Merge pull request #1 from GuidoBarzaghi/main

Final version

.gitignore

@@ -37,3 +37,7 @@ vignettes/*.pdf
 
 # R Environment Variables
 .Renviron
+
+# custom added
+*.html
+*.Rproj

README.Rmd

@@ -0,0 +1,221 @@
+---
+title: "Kleinendorst_et_al"
+author: "GuidoBarzaghi"
+date: "18/07/2021"
+output: rmarkdown::github_document
+---
+
+```{r setup, include=FALSE}
+knitr::opts_chunk$set(echo = TRUE, warning = FALSE, message = FALSE)
+```
+
+Fig.2
+```{r, fig.width=12}
+library(BSgenome.Mmusculus.UCSC.mm10)
+library(tidyverse)
+
+# Get list of ChIP confirmed JASPAR TFBSs
+JASPAR_TFBSs = readRDS("/g/krebs/krebs/analysis/SMF/MM/analysis/mapped_jaspar_ChIP_annotated.rds")
+JASPAR_TFBSs_ChIP = JASPAR_TFBSs[JASPAR_TFBSs$isBound == TRUE]
+
+UpstreamBins = GRanges(seqnames(JASPAR_TFBSs_ChIP), IRanges(start = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) - 35, end = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) - 25))
+UpstreamBinSeq = getSeq(BSgenome.Mmusculus.UCSC.mm10, UpstreamBins)
+TFBSBins = GRanges(seqnames(JASPAR_TFBSs_ChIP), IRanges(start = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) - 15, end = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) + 15))
+TFBSBinsSeq = getSeq(BSgenome.Mmusculus.UCSC.mm10, TFBSBins)
+DownstreamBins = GRanges(seqnames(JASPAR_TFBSs_ChIP), IRanges(start = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) + 25, end = end(JASPAR_TFBSs_ChIP) - (width(JASPAR_TFBSs_ChIP)/2) + 35))
+DownstreamBinSeq = getSeq(BSgenome.Mmusculus.UCSC.mm10, DownstreamBins)
+
+Covered_SE = vcountPattern(pattern = "DGCHN", subject = UpstreamBinSeq, fixed = F) > 0 & 
+  vcountPattern(pattern = "DGCHN", subject = TFBSBinsSeq, fixed = F) > 0 &
+  vcountPattern(pattern = "DGCHN", subject = DownstreamBinSeq, fixed = F) > 0
+
+UpstreamCoverage = vcountPattern(pattern = "GC", subject = UpstreamBinSeq) > 0 | vcountPattern(pattern = "CG", subject = UpstreamBinSeq) > 0
+TFBSCoverage = vcountPattern(pattern = "GC", subject = TFBSBinsSeq) > 0 | vcountPattern(pattern = "CG", subject = TFBSBinsSeq) > 0
+DownstreamCoverage = vcountPattern(pattern = "GC", subject = DownstreamBinSeq) > 0 | vcountPattern(pattern = "CG", subject = DownstreamBinSeq) > 0
+Covered_DE = UpstreamCoverage & TFBSCoverage & DownstreamCoverage
+
+JASPAR_TFBSs_ChIP %>%
+  as_tibble() %>%
+  mutate(TFBS = names(JASPAR_TFBSs_ChIP)) %>%
+  mutate(Covered_SE = Covered_SE, Covered_DE = Covered_DE) %>%
+  select(TFBS, name, Covered_SE, Covered_DE) -> TFBS_coverage_DF
+
+# N.b.: because I only ask for TFBSs to contain AT LEAST one cytosine, overcounting GCGs has no consequence
+
+# TF-wise
+TFBS_coverage_DF %>%
+  group_by(name) %>%
+  summarise(Count = sum(Covered_SE)) %>%
+  mutate(ExpType = "SE SMF") -> SE_TFBSs
+TFBS_coverage_DF %>%
+  group_by(name) %>%
+  summarise(Count = sum(Covered_DE)) %>%
+  mutate(ExpType = "DE SMF") -> DE_TFBSs
+TFBS_coverage_DF %>%
+  group_by(name) %>%
+  summarise(Count = n()) %>%
+  mutate(ExpType = "Total") -> Tot_TFBSs
+
+rbind(SE_TFBSs, DE_TFBSs, Tot_TFBSs) %>%
+  spread(ExpType, Count) %>%
+  gather(ExpType, nrTFBSs, -name, -Total) %>%
+  mutate(perc = (nrTFBSs / Total)*100) %>%
+  select(-nrTFBSs) %>%
+  spread(ExpType, perc) %>%
+  ggplot() +
+  geom_point(aes(x=`DE SMF`, y=`SE SMF`, size=Total)) +
+  ggrepel::geom_label_repel(aes(x=`DE SMF`, y=`SE SMF`, label=name), force = 3, box.padding = 0.5, max.overlaps = 15) +
+  xlab("% TFBS covered with DE SMF") +
+  ylab("% TFBS covered with SE SMF") +
+  theme_classic()
+```
+
+Fig.3
+```{r, fig.width=12, fig.height=6}
+library(SingleMoleculeFootprinting)
+library(BSgenome)
+library(tidyverse)
