diff --git a/.gitattributes b/.gitattributes
index cbc2434b98d336c508e665277afba4237ec7be16..38136aedacd55e466cd1487dccccad12baa7626a 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -2900,3 +2900,4 @@ assays/S7_A1_microscopy/dataset/Microscopic_images_shown_in_the_manuscript/Glu/3
 assays/S7_A1_microscopy/dataset/Microscopic_images_shown_in_the_manuscript/Glu/3141/Laser561-2.tif filter=lfs diff=lfs merge=lfs -text
 assays/S7_A1_microscopy/dataset/Microscopic_images_shown_in_the_manuscript/Glu/3238/Laser561-1.tif filter=lfs diff=lfs merge=lfs -text
 assays/S7_A1_microscopy/dataset/Microscopic_images_shown_in_the_manuscript/Glu/3239/Laser561-7.tif filter=lfs diff=lfs merge=lfs -text
+assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane-traffikcing-pathways-shows-increased-expression-in-khd4D-hyphae.html filter=lfs diff=lfs merge=lfs -text
diff --git a/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/README.md b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/README.md
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index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391
diff --git a/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/.gitkeep b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/.gitkeep
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index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391
diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_Goterm.pdf b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_Goterm.pdf
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rename from assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_Goterm.pdf
rename to assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_Goterm.pdf
diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_specific_goterm_cytoscape_network.cys b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_specific_goterm_cytoscape_network.cys
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rename to assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_specific_goterm_cytoscape_network.cys
diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_specific_goterm_gprofiler.txt b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/khd4D_hyphae_specific_targets/Khd4D_hyphae_specific_goterm_gprofiler.txt
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diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_network.pdf b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_network.pdf
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diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_specific_goterm_cytoscape_network.cys b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_specific_goterm_cytoscape_network.cys
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diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_specific_goterm_gProfiler.txt b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/GO_term_analysis/wt_hyphae_specific_targets/wt_hyphae_specific_goterm_gProfiler.txt
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diff --git a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/Differential_gene_expression_analysis/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd
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rename from assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/Differential_gene_expression_analysis/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd
rename to assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd
index 258453121192115885e042e3e15adba08ff5f360..9d098bd34f628c3831a8137424c1da6272146ec6 100644
--- a/assays/S6_A1_RNAseq_analysis_yeast_vs_hyphae/dataset/Differential_gene_expression_analysis/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd	
+++ b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane traffikcing pathways shows increased expression in khd4D hyphae.Rmd	
@@ -2,7 +2,11 @@
 title: "khd4D hyphae specific genes"
 author: "Srimeenakshi Sankaranarayanan"
 date: '2022-10-11'
-output: html_document
+output: 
+  html_document:
+    toc: yes
+    toc_float:
+      collapsed: true
 ---
 
 ```{r setup, include=FALSE}
@@ -34,12 +38,12 @@ library(gprofiler2)
 library(tidyr)
 library(stats)
 library(ggSankeyGrad)
+library(ggalluvial)
 ```
 
 
-## Import the theme for the paper
-
 ```{r theme, message=FALSE, warning=FALSE}
+## Import the theme for the paper
 #theme from Kathi
 myTheme1 <- theme_bw() +
     theme(axis.text = element_text(size = 8, colour="black"),
@@ -68,6 +72,7 @@ myTheme2 <- theme_bw() +
 ```
 
 
+This report includes analaysis comparing the genes that are differentially expressed during hyphae formation in Wt and khd4D cells. To test this, we will use the DESEq2 analysis comparing hyphae transcriptome to the yeast transcriptome (hyphae vs. yeast) in Wt and Khd4D cells. 
 
 ## Import the hyphal specific data sets
 
@@ -83,8 +88,7 @@ kD_FivsYe = readRDS("C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/DGE-bioinfo
 
 ```
 
-
-## Take only genes that are differentially expressed
+Here, only the regulated genes are considered for the analysis. 
 
