Last updated: 2021-02-25

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Genomic location enrichment

Significant loci with a p-value <= 0.01 and a absolute log2 fold-change lager 0.5 were tested for enrichment in annotated genomic feature using fisher exact test.

Ranges=rowData(dds_filt)

TotTagsofInterest=sum(Ranges$WaldPvalue_groupCD<=thresholdp & abs(Ranges$groupCD)>thresholdLFC)

Resall=data.frame()
index = Ranges$WaldPvalue_groupCD<=thresholdp& abs(Ranges$groupCD)>thresholdLFC
for (feat in unique(Ranges$feature)){
  tmp=table(Ranges$feature == feat, signif=index)
  resfish=fisher.test(tmp)
  res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
  Resall = rbind(Resall, res)
}
tmp=table(Ranges$tf_binding!="", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resall = rbind(Resall, res)
tmp=table(Ranges$cpg=="cpg", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resall = rbind(Resall, res)
colnames(Resall)=c("OR", "CI95L", "CI95U", "P")
rownames(Resall)=c(unique(Ranges$feature), "TF-binding", "CpG-island")
Resall$Beta = log(Resall$OR)
Resall$SE = (log(Resall$OR)-log(Resall$CI95L))/1.96
Resall$Padj=p.adjust(Resall$P, method = "bonferroni")

Resdown=data.frame()
index = Ranges$WaldPvalue_groupCD<=thresholdp & Ranges$groupCD<thresholdLFC
for (feat in unique(Ranges$feature)){
  tmp=table(Ranges$feature == feat, signif=index)
  resfish=fisher.test(tmp)
  res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
  Resdown = rbind(Resdown, res)
}
tmp=table(Ranges$tf_binding!="", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resdown = rbind(Resdown, res)
tmp=table(Ranges$cpg=="cpg", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resdown = rbind(Resdown, res)
colnames(Resdown)=c("OR", "CI95L", "CI95U", "P")
rownames(Resdown)=c(unique(Ranges$feature), "TF-binding", "CpG-island")
Resdown$Beta = log(Resdown$OR)
Resdown$SE = (log(Resdown$OR)-log(Resdown$CI95L))/1.96
Resdown$Padj=p.adjust(Resdown$P, method = "bonferroni")

Resup=data.frame()
index = Ranges$WaldPvalue_groupCD<=thresholdp & Ranges$groupCD>thresholdLFC
for (feat in unique(Ranges$feature)){
  tmp=table(Ranges$feature == feat, signif=index)
  resfish=fisher.test(tmp)
  res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
  Resup = rbind(Resup, res)
}
tmp=table(Ranges$tf_binding!="", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resup = rbind(Resup, res)
tmp=table(Ranges$cpg=="cpg", signif=index)
resfish=fisher.test(tmp)
res = c(resfish$estimate, unlist(resfish$conf.int), resfish$p.value)
Resup = rbind(Resup, res)
colnames(Resup)=c("OR", "CI95L", "CI95U", "P")
rownames(Resup)=c(unique(Ranges$feature), "TF-binding", "CpG-island")
Resup$Beta = log(Resup$OR)
Resup$SE = (log(Resup$OR)-log(Resup$CI95L))/1.96
Resup$Padj=p.adjust(Resup$P, method = "bonferroni")

par(mfrow=c(1,3))

multiORplot(Resall, Pval = "P", Padj = "Padj", beta="Beta",SE = "SE", pheno="All diff. methylated loci")
multiORplot(Resup, Pval = "P", Padj = "Padj", beta="Beta",SE = "SE", pheno="hypomethylated loci")
multiORplot(Resdown, Pval = "P", Padj = "Padj", beta="Beta",SE = "SE", pheno="Hypermethylated loci")