+
+Qinput = "/g/krebs/barzaghi/HTS/SMF/MM/2021-05-06-000000000-JFM6H_Lambda/aln/Qinput.txt"
+LambdaGenomeSeq = readDNAStringSet(filepath = "/g/krebs/barzaghi/analyses/misc/NP_rebuttal/LambdaGenome.fasta")
+names(LambdaGenomeSeq) = "J02459.1"
+QuasRprj = QuasR::qAlign(sampleFile = Qinput, genome = "/g/krebs/barzaghi/analyses/misc/NP_rebuttal/LambdaGenome.fasta", paired = 'fr', bisulfite = "undir")
+QuasRprj@aligner = "Rbowtie"
+Methylation = QuasR::qMeth(proj = QuasRprj, mode = "allC")
+
+GCs = FilterContextCytosines(MethGR = Methylation, genome = LambdaGenomeSeq, context = "GC")
+CGs = FilterContextCytosines(MethGR = Methylation, genome = LambdaGenomeSeq, context = "CG")
+
+GCs_collapsed = CollapseStrands(MethGR = GCs, context = "GC")
+CGs_collapsed = CollapseStrands(MethGR = CGs, context = "HCG")
+
+GCs_filtered = CoverageFilter(MethGR = GCs_collapsed, thr = 20)
+CGs_filtered = CoverageFilter(MethGR = CGs_collapsed, thr = 20)
+
+start(GCs_filtered) = start(GCs_filtered) - 2
+end(GCs_filtered) = end(GCs_filtered) + 1
+start(CGs_filtered) = start(CGs_filtered) - 1
+end(CGs_filtered) = end(CGs_filtered) + 2
+AllCs = c(GCs_filtered, CGs_filtered)
+
+KMERS = getSeq(LambdaGenomeSeq, AllCs)
+AllCs$Kmer_Context = KMERS
+
+AllCs %>%
+  as_tibble() %>%
+  filter(GenomicContext == "CG") %>%
+  select(-seqnames, -end, -strand, -width) %>%
+  gather(Measure, Value, -Kmer_Context, -GenomicContext, -start) %>%
+  separate(Measure, into = c("Sample", "Measure"), sep = "_", remove = TRUE) %>%
+  spread(Measure, Value) %>%
+  filter(str_detect(Sample, "CG")) -> EDA_DF_CGs
+AllCs %>%
+  as_tibble() %>%
+  filter(GenomicContext == "GC") %>%
+  select(-seqnames, -end, -strand, -width) %>%
+  gather(Measure, Value, -Kmer_Context, -GenomicContext, -start) %>%
+  separate(Measure, into = c("Sample", "Measure"), sep = "_", remove = TRUE) %>%
+  spread(Measure, Value)  %>%
+  filter(str_detect(Sample, "GC"))-> EDA_DF_GCs
+
+grep("Kmer_Context|GenomicContext", sort(unique(gsub("_Coverage$|_MethRate$", "", colnames(elementMetadata(AllCs))))), invert = TRUE, value = TRUE) %>%
+  data.frame() %>%
+  rename("Sample" = ".") %>%
+  mutate(Enzyme = gsub("[[:digit:]]|R", "", Sample), Conc = gsub("[[:alpha:]]", "", Sample)) %>%
+  mutate(Concentration = c(0,0.75,10,1.5,2,3,4,
+                           0,0.2,0.7,10,1.2,1.5,2,
+                           0.2,0.7,10,1.2,1.5,2)) %>%
+  mutate(Replicate = c(rep(1, 14), rep(2, 6))) %>%
+  select(-Conc) -> SamplesDict
+
+plyr::join(rbind(EDA_DF_CGs, EDA_DF_GCs), SamplesDict) -> Plotting_DF
+Plotting_DF %>%
+  group_by(Kmer_Context, Enzyme, Concentration) %>%
+  summarise(Median_MethRate = median(MethRate, na.rm = TRUE)) %>%
+  ungroup() -> Summarised_Plotting_DF
+
+Summarised_Plotting_DF %>%
+  group_by(Kmer_Context, Enzyme) %>%
+  arrange((Concentration)) %>%
+  mutate(MethRate_seqDiff = c(Median_MethRate[1], diff(Median_MethRate))) %>%
+  ungroup() %>%
+  mutate(Concentration = factor(Concentration, levels = rev(unique(Concentration)))) %>%
+  ggplot() +
+  geom_bar(aes(reorder(Kmer_Context, Median_MethRate, na.rm=TRUE), MethRate_seqDiff*100, fill=Concentration), stat='identity', position = "stack") +
+  facet_wrap(~Enzyme, scales = "free_x", labeller = as_labeller(c("GC" = "M.CviPI", "CG" = "M.SssI"))) +
+  xlab("") +
+  ylab("Methylation (%)") +
+  theme_classic() +
+  labs(fill = "Concentration\n[U/\u03bcg]") +
+  theme(axis.text.x = element_text(angle=90, vjust = 0.5), text = element_text(size = 25)) +
+  scale_y_continuous(breaks = c(0,100)) +
+  scale_fill_manual(values = rev(c(RColorBrewer::brewer.pal(name = "Reds", n=9), "black")))
