From d3fe54a943ce86223e2e34f0db8e0a7df70a3c00 Mon Sep 17 00:00:00 2001 From: Arthur Durante <arthur.durante@inrae.fr> Date: Tue, 24 Jan 2023 17:47:47 +0100 Subject: [PATCH 1/8] gitignore update --- .gitignore | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/.gitignore b/.gitignore index e0e9080f..41d73df4 100644 --- a/.gitignore +++ b/.gitignore @@ -2,4 +2,5 @@ .Rhistory .RData .Ruserdata -data/processed/* \ No newline at end of file +data/processed/ +data/raw/ \ No newline at end of file -- GitLab From a6b6f948d18d12877cc0ae6845b40e8a1a95c557 Mon Sep 17 00:00:00 2001 From: Arthur Durante <arthur.durante@inrae.fr> Date: Tue, 24 Jan 2023 17:49:08 +0100 Subject: [PATCH 2/8] Normalisation of transcriptome work in progress --- 00_initialisation_and_setup.R | 1 + 01_metadata_cleaning.R | 3 + 01_transcriptome_processing.R | 2 - 02_transcriptome_normalisation.R | 153 ++++ .../PCA_batch_before_normalisation.png | Bin 0 -> 30475 bytes .../boxplot_selset_beforenormalisation.png | Bin 0 -> 113836 bytes .../proportion_of_na_after_na_filtering.png | Bin 0 -> 31498 bytes .../proportion_of_na_before_filtering.png | Bin 0 -> 31517 bytes renv.lock | 654 ++++++++++++++++++ 9 files changed, 811 insertions(+), 2 deletions(-) create mode 100644 01_metadata_cleaning.R delete mode 100644 01_transcriptome_processing.R create mode 100644 02_transcriptome_normalisation.R create mode 100644 plot/transcriptome_normalisation/PCA_batch_before_normalisation.png create mode 100644 plot/transcriptome_normalisation/boxplot_selset_beforenormalisation.png create mode 100644 plot/transcriptome_normalisation/proportion_of_na_after_na_filtering.png create mode 100644 plot/transcriptome_normalisation/proportion_of_na_before_filtering.png diff --git a/00_initialisation_and_setup.R b/00_initialisation_and_setup.R index 91f84c57..eadea6ea 100644 --- a/00_initialisation_and_setup.R +++ b/00_initialisation_and_setup.R @@ -5,3 +5,4 @@ renv::init() renv::dependencies() #command used to compare renv lock & renv library, check what package isn't saved in lock renv::status() + diff --git a/01_metadata_cleaning.R b/01_metadata_cleaning.R new file mode 100644 index 00000000..95f5a414 --- /dev/null +++ b/01_metadata_cleaning.R @@ -0,0 +1,3 @@ +# loading transcrptomic data obtained by Get-Trix +library(data.table) +transcriptome <- fread("data/raw/MA0050_All_All_pData.csv") diff --git a/01_transcriptome_processing.R b/01_transcriptome_processing.R deleted file mode 100644 index f5b27be1..00000000 --- a/01_transcriptome_processing.R +++ /dev/null @@ -1,2 +0,0 @@ -# loading transcrptomic data obtained by Get-Trix - diff --git a/02_transcriptome_normalisation.R b/02_transcriptome_normalisation.R new file mode 100644 index 00000000..f205b8cb --- /dev/null +++ b/02_transcriptome_normalisation.R @@ -0,0 +1,153 @@ +# loading transcriptomic data obtained by Get-Trix + +# package loading ----------- + +library(data.table) +library(ggplot2) +library(patchwork) +library(mixOmics) + +# function definitions --------------- + +expboxplot <- function(expmat, colors, title = "") { + oldPar <- par(mar = c(0.1, 2, 2, 0.2), mgp = c(1.5, 0.5, 0)) + nbox <- ncol(expmat) + boxplot( + expmat, + col = colors, + pch = 19, outcex = 0.1, xaxt = "n", xlim = c(nbox * 0.035, nbox * 0.965), + boxlwd = 0.2 + ) + title(paste0(title, " (", nrow(expmat), " probes)"), line = 0.5) + par(oldPar) +} + +replace_na_by_rowmean <- function(x) { + t(apply(x, 1, function(y) { + y[is.na(y)] <- mean(y, na.rm = TRUE) + y + })) +} + + + +# data loading ---------- + +samples <- fread("data/raw/MA0050_All_All_pData.csv") +probes <- fread("data/raw/MA0050_All_All_fData.csv") + +# selset: denoised data, worset: normalised data by TriX, imperfect normalisation +transcriptomes <- fread("data/raw/MA0050_All_All_SelSetNormWash.csv", dec = ",")[, -1] +transcriptomes <- as.matrix(transcriptomes) + +# before normalisation ----------- +my_colours <- setNames( + rainbow(10, s = 0.5), + unique(samples$Manip) +) + +## boxplot of probes values per samples -------- +png( + "plot/transcriptome_normalisation/boxplot_selset_beforenormalisation.png", + width = 2500, height = 800, pointsize = 16 +) +expboxplot(transcriptomes, colors = my_colours[samples$Manip], title = "SelSet") +dev.off() + +## proportions of NA --------- +na_by_row <- data.table( + rank = seq_len(nrow(transcriptomes)), + na_prop = (apply(transcriptomes, 1, function(x) sum(is.na(x)) / length(x))) |> + sort(x = _, decreasing = TRUE) +) +na_by_col <- data.table( + rank = seq_len(ncol(transcriptomes)), + na_prop = (apply(transcriptomes, 2, function(x) sum(is.na(x)) / length(x))) |> + sort(x = _, decreasing = TRUE) +) +plot_row <- ggplot(na_by_row, aes(x = rank, y = na_prop)) + + geom_line(colour = "dodgerblue", size = 1.5) + + scale_x_continuous(breaks = scales::pretty_breaks(n = 3)) + + labs(title = "Proportion of NA\nper probe", x = "Rank", y = "Proportion of NA") + + coord_cartesian(ylim = c(0, 1)) + + theme_bw(base_size = 14) +plot_col <- ggplot(na_by_col, aes(x = rank, y = na_prop)) + + geom_line(colour = "darkred", size = 1.5) + + coord_cartesian(ylim = c(0, 1)) + + labs(title = "Proportion of NA\nper sample", x = "Rank", y = "Proportion of NA") + + theme_bw(base_size = 14) + +ggsave( + "plot/transcriptome_normalisation/proportion_of_na_before_filtering.png", + plot = plot_row + plot_col, + width = 6, height = 3.5 +) + +## removing probes with too many missing values ------- +transcriptomes <- transcriptomes[apply(transcriptomes, 1, function(x) sum(is.na(x)) / length(x)) < 0.01, ] + +na_by_row <- data.table( + rank = seq_len(nrow(transcriptomes)), + na_prop = (apply(transcriptomes, 1, function(x) sum(is.na(x)) / length(x))) |> + sort(x = _, decreasing = TRUE) +) +na_by_col <- data.table( + rank = seq_len(ncol(transcriptomes)), + na_prop = (apply(transcriptomes, 2, function(x) sum(is.na(x)) / length(x))) |> + sort(x = _, decreasing = TRUE) +) +plot_row <- ggplot(na_by_row, aes(x = rank, y = na_prop)) + + geom_line(colour = "dodgerblue", size = 1.5) + + scale_x_continuous(breaks = scales::pretty_breaks(n = 3)) + + labs(title = "Proportion of NA\nper probe", x = "Rank", y = "Proportion of NA") + + coord_cartesian(ylim = c(0, 1)) + + theme_bw(base_size = 14) +plot_col <- ggplot(na_by_col, aes(x = rank, y = na_prop)) + + geom_line(colour = "darkred", size = 1.5) + + coord_cartesian(ylim = c(0, 1)) + + labs(title = "Proportion of NA\nper sample", x = "Rank", y = "Proportion of NA") + + theme_bw(base_size = 14) + +ggsave( + "plot/transcriptome_normalisation/proportion_of_na_after_na_filtering.png", + plot = plot_row + plot_col, + width = 6, height = 3.5 +) + +## replacing the few missing NA for PCA --------- +transcriptomes_nona <- replace_na_by_rowmean(transcriptomes) + +## PCA by batch ------------ + +pca_nona <- pca(t(transcriptomes_nona), ncomp = 4, center = TRUE, scale = TRUE) + +png( + "plot/transcriptome_normalisation/PCA_batch_before_normalisation.png", + width = 800, height = 800 +) +plotscale <- 3 +plotIndiv( + pca_nona, + group = samples$Manip, + legend = TRUE, legend.title = "Batch", + ind.names = FALSE, + size.title = rel(2) * plotscale, + size.subtitle = rel(1.5) * plotscale, + size.xlabel = rel(1) * plotscale, + size.ylabel = rel(1) * plotscale, + size.axis = rel(0.8) * plotscale, + size.legend = rel(1) * plotscale, + size.legend.title = rel(1.1) * plotscale +) +dev.off() + + +# TODO +# installer sva +# charger le fichier trx_metadata_gd avec fread +# faire tourner sva::ComBat en retirant le batch, sur un model sex + cond +# faire les plots ACP et boxplot sur les