GO-term Enrichment

Significant loci and differentially methylated regions with a p-value <= 0.01 and an absolute log2 fold-change lager 0.5 were tested for enrichment among GO-terms Molecular Function, Cellular Compartment and Biological Processes, KEGG pathways, Transcription factor Binding sites, Human Protein Atlas Tissue Expression, Human Phenotypes.

getGOresults = function(geneset, genereference){
  resgo = gost(geneset, organism = "hsapiens",
               correction_method = "fdr",
               domain_scope = "custom",
               sources = c("GO:BP", "GO:MF", "GO:CC", "KEGG", "TF", "HP", "HPA"),
               custom_bg = genereference)
  if(length(resgo) != 0){
    return(resgo)
  } else {
    print("no significant results")
    return(NULL)
  }  
}

gene_univers = getuniquegenes(as.data.frame(rowRanges(dds_filt))$gene)

# idx = (results_Deseq$pvalue <= thresholdp) & (results_Deseq$log2FoldChange > thresholdLFC)
# genes_pos_beta = getuniquegenes(as.data.frame(rowRanges(dds_filt))$gene[idx])
# 
# idx = (results_Deseq$pvalue <= thresholdp) & (results_Deseq$log2FoldChange <= -thresholdLFC)
# genes_neg_beta = getuniquegenes(as.data.frame(rowRanges(dds_filt))$gene[idx])

idx = (results_Deseq$pvalue <= thresholdp & 
         (abs(results_Deseq$log2FoldChange) > thresholdLFC))

genes_reg = getuniquegenes(as.data.frame(rowRanges(dds_filt))$gene[idx])


dmr_genes = unique(resultsdmr_table$name[resultsdmr_table$p.value<=thresholdp & 
                   abs(resultsdmr_table$value)>=thresholdLFC])


Genes_of_interset = list("01_dmregions" = dmr_genes,  
                         "02_dmtag" = genes_reg
                         # , 
                         # "03_hypermethlated" = genes_neg_beta, 
                         # "04_hypomethlated" = genes_pos_beta
                         )

gostres = getGOresults(Genes_of_interset, gene_univers)

gostplot(gostres, capped = TRUE, interactive = T)
p = gostplot(gostres, capped = TRUE, interactive = F)

toptab = gostres$result

pp = publish_gostplot(p, filename = paste0(Home,"/output/gostres.pdf"))
The image is saved to C:/Users/chiocchetti/Projects/femNATCD_MethSeq/output/gostres.pdf
write.xlsx2(toptab, file = paste0(Home,"/output/GOres.xlsx"), sheetName = "GO_enrichment")

Kang Brain Module Enrichments

Gene sets identified to be deferentially methylated with a p-value <= 0.01 and an absolute log2 fold-change larger 0.5 were tested for enrichment among gene-modules coregulated during Brain expression.

# define Reference Universe 

KangUnivers<- read.table(paste0(Home,"/data/KangUnivers.txt"), sep="\t", header=T)
colnames(KangUnivers)<-c("EntrezId","Symbol")

Kang_genes<-read.table(paste0(Home,"/data/Kang_dataset_genesMod_version2.txt"),sep="\t",header=TRUE)

#3)Generate Gene universe to be used for single gene lists
tmp=merge(KangUnivers,Kang_genes,by.y="EntrezGene",by.x="EntrezId",all=TRUE) #18826
KangUni_Final<-tmp[duplicated(tmp$EntrezId)==FALSE,] #18675


# Local analysis gene universe
Annotation_list<-data.frame(Symbol = gene_univers)

# match modules 
Annotation_list$Module = Kang_genes$Module[match(Annotation_list$Symbol,Kang_genes$symbol)]

# check if overlapping in gene universes
Annotation_list$univers = Annotation_list$Symbol %in% KangUni_Final$Symbol

# drop duplicates 
Annotation_list = Annotation_list[duplicated(Annotation_list$Symbol)==FALSE,]

# selct only genes that have been detected on both datasets
Annotation_list = Annotation_list[Annotation_list$univers==T,] 