 ```{r}
 
@@ -142,97 +146,10 @@ sp1new
 
 ```
 
-## compare geneIDs that are DEGs
-
-```{r}
-
-# make Df only with DEGs
-wt_yevFi.dg = wt_yevFi.BM %>% filter(grepl('up|down', up_down))
-
-kD_yevFi.dg = kD_FivsYe.BM %>% filter(grepl('up|down', up_down))
-
-#  make geneiD column
-wt_yevFi.dg = tibble::rownames_to_column(wt_yevFi.dg, "GeneID")
-
-kD_yevFi.dg = tibble::rownames_to_column(kD_yevFi.dg, "GeneID")
-
-venn.list=list(wt=wt_yevFi.dg$GeneID,kD=kD_yevFi.dg$GeneID)
-
-#make venndiagram
-veNn1 = ggvenn(venn.list,c("wt", "kD"), fill_color = c("gray32", "dodgerblue4")) +
-      ggtitle("Overlap between DEGs in wt and khd4D hyphae")+
-      myTheme1
-veNn1
-
-#separate unique genes in wt and kD
-wtvsKd= list(
-  wt.unique = wt_yevFi.dg$GeneID[wt_yevFi.dg$GeneID %in% setdiff(wt_yevFi.dg$GeneID, kD_yevFi.dg$GeneID)],
-  kD.unique = kD_yevFi.dg$GeneID[kD_yevFi.dg$GeneID %in% setdiff(kD_yevFi.dg$GeneID, wt_yevFi.dg$GeneID)]
-)
-
-
-wt.uni=as.data.frame(wtvsKd$wt.unique)
-
-kd.uni=as.data.frame(wtvsKd$kD.unique)
-
-
-# select only upregulated genes
-sel.upWt= as.vector(wt_yevFi.dg$GeneID %in% wt.uni$`wtvsKd$wt.unique`)
-
-table(sel.upWt)
-
-up.wt.uni=subset(wt_yevFi.dg, sel.upWt)
 
-up.wt.hyphae=subset(up.wt.uni, up_down== "up")
+## Compare gene expression changes between Wt and khd4D cells during hyphae formation
 
-
-# for Khd4 select only upregulated genes
-sel.upKd= as.vector(kD_yevFi.dg$GeneID %in% kd.uni$`wtvsKd$kD.unique`)
-
-table(sel.upKd)
-
-up.kD.uni=subset(kD_yevFi.dg, sel.upKd)
-
-up.kD.hyphae=subset(up.kD.uni, up_down== "up")
-
-#volcano plot
-#wildtype
-wt.uni$l2F=lookup(wt.uni$`wtvsKd$wt.unique`, wt_yevFi.dg$GeneID, wt_yevFi.dg$log2FoldChange )
-wt.uni$pval=lookup(wt.uni$`wtvsKd$wt.unique`, wt_yevFi.dg$GeneID, wt_yevFi.dg$padj )
-
-#khd4D
-kd.uni$l2F=lookup(kd.uni$`wtvsKd$kD.unique`, kD_yevFi.dg$GeneID, kD_yevFi.dg$log2FoldChange)
-kd.uni$pval=lookup(kd.uni$`wtvsKd$kD.unique`, kD_yevFi.dg$GeneID, kD_yevFi.dg$padj)
-
-#ggplot volcano wt
-wtL2F=ggplot(wt.uni,aes(x=wt.uni$l2F, y=-log10(wt.uni$pval)))+
-  geom_point(size=2,colour="darkgrey")+
-  coord_cartesian(ylim=c(0,50), xlim=c(-4,4))+
-  scale_x_continuous(breaks=seq(-4,4, 2))+
-  stat_quadrant_counts(size=8)+
-  myTheme1
-
-#ggplot volcano kD
-kDL2F=ggplot(kd.uni,aes(x=kd.uni$l2F, y=-log10(kd.uni$pval)))+
-  geom_point(size=2,colour="darkgrey")+
-  coord_cartesian(ylim=c(0,50), xlim=c(-4,4))+
-  scale_x_continuous(breaks=seq(-4,4, 2))+
-  stat_quadrant_counts(size=8)+
-  myTheme1
-
-figEV6IJ<-ggarrange(wtL2F, kDL2F,
-          labels = c("A", "B"),
-          ncol = 2, nrow = 1)
-
-
-figEV6IJ
-
-#ggsave(figEV6IJ, path="C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/TRIBE_data/Editing_events_all/Khd4/bedgraphs", filename="figEV6IJ.pdf", height=10, width=15,  unit="cm")
-```
-
-
-# Alluvial plot
-```{r, Alluvial plot}
+```{r}
 #wt_hyphae specific
 #wt_yevFi.BM 
 #kD_hyphae specific
@@ -260,53 +177,34 @@ df.allu=genes_all[,c(2,3)]
 
 
 dat.allu=as.data.frame(table(melt(df.allu))) # dataframe alluvial
+```
 
-# alluvial plot
-#p=ggplot(dat.allu, aes(y=Freq, axis1=wt, axis2=kd)) +
-  #geom_alluvium(aes(fill=wt, fill=kd, width = 1/12)) +
-  #scale_fill_manual(values = c("up" = "#104E8B", "down" = "#68838B", "not" = "#BEBEBE")) +
-  #geom_stratum(width = 1/12, fill = "black", color = "grey") +
-  #geom_label(stat = "stratum", aes(label = after_stat(stratum)))+
- #myTheme1
 
-# lets try a different package.
-library(ggSankeyGrad)
+```{r Alluvial plot}
+
+kD_lo.ga = ggplot(dat.allu, aes(y = Freq, axis1 = factor(wt, levels=c("up", "not", "down")), axis2 = factor(kd, levels=c("up", "not", "down")))) +
+  geom_alluvium(aes(fill = wt), width = 1/24) + 
+  scale_fill_manual(values = c("up" = "#104E8B", "down" = "#68838B", "not" = "#BEBEBE")) +
+  geom_stratum(width = 1/24, color = "grey") +
+  geom_label(stat = "stratum", aes(label = after_stat(stratum))) +
+  myTheme1
 
-dat.allu$col2=c("#68838B",
-"#BEBEBE",
-"#104E8B",
-"#68838B",
-"#BEBEBE",
-"#104E8B",
-"#68838B",
-"#BEBEBE",
-"#104E8B"
-)
-
-dat.allu$col1=c("#68838B",
-"#68838B",
-"#68838B",
-"#BEBEBE",
-"#BEBEBE",
-"#BEBEBE",
-"#104E8B",
-"#104E8B",
-"#104E8B"
-)
-
-kD_lo.ga=with(dat.allu, ggSankeyGrad(c1=wt,
-                            c2=kd,
-                            col1=col1,
-                            col2=col2,
-                            values = Freq,
-                            label=TRUE))+myTheme1# please check the colour pattern
 kD_lo.ga
 
+
+
 #ggsave(kD_lo.ga, path="C:/Users/Sri/Documents/Khd4/DGE_analysis_SM_PhD/DGE-bioinfofolder/20220919_yeastvhyphae/ggplots", filename="kD_lo.ga_sanskeyplot_new1.pdf", width=10, height = 5, dpi = 300,unit="cm")
 ```
 
