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index 2635f9cbbd98377405c62ee003f1d6b4b772e7b2..a254b06dd6dc28708e27a74eef85cfceb3ad5604 100644
--- a/.gitattributes
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diff --git a/runs/deseq2/CANA1_RNAseq_PCA.RDS b/runs/deseq2/CANA1_RNAseq_PCA.RDS
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diff --git a/runs/deseq2/Image.RData b/runs/deseq2/Image.RData
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diff --git a/runs/deseq2/allgenes.csv b/runs/deseq2/allgenes.csv
new file mode 100644
index 0000000000000000000000000000000000000000..88c117724d072337cf1161cad67b377581d5c91e
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diff --git a/runs/deseq2/sample_table.csv b/runs/deseq2/sample_table.csv
new file mode 100644
index 0000000000000000000000000000000000000000..300e589a4b668b392126bb57604ce9258a1b6e91
--- /dev/null
+++ b/runs/deseq2/sample_table.csv
@@ -0,0 +1,16 @@
+Sample,Filename,tissue,genotype,#,Plant,group
+Muwl1,3698_A_run535_TTCTCCGA_S199_L007.htseq,whiteleaf,MU,11,B11d,MU_whiteleaf
+Muwl2,3698_B_run535_TACGCAGG_S200_L007.htseq,whiteleaf,MU,14,B14d,MU_whiteleaf
+Muwl3,3698_C_run531_GCGTACGT_S248_L007.htseq,whiteleaf,MU,32,B11c,MU_whiteleaf
+Mugl1,3698_D_run531_CTTATGGA_S249_L007.htseq,greenleaf,MU,10,B11d,MU_greenleaf
+Mugl2,3698_E_run531_CTACGCTT_S250_L007.htseq,greenleaf,MU,19,B14a,MU_greenleaf
+Mugl3,3698_F_run535_CCGTTCAT_S201_L007.htseq,greenleaf,MU,20,B11c,MU_greenleaf
+Wtgl1,3698_G_run535_CAGTATTA_S202_L007.htseq,greenleaf,WT,9,B17c,WT_greenleaf
+Wtgl2,3698_H_run531_ATCCGTGC_S253_L007.htseq,greenleaf,WT,21,B17a,WT_greenleaf
+Wtgl3,3698_I_run535_TTCGACTA_S203_L007.htseq,greenleaf,WT,23,I06c,WT_greenleaf
+Mus1,3698_J_run535_TACATGTA_S204_L007.htseq,stem,MU,111,B14a,MU_stem
+Mus2,3698_K_run535_GCGCTTGA_S205_L007.htseq,stem,MU,125,B14d,MU_stem
+Mus3,3698_L_run531_CTGCGTGA_S257_L007.htseq,stem,MU,131,B12d,MU_stem
+Wts1,3698_M_run535_CTAACACG_S206_L007.htseq,stem,WT,115,I05b,WT_stem
+Wts2,3698_N_run531_CCGTAGGA_S259_L007.htseq,stem,WT,127,B05b,WT_stem
+Wts3,3698_O_run535_CAGGTGTC_S207_L007.htseq,stem,WT,128,B06c,WT_stem
diff --git a/studies/canal-1_2017_GH/isa.study.xlsx b/studies/canal-1_2017_GH/isa.study.xlsx
index 18c5cd4b0f68f0189dd63a94d205b0f71312f10a..8a6de5a62cf283042c92b7f2c0b3ac045927e308 100644
Binary files a/studies/canal-1_2017_GH/isa.study.xlsx and b/studies/canal-1_2017_GH/isa.study.xlsx differ
diff --git a/studies/canal-1_2018_GH/isa.study.xlsx b/studies/canal-1_2018_GH/isa.study.xlsx
index 0969923b8a40dca63525ed1c3b6e40d9438ca474..08bbeee2aef31c476743cc50d8c8a7224a718cd3 100644
Binary files a/studies/canal-1_2018_GH/isa.study.xlsx and b/studies/canal-1_2018_GH/isa.study.xlsx differ
diff --git a/studies/canal-1_2019_GH/isa.study.xlsx b/studies/canal-1_2019_GH/isa.study.xlsx
index 64f4ac196762245a85a459097f6b07b0b71ef5f6..fef2df1d5410fc1cdb51764e0d76d98dad7e55ce 100644
Binary files a/studies/canal-1_2019_GH/isa.study.xlsx and b/studies/canal-1_2019_GH/isa.study.xlsx differ
diff --git a/workflows/RNA_deseq2/Deseq2.R b/workflows/RNA_deseq2/Deseq2.R
new file mode 100644
index 0000000000000000000000000000000000000000..deeea6935751f289d13d47e2b12f40d12e569dae
