_fixed() +
theme_bw() +
theme(legend.title = element_blank()) +
theme(legend.text = element_text(face="bold", size=16)) +
theme(axis.text = element_text(size=14)) +
theme(axis.title = element_text(size=14))
p2 + geom_convexhull(aes(fill = RearingCondition, color = RearingCondition), alpha = 0.5) +
scale_fill_manual(values = c("blue", "red"))+
ggtitle("PCA on S. gregaria Head tissues", subtitle = "vst transformation") 
select <- order(rowMeans(counts(shigeru,normalized=TRUE)),
decreasing=TRUE)[1:12]
df <- as.data.frame(colData(shigeru)[,c("RearingCondition","Tissue")])
# Count matrix
pheatmap(assay(shigeru_ntd)[select,], cluster_rows=FALSE, show_rownames=FALSE,
cluster_cols=FALSE, annotation_col=df, main = "Count Matrix after norm transformation")
pheatmap(assay(shigeru_vst)[select,], cluster_rows=FALSE, show_rownames=FALSE,
cluster_cols=FALSE, annotation_col=df, main = "Count Matrix after vst transformation")
pheatmap(assay(shigeru_rlog)[select,], cluster_rows=FALSE, show_rownames=FALSE,
cluster_cols=FALSE, annotation_col=df, main = "Count Matrix after rlog transformation")
# calculate between-sample distance matrix
metadata <- sampletable[,c("RearingCondition", "Tissue")]
rownames(metadata) <- sampletable$SampleName
sampleDistMatrix.rlog <- as.matrix(dist(t(assay(shigeru_rlog))))
pheatmap(sampleDistMatrix.rlog, annotation_col=metadata, main = "Head tissue heatmap distance matrix, rlog transformation")
sampleDistMatrix.vst<- as.matrix(dist(t(assay(shigeru_vst))))
pheatmap(sampleDistMatrix.vst, annotation_col=metadata, main = "Head tissue heatmap distance matrix, rlog transformation")
# Ma plot parameters after shrinkage
de_shrink <- lfcShrink(dds = shigeru, coef="RearingCondition_Crowded_vs_Isolated", type="apeglm")
#head(de_shrink)
maplot <-ggmaplot(de_shrink, fdr = 0.05, fc = 1, size = 2, palette = c("#B31B21", "#1465AC", "darkgray"), genenames = as.vector(rownames(de_shrink$name)), top = 0,legend="top",label.select = NULL) +
coord_cartesian(xlim = c(0, 20)) +
scale_y_continuous(limits=c(-12, 12)) +
theme(axis.text.x = element_text(size=14),axis.text.y = element_text(size=13),axis.title.x = element_text(size=4),axis.title.y = element_text(size=17),axis.line = element_line(size = 1, colour="gray20"),axis.ticks = element_line(size = 1, colour="gray20")) +
guides(color = guide_legend(override.aes = list(size = c(3,3,3)))) +
theme(legend.position = c(0.70, 0.12),legend.text=element_text(size=14,face="bold"),legend.background = element_rect(fill="transparent")) +
theme(plot.title = element_text(size=18, colour="gray30", face="bold",hjust=0.06, vjust=-5)) +
labs(title="MA-plot for the shrunken log2 fold changes in the Head tissues")
maplot
#Volcano plot
keyvals <-ifelse(
res_shigeru$log2FoldChange >= 1 & res_shigeru$padj <= 0.05, '#B31B21',
ifelse(res_shigeru$log2FoldChange <= -1 & res_shigeru$padj <= 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(res_shigeru,
lab = rownames(res_shigeru),
x = 'log2FoldChange',
y = 'padj',
pCutoff = 0.05,
FCcutoff = 1,
pointSize = 2,
labSize = 3,
colAlpha = 4/5,
colCustom = keyvals,
drawConnectors = TRUE)
Figure XX: XXX
piceifrons
cancellata
americana
cubense
nitens
STRATEGY 2: Own RefSeq genome
DEseq2
This follows the same code as for STRATEGY 1 except that we will change the RefSeq to the transcript species genome path.