# final reference 
UniversalGeneset=Annotation_list$Symbol

# define Gene lists to test 
# sort and order Modules to be teste 

Modules=unique(Annotation_list$Module)
Modules = Modules[! Modules %in% c(NA, "")]
Modules = Modules[order(as.numeric(gsub("M","",Modules)))]

GL_all=list()

for(i in Modules){
  GL_all[[i]]=Annotation_list$Symbol[Annotation_list$Module%in%i]
}
GL_all[["M_all"]]=Kang_genes$symbol[Kang_genes$Module %in% Modules]


GOI1 = Genes_of_interset

Resultsall=list()
for(j in names(GOI1)){
  Res = data.frame()
  for(i in names(GL_all)){
    Modulegene=GL_all[[i]]
    Factorgene=GOI1[[j]]
    Testframe<-fisher.test(table(factor(UniversalGeneset %in% Factorgene,levels=c("TRUE","FALSE")),
                                 factor(UniversalGeneset %in% Modulegene,levels=c("TRUE","FALSE"))))
    beta=log(Testframe$estimate)
    Res[i, "beta"] =beta
    Res[i, "SE"]=abs(beta-log(Testframe$conf.int[1]))/1.96
    Res[i, "Pval"]=Testframe$p.value
    Res[i, "OR"]=(Testframe$estimate)
    Res[i, "ORL"]=(Testframe$conf.int[1])
    Res[i, "ORU"]=(Testframe$conf.int[2])
  }
  Res$Padj = p.adjust(Res$Pval, method = "bonferroni")
  Resultsall[[j]] = Res
  
}
par(mfrow = c(2,2))
for (i in names(Resultsall)){ 
  multiORplot(datatoplot = Resultsall[[i]], pheno=i)
}

Modsig = c()

for(r in names(Resultsall)){
  a=rownames(Resultsall[[r]])[Resultsall[[r]]$Padj<=0.05]
  Modsig = c(Modsig,a)
}

# show brains TODO
Modisg=unique(Modsig)
load(paste0(Home,"/data/Kang_DataPreprocessing.RData")) #Load the Kang expression data of all genes 
datExprPlot=matriz #Expression data of Kang loaded as Rdata object DataPreprocessing.RData

SEM

dropfact=c("site")


Patdata=as.data.frame(colData(dds_filt))

load(paste0(Home, "/output/envFact.RData"))
load(paste0(Home, "/output/modelFact.RData"))

envFact=envFact[!envFact %in% dropfact] 
modelFact=modelFact[!modelFact %in% dropfact] 

EpiMarker = c()

# TopHit
Patdata$Epi_TopHit=log2_cpm[base::which.min(results_Deseq$pvalue),]

# 1PC of all diff met
tmp=glmpca(log2_cpm[base::which(results_Deseq$pvalue<=thresholdp),], 1)

Patdata$Epi_all= tmp$factors$dim1
  
EpiMarker = c(EpiMarker, "Epi_TopHit", "Epi_all")

#Brain Modules

Epitestset=GL_all[Modisg]

for(n in names(Epitestset)){
  index=gettaglistforgenelist(genelist = Epitestset[[n]], dds_filt)
  index = base::intersect(index, base::which(results_Deseq$pvalue<=thresholdp))
  # get eigenvalue
  epiname=paste0("Epi_",n)
  tmp=glmpca(log2_cpm[index,], 1)
  Patdata[,epiname]= tmp$factors$dim1
  EpiMarker = c(EpiMarker, epiname)
}

cormat = cor(apply(Patdata[,c("group", envFact, modelFact, EpiMarker)] %>% mutate_all(as.numeric), 2, minmax_scaling),
             use = "pairwise.complete.obs")

par(mfrow=c(1,2))
corrplot(cormat, main="correlations")
corrplot(cormat, order = "hclust", main="scaled correlations")

fullmodEnv=paste(envFact,modelFact, sep = "+", collapse = "+")