--- /dev/null
+++ b/workflows/RNA_deseq2/Deseq2.R
@@ -0,0 +1,188 @@
+# Library -----------------------------------------------------------------
+
+library(tidyverse)
+library(here)
+library("DESeq2")
+library("ggplot2")
+library("magrittr")
+library("dplyr")
+library("pheatmap")
+library("RColorBrewer")
+library("apeglm")
+library("data.table")
+library("topGO")
+library("GOplot")
+library("ashr")
+library(readxl)
+
+# Directories -------------------------------------------------------------
+sampleDirectory <- here("runs/LSTrAP/output/sly/htseq/sly/")
+study <- here("studies/canal-1_2017_GH/")
+assay <- here("assays/canal-1_Transcriptomics/")
+
+setwd(here())
+
+out <- here("runs/deseq2")
+
+if (file.exists(out)) {
+  cat("The folder already exists")
+} else {
+  dir.create(out)
+}
+
+# Sample loading ----------------------------------------------------------
+
+isa_study <- read_xlsx(here(study, "isa.study.xlsx"))
+isa_ext <- read_xlsx(here(assay, "isa.assay.xlsx"), sheet = 1)
+isa_ana <- read_xlsx(here(assay, "isa.assay.xlsx"), sheet = 3)
+sampleTable <- read_csv(here(assay, "dataset/CANA1_2017_RNAseq_samplelist.csv"))
+
+isa_study_tidy <- isa_study %>% 
+  dplyr::select(source_name_study = `Source Name`,
+                tissue_group = `Characteristic [organism part]`,
+                leaf_col = `Characteristic [leaf color]`,
+                genotype = `Characteristic [genotype]`,
+                sample_name_study = `Sample Name`)
+
+isa_ext_tidy <- isa_ext %>% 
+  dplyr::select(source_name_ext = `Source Name`,
+                Sample = `Characteristic [sample name]`,
+                num = `Parameter [original sample number]`,
+                sample_name_ext = `Sample Name`)
+
+isa_ana_tidy <- isa_ana %>%
+  dplyr::select(source_name_ana = `Source Name`,
+                Filename = `Derived Data File`)
+
+sampleTable <- isa_ext_tidy %>% 
+  left_join(isa_study_tidy, by = c("source_name_ext" = "sample_name_study")) %>% 
+  full_join(isa_ana_tidy, by = c("sample_name_ext" = "source_name_ana")) %>% 
+  mutate(genotype = if_else(str_detect(genotype, "M82"),
+                            "WT",
+                            "MU"),
+         tissue = if_else(str_detect(tissue_group, "leaf"),
+                          str_c(leaf_col, tissue_group),
+                          tissue_group),
+         group = str_c(genotype, tissue, sep = "_")) %>% 
+  arrange(tissue_group, genotype, dplyr::desc(leaf_col)) %>% 
+  dplyr::select(Sample,
+                Filename,
+                tissue,
+                genotype,
+                `#` =num,
+                Plant = source_name_study,
+                group) %>% 
+  as.data.frame()
+
+# Load data - creates a DESeqDataSet object
+dds <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable,
+                                  directory = sampleDirectory,
+                                  design = ~ group)
+
+
+# Deseq pipeline ----------------------------------------------------------
+
+# Alternative designs
+# Controlling for tissue of original, what is the effect of MU vs. WT?
+# design=~tissue + genotype ( + interaction?)