données normalisés +# exporter les données normalisés avec fwrite + + + diff --git a/plot/transcriptome_normalisation/PCA_batch_before_normalisation.png b/plot/transcriptome_normalisation/PCA_batch_before_normalisation.png new file mode 100644 index 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Subject: [PATCH 3/8] normalisation of transcriptomic data with sex and condition included in the models, pca and boxplot from the data --- 02_transcriptome_normalisation.R | 56 ++- .../PCA_batch_after_normalisation.png | Bin 0 -> 31382 bytes .../PCA_batch_before_normalisation.png | Bin 30475 -> 31109 bytes .../boxplot_selset_after_normalisation.png | Bin 0 -> 121155 bytes renv.lock | 461 ++++++++++++++++++ 5 files changed, 510 insertions(+), 7 deletions(-) create mode 100644 plot/transcriptome_normalisation/PCA_batch_after_normalisation.png create mode 100644 plot/transcriptome_normalisation/boxplot_selset_after_normalisation.png diff --git a/02_transcriptome_normalisation.R b/02_transcriptome_normalisation.R index f205b8cb..3d58b87a 100644 --- a/02_transcriptome_normalisation.R +++ b/02_transcriptome_normalisation.R @@ -6,6 +6,7 @@ library(data.table) library(ggplot2) library(patchwork) library(mixOmics) +library(sva) # function definitions --------------- @@ -35,6 +36,7 @@ replace_na_by_rowmean <- function(x) { samples <- fread("data/raw/MA0050_All_All_pData.csv") probes <- fread("data/raw/MA0050_All_All_fData.csv") +metadata <- fread("data/raw/trx_metadata_GD.tsv") # selset: denoised data, worset: normalised data by TriX, imperfect normalisation transcriptomes <- fread("data/raw/MA0050_All_All_SelSetNormWash.csv", dec = ",")[, -1] @@ -131,7 +133,8 @@ plotIndiv( group = samples$Manip, legend = TRUE, legend.title = "Batch", ind.names = FALSE, - size.title = rel(2) * plotscale, + title = "pca from raw data", + size.title = rel(1) * plotscale, size.subtitle = rel(1.5) * plotscale, size.xlabel = rel(1) * plotscale, size.ylabel = rel(1) * plotscale, @@ -141,13 +144,52 @@ plotIndiv( ) dev.off() +## normalisation & metadata ------- + +model_4_pca <- model.matrix(~ sex + cond, data = metadata) +normalised_set <- ComBat(transcriptomes_nona, batch = samples$Manip, mod = model_4_pca) +fwrite(normalised_set, file = "data/raw/normalised_transcriptomic_data.csv") + +## pca & boxplotfrom normalised data ------------- + +pca_norm <- pca(t(normalised_set), ncomp = 4, center = TRUE, scale = TRUE) + +png( + "plot/transcriptome_normalisation/PCA_batch_after_normalisation.png", + width = 800, height = 800 +) +plotscale <- 3 +plotIndiv( + pca_norm, + group = samples$Manip, + legend = TRUE, legend.title = "Batch", + ind.names = FALSE, + title = "pca from normalised data", + size.title = rel(1) * plotscale, + size.subtitle = rel(1.5) * plotscale, + size.xlabel = rel(1) * plotscale, + size.ylabel = rel(1) * plotscale, + size.axis = rel(0.8) * plotscale, + size.legend = rel(1) * plotscale, + size.legend.title = rel(1.1) * plotscale +) +dev.off() + +png( + "plot/transcriptome_normalisation/boxplot_selset_after_normalisation.png", + width = 2500, height = 800, pointsize = 16 +) +expboxplot(normalised_set, colors = my_colours[samples$Manip], title = "normalised data") +dev.off() + +# TO DO +# installer sva (done) +# charger le fichier trx_metadata_gd avec fread 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"Bioconductor", + "git_url": "https://git.bioconductor.org/packages/Biobase", + "git_branch": "RELEASE_3_16", + "git_last_commit": "767f2f3", + "git_last_commit_date": "2022-11-01", + "Hash": "96e8b620897cc9a03deff4097fa8b265", + "Requirements": [ + "BiocGenerics" + ] + }, + "BiocGenerics": { + "Package": "BiocGenerics", + "Version": "0.44.