Dataset = Patdata[,c("group", envFact,modelFact,EpiMarker)]

model = paste0("group ~ Epi+", fullmodEnv,"
Epi ~" , fullmodEnv,"
Epi~~Epi
group~~group
")

Netlist = list()

for (marker in EpiMarker) {
  Dataset$Epi = Dataset[,marker]
  Datasetscaled = Dataset %>% mutate_if(is.numeric, minmax_scaling)
  Datasetscaled = Datasetscaled %>% mutate_if(is.factor, ~ as.numeric(.)-1)
  
  fit<-lavaan(model,data=Datasetscaled)
  
  sink(paste0(Home,"/output/SEM_summary_group",marker,".txt"))
  summary(fit)
  fitMeasures(fit)
  sink()
  
  #SOURCE FOR PLOT https://stackoverflow.com/questions/51270032/how-can-i-display-only-significant-path-lines-on-a-path-diagram-r-lavaan-sem
  restab=lavaan::standardizedSolution(fit) %>% dplyr::filter(!is.na(pvalue)) %>% 
    arrange(desc(pvalue)) %>% mutate_if("is.numeric","round",3) %>% 
    dplyr::select(-ci.lower,-ci.upper,-z)
  
  pvalue_cutoff <- 0.05
  
  obj <- semPlot:::semPlotModel(fit)
  original_Pars <- obj@Pars
  check_Pars <- obj@Pars %>% dplyr:::filter(!(edge %in% c("int","<->") | lhs == rhs)) # this is the list of parameter to sift thru
  keep_Pars <- obj@Pars %>% dplyr:::filter(edge %in% c("int","<->") | lhs == rhs) # this is the list of parameter to keep asis
  
  test_against <- lavaan::standardizedSolution(fit) %>% dplyr::filter(pvalue < pvalue_cutoff, rhs != lhs)
  
  # for some reason, the rhs and lhs are reversed in the standardizedSolution() output, for some of the values
  # I'll have to reverse it myself, and test against both orders
  
  test_against_rev <- test_against %>% dplyr::rename(rhs2 = lhs, lhs = rhs) %>% dplyr::rename(rhs = rhs2)
  
  checked_Pars <-
    check_Pars %>% semi_join(test_against, by = c("lhs", "rhs")) %>% bind_rows(
      check_Pars %>% semi_join(test_against_rev, by = c("lhs", "rhs"))
    )
  
  obj@Pars <- keep_Pars %>% bind_rows(checked_Pars) %>% 
    mutate_if("is.numeric","round",3) %>% 
    mutate_at(c("lhs","rhs"),~gsub("Epi", marker,.))
  
  obj@Vars = obj@Vars %>% mutate_at(c("name"),~gsub("Epi", marker,.))
  
  DF = obj@Pars
  DF = DF[DF$lhs!=DF$rhs,]
  DF = DF[abs(DF$std)>0.1,]
  
  DF = DF[DF$edge == "~>",] # only include directly modelled effects in figure 
  
  nodes <- data.frame(id=obj@Vars$name, label = obj@Vars$name)
  nodes$color<-Dark8[8]
  nodes$color[nodes$label == "group"] = Dark8[3]
  nodes$color[nodes$label == marker] = Dark8[4]
  nodes$color[nodes$label %in% envFact] = Dark8[5]
  
  edges <- data.frame(from = DF$lhs, 
                      to = DF$rhs, 
                      width=abs(DF$std), 
                      arrows ="to")
  
  edges$dashes = F
  edges$label =  DF$std
  edges$color=Dark8[1:2][factor(sign(DF$std), levels=c(-1,0,1),labels=c(1,2,2))]
  plotnet<- visNetwork(nodes, edges,
                       main=list(text=marker,
                                 style="font-family:arial;font-size:20px;text-align:center"),
                       submain=list(text="significant paths", style="font-family:arial;text-align:center")) %>% 
    visEdges(arrows =list(to = list(enabled = TRUE, scaleFactor = 0.7))) %>%
    visPhysics(enabled = T, solver = "forceAtlas2Based")
  