+```
+
+Fig.6
+```{r, fig.width=12, fig.height=6}
+library(SingleMoleculeFootprinting) # for this use the dev version https://github.com/Krebslabrep/SingleMoleculeFootprinting/tree/dev
+library(ggplot2)
+library(tidyverse)
+library(BSgenome.Mmusculus.UCSC.mm10)
+
+Qinput = "/g/krebs/barzaghi/HTS/SMF/MM/QuasR_input_files/QuasR_input_AllCanWGpooled_dprm.txt"
+
+MiSeq_Samples = grep("NO.*_MiSeq$", readr::read_delim(Qinput, "\t")$SampleName, value = TRUE)
+NextSeq_Sample = "SMF_MM_TKO_as_NO_R_NextSeq"
+
+chr19 = GRanges(seqinfo(BSgenome.Mmusculus.UCSC.mm10))[19]
+
+CallContextMethylation(sampleSheet = Qinput,
+                       sample = MiSeq_Samples,
+                       genome = BSgenome.Mmusculus.UCSC.mm10,
+                       RegionOfInterest = chr19,
+                       coverage = 1,
+                       returnSM = FALSE) -> MiSeq
+CallContextMethylation(sampleSheet = Qinput,
+                       sample = NextSeq_Sample,
+                       genome = BSgenome.Mmusculus.UCSC.mm10,
+                       RegionOfInterest = chr19,
+                       coverage = 20,
+                       returnSM = FALSE) -> NextSeq
+
+LowCoverageMethRateDistribution(LowCoverage = MiSeq$DGCHN,
+                                LowCoverage_samples = MiSeq_Samples,
+                                HighCoverage = NextSeq$DGCHN,
+                                HighCoverage_samples = NextSeq_Sample,
+                                bins = 50, returnDF = TRUE, returnPlot = TRUE, MSE = TRUE, return_MSE_DF = TRUE, return_MSE_plot = TRUE) -> Results
+
+Results$MethylationDistribution_DF %>%
+  mutate(`Sample quality` = ifelse(Sample %in% c("SMF_MM_NP_NO_R3_MiSeq", "SMF_MM_NP_NO_R4_MiSeq"), "bad", "Good")) %>%
+  ggplot(aes(x=ExpectedMeth*100,y=ObservedMeth*100, group=interaction(Sample,Coverage), color=`Sample quality`)) +
+  geom_line(aes(linetype=Coverage, size=Coverage)) +
+  ylab("Observed Methylation (%)") +
+  xlab("Expected Methylation (%)") +
+  theme_classic() +
+  guides(color = guide_legend(override.aes = list(size = 1)), linetype = guide_legend(override.aes = list(size = 1))) +
+  theme(text = element_text(size = 15), legend.key.width = unit(5, "mm")) +
+  scale_x_continuous(breaks = c(0,100)) +
+  scale_y_continuous(breaks = c(0,100)) +
+  scale_size_manual("type", values = c(2.5, 1), guide = "none") -> pl1
+
+Results$MSE_DF %>%
+  mutate(`Sample quality` = ifelse(Sample %in% c("SMF_MM_NP_NO_R3_MiSeq", "SMF_MM_NP_NO_R4_MiSeq"), "bad", "Good")) %>%
+  filter(Sample != "SMF_MM_TKO_as_NO_R_NextSeq") %>%
+  mutate(Sample = gsub("_", " ",
+                       gsub("NP", "Neural_progenitor",
+                            gsub("ES", "mESC",
+                                 gsub("_MiSeq$|_NO|SMF_|MM_", "", Sample))))) %>%
+  ggplot(aes(Sample, MSE, fill=`Sample quality`)) +
+    geom_bar(stat = 'identity') +
+    ylab("Mean squared error\n(MSE)") +
+    xlab("") +
+    theme_classic() +
+    scale_y_continuous(breaks = c(0, 0.06)) +
+    theme(axis.text.x = element_text(angle = 90, vjust = 0.5), text = element_text(size = 15)) -> pl2
+  
+cowplot::plot_grid(pl1, pl2, nrow = 1, align = "none")
+```
+
+Fig.7
+
+The right panels in Fig.7 was produced as shown in the [vignette](https://www.bioconductor.org/packages/release/bioc/vignettes/SingleMoleculeFootprinting/inst/doc/SingleMoleculeFootprinting.html) of the [SingleMoleculeFootprinting](https://www.bioconductor.org/packages/release/bioc/html/SingleMoleculeFootprinting.html) Bioconductor package. The left panel of the same figure was produced using analogous code on a bait capture dataset.