+## Gene Ontology term analysis
+ 
+Next, the GO terms enriched in genes specifically upregulated in either WT or khd4Δ cells during hyphae formation were analyzed to identify the biological processes underlying the phenotype.
+
 
-```{r, Alluvial plot}
+### Genes upregulated only in Wt hyphae
+
+
+```{r, Go analysis Wt}
 #gprofiler 
 kD_upuni=df.allu[df.allu$wt== "not" & df.allu$kd== "up", ]
 kD_upuni$gene.id=rownames(kD_upuni)
@@ -326,15 +224,18 @@ Go_wtupuni= gost(query = goWt$gene.id,
 #visualization
 vis.wtgO<- gostplot(Go_wtupuni, capped = TRUE, interactive = TRUE)
 vis.wtgO
+```
 
+### Genes upregulated only in Wt hyphae
 
+```{r, Go analysis KD}
 Go_kdupuni= gost(query = goKD$gene.id, 
                 organism = "umaydis", ordered_query = FALSE, 
                 multi_query = FALSE, significant = TRUE, exclude_iea = FALSE, 
                 measure_underrepresentation = FALSE, evcodes = TRUE, 
                 user_threshold = 0.05, correction_method = "g_SCS", 
                 domain_scope = "known", custom_bg = NULL, 
-                numeric_ns = "", sources = NULL, as_short_link = TRUE)
+                numeric_ns = "", sources = NULL, as_short_link = FALSE)
 
 #visualization
 vis.kDgO<- gostplot(Go_kdupuni, capped = TRUE, interactive = TRUE)
@@ -344,45 +245,9 @@ vis.kDgO
 ```
 
 
-To find genes that goes down or yeast specific unique to wt and khd4D cells
-
 ```{r}
-
-wt_downuni=df.allu[df.allu$wt== "down" & df.allu$kd== "not", ]
-wt_downuni$gene.id=rownames(wt_downuni)
-goWt=as.list(wt_downuni)
-
-Go_wtupuni= gost(query = goWt$gene.id, 
-                organism = "umaydis", ordered_query = FALSE, 
-                multi_query = FALSE, significant = TRUE, exclude_iea = FALSE, 
-                measure_underrepresentation = FALSE, evcodes = TRUE, 
-                user_threshold = 0.05, correction_method = "g_SCS", 
-                domain_scope = "known", custom_bg = NULL, 
-                numeric_ns = "", sources = NULL, as_short_link = TRUE)
-#visualization
-vis.wtgO<- gostplot(Go_wtupuni, capped = TRUE, interactive = TRUE)
-vis.wtgO
-
-
-
-#gprofiler 
-kD_downuni=df.allu[df.allu$wt== "not" & df.allu$kd== "down", ]
-kD_downuni$gene.id=rownames(kD_downuni)
-goKD=as.list(kD_downuni)
-
-Go_kdupuni= gost(query = goKD$gene.id, 
-                organism = "umaydis", ordered_query = FALSE, 
-                multi_query = FALSE, significant = TRUE, exclude_iea = FALSE, 
-                measure_underrepresentation = FALSE, evcodes = TRUE, 
-                user_threshold = 0.05, correction_method = "g_SCS", 
-                domain_scope = "known", custom_bg = NULL, 
-                numeric_ns = "", sources = NULL, as_short_link = TRUE)
-
-#visualization
-vis.kDgO<- gostplot(Go_kdupuni, capped = TRUE, interactive = TRUE)
-vis.kDgO
-
-
+sessionInfo()
 ```
 
 
+
diff --git a/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane-traffikcing-pathways-shows-increased-expression-in-khd4D-hyphae.html b/assays/S7_A1_RNAseq_analysis_Wt_Khd4D_hyphae_specific_genes/dataset/Membrane-traffikcing-pathways-shows-increased-expression-in-khd4D-hyphae.html
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+oid sha256:bd0bf4ab65f972377263054ae453f43dd27b2fd0a78aa79bd45adc1c599031f6
+size 4901027
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