  Netlist[[marker]] = plotnet
  htmlfile = paste0(Home,"/output/SEMplot_",marker,".html")
  visSave(plotnet, htmlfile)
  webshot(paste0(Home,"/output/SEMplot_",marker,".html"), zoom = 1, 
          file = paste0(Home,"/output/SEMplot_",marker,".png"))
}
rmd_paths <-paste0(tempfile(c(names(Netlist))),".Rmd")
names(rmd_paths) <- names(Netlist)

for (n in names(rmd_paths)) {
    sink(file = rmd_paths[n])
    cat("  \n",
        "```{r, echo = FALSE}",
            "Netlist[[n]]",
        "```",
        sep = "  \n")
    sink()
}

Interactive results SEM analysis

only direct effects with a significant standardized effect of p<0.05 are shown.

    for (n in names(rmd_paths)) {
        cat(knitr::knit_child(rmd_paths[[n]],
                              quiet= TRUE))
        file.remove(rmd_paths[[n]])
    }

sessionInfo()
R version 4.0.3 (2020-10-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

Random number generation:
 RNG:     Mersenne-Twister 
 Normal:  Inversion 
 Sample:  Rounding 
 
locale:
[1] LC_COLLATE=German_Germany.1252  LC_CTYPE=German_Germany.1252   
[3] LC_MONETARY=German_Germany.1252 LC_NUMERIC=C                   
[5] LC_TIME=German_Germany.1252    

attached base packages:
[1] parallel  stats4    stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] RCircos_1.2.1               compareGroups_4.4.6        
 [3] webshot_0.5.2               visNetwork_2.0.9           
 [5] org.Hs.eg.db_3.12.0         AnnotationDbi_1.52.0       
 [7] xlsx_0.6.5                  gprofiler2_0.2.0           
 [9] BiocParallel_1.24.1         kableExtra_1.3.1           
[11] glmpca_0.2.0                knitr_1.30                 
[13] DESeq2_1.30.0               SummarizedExperiment_1.20.0
[15] Biobase_2.50.0              MatrixGenerics_1.2.0       
[17] matrixStats_0.57.0          GenomicRanges_1.42.0       
[19] GenomeInfoDb_1.26.2         IRanges_2.24.1             
[21] S4Vectors_0.28.1            BiocGenerics_0.36.0        
[23] forcats_0.5.0               stringr_1.4.0              
[25] dplyr_1.0.2                 purrr_0.3.4                
[27] readr_1.4.0                 tidyr_1.1.2                
[29] tibble_3.0.4                tidyverse_1.3.0            
[31] semPlot_1.1.2               lavaan_0.6-7               
[33] ggplot2_3.3.3               gplots_3.1.1               
[35] corrplot_0.84               RColorBrewer_1.1-2         
[37] workflowr_1.6.2            