DEGs
gregaria
piceifrons
cancellata
americana
cubense
nitens
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] grid stats4 stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] ggConvexHull_0.1.0 ggdist_3.3.2
[3] vsn_3.72.0 cowplot_1.1.3
[5] sva_3.52.0 BiocParallel_1.38.0
[7] genefilter_1.86.0 mgcv_1.9-1
[9] nlme_3.1-166 clusterProfiler_4.12.6
[11] pheatmap_1.0.12 SARTools_1.8.1
[13] kableExtra_1.4.0 edgeR_4.2.2
[15] limma_3.60.6 ashr_2.2-63
[17] EnhancedVolcano_1.22.0 ggrepel_0.9.6
[19] ggpubr_0.6.0 apeglm_1.26.1
[21] circlize_0.4.16 RColorBrewer_1.1-3
[23] ComplexHeatmap_2.20.0 reshape2_1.4.4
[25] ggthemes_5.1.0 plotly_4.10.4
[27] DT_0.33 data.table_1.16.2
[29] lubridate_1.9.3 forcats_1.0.0
[31] stringr_1.5.1 dplyr_1.1.4
[33] purrr_1.0.2 readr_2.1.5
[35] tidyr_1.3.1 tibble_3.2.1
[37] ggplot2_3.5.1 tidyverse_2.0.0
[39] rmdformats_1.0.4 knitr_1.48
[41] txdbmaker_1.0.1 GenomicFeatures_1.56.0
[43] AnnotationDbi_1.66.0 tximport_1.32.0
[45] DESeq2_1.44.0 SummarizedExperiment_1.34.0
[47] Biobase_2.64.0 MatrixGenerics_1.16.0
[49] matrixStats_1.4.1 GenomicRanges_1.56.2
[51] GenomeInfoDb_1.40.1 IRanges_2.38.1
[53] S4Vectors_0.42.1 BiocGenerics_0.50.0
loaded via a namespace (and not attached):
[1] fs_1.6.5 bitops_1.0-9 enrichplot_1.24.4
[4] httr_1.4.7 doParallel_1.0.17 numDeriv_2016.8-1.1
[7] tools_4.4.1 backports_1.5.0 utf8_1.2.4
[10] R6_2.5.1 lazyeval_0.2.2 GetoptLong_1.0.5
[13] withr_3.0.2 prettyunits_1.2.0 GGally_2.2.1
[16] gridExtra_2.3 preprocessCore_1.66.0 cli_3.6.3
[19] scatterpie_0.2.4 labeling_0.4.3 sass_0.4.9
[22] SQUAREM_2021.1 mvtnorm_1.3-1 mixsqp_0.3-54
[25] Rsamtools_2.20.0 systemfonts_1.1.0 yulab.utils_0.1.7
[28] gson_0.1.0 DOSE_3.30.5 svglite_2.1.3
[31] R.utils_2.12.3 invgamma_1.1 bbmle_1.0.25.1
[34] rstudioapi_0.17.1 RSQLite_2.3.7 gridGraphics_0.5-1
[37] generics_0.1.3 shape_1.4.6.1 BiocIO_1.14.0
[40] distributional_0.5.0 car_3.1-3 GO.db_3.19.1
[43] Matrix_1.7-1 fansi_1.0.6 abind_1.4-8
[46] R.methodsS3_1.8.2 lifecycle_1.0.4 yaml_2.3.10
[49] carData_3.0-5 qvalue_2.36.0 SparseArray_1.4.8
[52] BiocFileCache_2.12.0 blob_1.2.4 promises_1.3.0
[55] crayon_1.5.3 bdsmatrix_1.3-7 lattice_0.22-6
[58] annotate_1.82.0 KEGGREST_1.44.1 pillar_1.9.0
[61] fgsea_1.30.0 rjson_0.2.23 codetools_0.2-20
[64] fastmatch_1.1-4 glue_1.8.0 ggfun_0.1.7
[67] treeio_1.28.0 vctrs_0.6.5 png_0.1-8
[70] gtable_0.3.6 emdbook_1.3.13 cachem_1.1.0
[73] xfun_0.49 S4Arrays_1.4.1 tidygraph_1.3.1
[76] coda_0.19-4.1 survival_3.7-0 iterators_1.0.14
[79] statmod_1.5.0 ggtree_3.12.0 bit64_4.5.2
[82] progress_1.2.3 filelock_1.0.3 rprojroot_2.0.4
[85] bslib_0.8.0 affyio_1.74.0 irlba_2.3.5.1
[88] colorspace_2.1-1 DBI_1.2.3 tidyselect_1.2.1
[91] bit_4.5.0 compiler_4.4.1 curl_5.2.3
[94] git2r_0.35.0 httr2_1.0.5 xml2_1.3.6
[97] ggdendro_0.2.0 DelayedArray_0.30.1 shadowtext_0.1.4
[100] bookdown_0.41 rtracklayer_1.64.0 scales_1.3.0
[103] hexbin_1.28.4 affy_1.82.0 rappdirs_0.3.3
[106] digest_0.6.37 rmarkdown_2.28 XVector_0.44.0
[109] htmltools_0.5.8.1 pkgconfig_2.0.3 highr_0.11
[112] dbplyr_2.5.0 fastmap_1.2.0 rlang_1.1.4
[115] GlobalOptions_0.1.2 htmlwidgets_1.6.4 UCSC.utils_1.0.0
[118] farver_2.1.2 jquerylib_0.1.4 jsonlite_1.8.9
[121] GOSemSim_2.30.2 R.oo_1.26.0 RCurl_1.98-1.16
[124] magrittr_2.0.3 ggplotify_0.1.2 Formula_1.2-5
[127] GenomeInfoDbData_1.2.12 patchwork_1.3.0 munsell_0.5.1
[130] Rcpp_1.0.13 ape_5.8 viridis_0.6.5
[133] stringi_1.8.4 ggraph_2.2.1 zlibbioc_1.50.0
[136] MASS_7.3-61 plyr_1.8.9 ggstats_0.7.0
[139] parallel_4.4.1 graphlayouts_1.2.0 Biostrings_2.72.1
[142] splines_4.4.1 hms_1.1.3 locfit_1.5-9.10
[145] igraph_2.1.1 ggsignif_0.6.4 biomaRt_2.60.1
[148] XML_3.99-0.17 evaluate_1.0.1 BiocManager_1.30.25
[151] tweenr_2.0.3 tzdb_0.4.0 foreach_1.5.2
[154] httpuv_1.6.15 polyclip_1.10-7 clue_0.3-65
[157] ggforce_0.4.2 broom_1.0.7 xtable_1.8-4
[160] restfulr_0.0.15 tidytree_0.4.6 rstatix_0.7.2
[163] later_1.3.2 viridisLite_0.4.2 truncnorm_1.0-9
[166] aplot_0.2.3 memoise_2.0.1 GenomicAlignments_1.40.0
[169] cluster_2.1.6 workflowr_1.7.1 timechange_0.3.0