+
+#Prefilter data
+nrow(dds) #number of rows before filtering
+dds <- dds[ rowSums(counts(dds)) > 1, ] #only use rows with genes which are expressed at all (could also be used to filter for genes with general low counts but already done by DESeq)
+nrow(dds) #number of rows after filtering
+
+# Check factor levels
+levels(dds$group)
+dds$group=relevel(dds$group,"WT_greenleaf") #relevel factors to set wildtype green leaves as base
+levels(dds$group)# levels should be changed now
+# if not using group, could check levels of dds$tissue, dds$genotype
+
+# regularized log transformation
+#rld <- rlog(dds, blind = TRUE)
+# VST
+vds=vst(dds)
+
+# Plot PCA
+plotPCA(vds,intgroup=("group")) # or plot rld
+
+ggsave(here(out,"CANA1_RNAseq_PCA.jpg"))
+
+saveRDS(last_plot(), here(out,"CANA1_RNAseq_PCA.RDS"))
+
+# Running the differential expression pipeline
+dds <- DESeq(dds)
+
+# Wald test for the GLM coefficients
+# I think this step is redundant with DESeq
+#dds <- nbinomWaldTest(dds)
+
+# Change the pvalue - adjust the alpha used in independent filtering
+deseqres<- results(dds,alpha=0.1)
+summary(deseqres)
+
+# use contrasts to extract your desired comparisons
+
+deseqres_WTglB_vs_MUgl=results(dds,alpha=0.1,contrast=c("group","MU_greenleaf", "WT_greenleaf"))
+deseqres_WTglB_vs_MUwl=results(dds,alpha=0.1,contrast = c("group","MU_whiteleaf","WT_greenleaf"))
+resultsNames(dds) # to see which comparisons were made
+deseq_lfcshrink_WTglB_vs_MUwl <- lfcShrink(dds = dds, res =deseqres_WTglB_vs_MUwl, coef = "group_MU_whiteleaf_vs_WT_greenleaf",type = "apeglm")
+deseq_lfcshrink_WTglB_vs_MUgl <- lfcShrink(dds = dds, res = deseqres_WTglB_vs_MUgl, coef = "group_MU_greenleaf_vs_WT_greenleaf",type = "apeglm")
+
+#Relevel to MU_greenleaf
+dds$group=relevel(dds$group,"MU_greenleaf") #relevel factors to set wildtype green leaves as base
+levels(dds$group)# levels should be changed now
+dds <- nbinomWaldTest(dds)
+deseqres_MUglB_vs_MUwl=results(dds,alpha=0.1,contrast=c("group","MU_whiteleaf","MU_greenleaf"))
+resultsNames(dds)
+deseq_lfcshrink_MUglB_vs_MUwl <- lfcShrink(dds = dds, res = deseqres_MUglB_vs_MUwl, coef = "group_MU_whiteleaf_vs_MU_greenleaf",type = "apeglm")
+
+#Relevel to stems
+dds$group=relevel(dds$group,"WT_stem") #relevel factors to set wildtype green leaves as base
+levels(dds$group)# levels should be changed now
+dds <- nbinomWaldTest(dds)
+deseqres_WTsB_vs_MUs=results(dds,alpha=0.1,contrast=c("group","MU_stem","WT_stem"))
+resultsNames(dds)
+deseq_lfcshrink_WTsB_vs_MUs <- lfcShrink(dds = dds, res = deseqres_WTsB_vs_MUs,  coef = "group_MU_stem_vs_WT_stem",type = "apeglm")
+
+# Summary (numbers, outliers, dropped genes)
+summary(deseqres_MUglB_vs_MUwl)
+summary(deseqres_WTglB_vs_MUgl)
+summary(deseqres_WTglB_vs_MUwl)
+summary(deseqres_WTsB_vs_MUs)
+
+#START PIPELINE#
+#Make deseq tables and files for all comparisons
+# Prepare data table
+df_matrix <- data.frame(counts(dds,normalized=TRUE))
+
+# Prepare statistics table
+
+df_out <- data.frame(log2FC_WTglB_vs_MUgl = deseq_lfcshrink_WTglB_vs_MUgl$log2FoldChange,
+                     padj_WTglB_vs_MUgl = deseq_lfcshrink_WTglB_vs_MUgl$padj,
+                     log2FC_WTglB_vs_MUwl = deseq_lfcshrink_WTglB_vs_MUwl$log2FoldChange,
+                     padj_WTglB_vs_MUwl = deseq_lfcshrink_WTglB_vs_MUwl$padj,
+                     log2FC_MUglB_vs_MUwl = deseq_lfcshrink_MUglB_vs_MUwl$log2FoldChange,
+                     padj_MUglB_vs_MUwl = deseq_lfcshrink_MUglB_vs_MUwl$padj,
+                     log2FC_WTsB_vs_MUs = deseq_lfcshrink_WTsB_vs_MUs$log2FoldChange,
+                     padj_WTsB_vs_MUs = deseq_lfcshrink_WTsB_vs_MUs$padj,
+                     row.names = rownames(deseq_lfcshrink_WTglB_vs_MUgl)
+                     )
+
+df_write <- merge(df_matrix, df_out, by=0, all=TRUE)
+
+allgenes <- df_write
+
+write_csv(allgenes, file = here(out, "allgenes.csv"))
+write_csv(sampleTable, here(out, "sample_table.csv"))
+
+# Log used code ------------------------------------------------------------
+
+file_name <- sys.frame(1)$ofile
+
+file.copy(file_name,
+          to = here(out, "executing.R"),overwrite = T)
+
+
+# Temporary .RData file to avoid having to rerun code ---------------------
+
+save.image(here(out, "Image.RData"))