loaded via a namespace (and not attached):
  [1] tidyselect_1.1.0       lme4_1.1-26            RSQLite_2.2.2         
  [4] htmlwidgets_1.5.3      grid_4.0.3             munsell_0.5.0         
  [7] chron_2.3-56           statmod_1.4.35         withr_2.4.1           
 [10] colorspace_2.0-0       uuid_0.1-4             OpenMx_2.18.1         
 [13] rstudioapi_0.13        officer_0.3.16         rJava_0.9-13          
 [16] huge_1.3.4.1           mi_1.0                 git2r_0.28.0          
 [19] GenomeInfoDbData_1.2.4 mnormt_2.0.2           farver_2.0.3          
 [22] bit64_4.0.5            rprojroot_2.0.2        coda_0.19-4           
 [25] vctrs_0.3.6            generics_0.1.0         xfun_0.20             
 [28] R6_2.5.0               arm_1.11-2             locfit_1.5-9.4        
 [31] bitops_1.0-6           cachem_1.0.1           DelayedArray_0.16.0   
 [34] assertthat_0.2.1       promises_1.1.1         scales_1.1.1          
 [37] nnet_7.3-15            gtable_0.3.0           processx_3.4.5        
 [40] rlang_0.4.10           genefilter_1.72.0      systemfonts_0.3.2     
 [43] splines_4.0.3          lazyeval_0.2.2         broom_0.7.3           
 [46] checkmate_2.0.0        yaml_2.2.1             reshape2_1.4.4        
 [49] abind_1.4-5            modelr_0.1.8           crosstalk_1.1.1       
 [52] d3Network_0.5.2.1      backports_1.2.0        httpuv_1.5.5          
 [55] HardyWeinberg_1.7.1    Hmisc_4.4-2            tools_4.0.3           
 [58] psych_2.0.12           ellipsis_0.3.1         Rsolnp_1.16           
 [61] Rcpp_1.0.5             plyr_1.8.6             base64enc_0.1-3       
 [64] zlibbioc_1.36.0        RCurl_1.98-1.2         rockchalk_1.8.144     
 [67] ps_1.5.0               rpart_4.1-15           pbapply_1.4-3         
 [70] qgraph_1.6.5           haven_2.3.1            cluster_2.1.0         
 [73] fs_1.5.0               magrittr_2.0.1         data.table_1.13.6     
 [76] openxlsx_4.2.3         flextable_0.6.2        reprex_1.0.0          
 [79] truncnorm_1.0-8        tmvnsim_1.0-2          matrixcalc_1.0-3      
 [82] whisker_0.4            mime_0.9               xlsxjars_0.6.1        
 [85] hms_1.0.0              evaluate_0.14          xtable_1.8-4          
 [88] XML_3.99-0.5           jpeg_0.1-8.1           readxl_1.3.1          
 [91] gridExtra_2.3          compiler_4.0.3         mice_3.12.0           
 [94] writexl_1.3.1          KernSmooth_2.23-18     crayon_1.3.4          
 [97] minqa_1.2.4            htmltools_0.5.1.1      corpcor_1.6.9         
[100] later_1.1.0.1          Formula_1.2-4          geneplotter_1.68.0    
[103] lubridate_1.7.9.2      DBI_1.1.1              kutils_1.70           
[106] dbplyr_2.0.0           MASS_7.3-53            boot_1.3-26           
[109] Matrix_1.2-18          cli_2.2.0              igraph_1.2.6          
[112] BDgraph_2.63           pkgconfig_2.0.3        sem_3.1-11            
[115] foreign_0.8-81         plotly_4.9.3           xml2_1.3.2            
[118] pbivnorm_0.6.0         annotate_1.68.0        XVector_0.30.0        
[121] rvest_0.3.6            callr_3.5.1            digest_0.6.27         
[124] rmarkdown_2.6          cellranger_1.1.0       htmlTable_2.1.0       
[127] lisrelToR_0.1.4        gdtools_0.2.3          shiny_1.6.0           
[130] gtools_3.8.2           rjson_0.2.20           nloptr_1.2.2.2        
[133] lifecycle_0.2.0        nlme_3.1-151           glasso_1.11           
[136] jsonlite_1.7.2         carData_3.0-4          viridisLite_0.3.0     
[139] fansi_0.4.1            pillar_1.4.7           lattice_0.20-41       
[142] fastmap_1.1.0          httr_1.4.2             survival_3.2-7        
[145] glue_1.4.2             zip_2.1.1              fdrtool_1.2.16        
[148] png_0.1-7              bit_4.0.4              stringi_1.5.3         
[151] regsem_1.6.2           blob_1.2.1             latticeExtra_0.6-29   
[154] caTools_1.18.1         memoise_2.0.0