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/BiocGenerics", + "git_branch": "RELEASE_3_16", + "git_last_commit": "d7cd9c1", + "git_last_commit_date": "2022-11-01", + "Hash": "0de19224c2cd94f48fbc0d0bc663ce3b", + "Requirements": [] + }, "BiocManager": { "Package": "BiocManager", "Version": "1.30.19", @@ -42,6 +85,85 @@ "snow" ] }, + "Biostrings": { + "Package": "Biostrings", + "Version": "2.66.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/Biostrings", + "git_branch": "RELEASE_3_16", + "git_last_commit": "3470ca7", + "git_last_commit_date": "2022-11-01", + "Hash": "5666d0758c6dea426c81b3f644c28eaf", + "Requirements": [ + "BiocGenerics", + "GenomeInfoDb", + "IRanges", + "S4Vectors", + "XVector", + "crayon" + ] + }, + "DBI": { + "Package": "DBI", + "Version": "1.1.3", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "b2866e62bab9378c3cc9476a1954226b", + "Requirements": [] + }, + "GenomeInfoDb": { + "Package": "GenomeInfoDb", + "Version": "1.34.6", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/GenomeInfoDb", + "git_branch": "RELEASE_3_16", + "git_last_commit": "a6b9afe", + "git_last_commit_date": "2023-01-02", + "Hash": "650861279ec771af3dd9fa389a1812b7", + "Requirements": [ + "BiocGenerics", + "GenomeInfoDbData", + "IRanges", + "RCurl", + "S4Vectors" + ] + }, + "GenomeInfoDbData": { + "Package": "GenomeInfoDbData", + "Version": "1.2.9", + "Source": "Bioconductor", + "Hash": "618b1efac0f8b8c130afac3e0eafd47c", + "Requirements": [] + }, + "IRanges": { + "Package": "IRanges", + "Version": "2.32.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/IRanges", + "git_branch": "RELEASE_3_16", + "git_last_commit": "2b5c9fc", + "git_last_commit_date": "2022-11-01", + "Hash": "c4d63bc50829c9d3ac6d4500bea17b06", + "Requirements": [ + "BiocGenerics", + "S4Vectors" + ] + }, + "KEGGREST": { + "Package": "KEGGREST", + "Version": "1.38.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/KEGGREST", + "git_branch": "RELEASE_3_16", + "git_last_commit": "4dfbff9", + "git_last_commit_date": "2022-11-01", + "Hash": "c868da6f0062c0b977823dec78e92b67", + "Requirements": [ + "Biostrings", + "httr", + "png" + ] + }, "MASS": { "Package": "MASS", "Version": "7.3-58.1", @@ -76,6 +198,32 @@ "Hash": "45f0398006e83a5b10b72a90663d8d8c", "Requirements": [] }, + "RCurl": { + "Package": "RCurl", + "Version": "1.98-1.9", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "ea2f19dade6bd911b29b9bbb115d08f6", + "Requirements": [ + "bitops" + ] + }, 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"crayon": { + "Package": "crayon", + "Version": "1.5.2", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "e8a1e41acf02548751f45c718d55aa6a", + "Requirements": [] + }, + "curl": { + "Package": "curl", + "Version": "5.0.0", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "e4f97056611e8e6b8b852d13b7400cf1", + "Requirements": [] + }, "data.table": { "Package": "data.table", "Version": "1.14.6", @@ -174,6 +452,21 @@ "vctrs" ] }, + "edgeR": { + "Package": "edgeR", + "Version": "3.40.2", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/edgeR", + "git_branch": "RELEASE_3_16", + "git_last_commit": "ddb1cb9", + "git_last_commit_date": "2023-01-19", + "Hash": "e5ea1fc0a6bb6213941c8be214bf493d", + "Requirements": [ + "Rcpp", + "limma", + "locfit" + ] + }, "ellipse": { "Package": "ellipse", "Version": "0.4.3", @@ -208,6 +501,14 @@ "Hash": "8106d78941f34855c440ddb946b8f7a5", "Requirements": [] }, + "fastmap": { + "Package": "fastmap", + "Version": "1.1.0", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "77bd60a6157420d4ffa93b27cf6a58b8", + "Requirements": [] + }, "formatR": { "Package": "formatR", "Version": "1.14", @@ -235,6 +536,23 @@ "Hash": "0d9bf02413ddc2bbe8da9ce369dcdd2b", "Requirements": [] }, + "genefilter": { + "Package": "genefilter", + "Version": "1.80.3", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/genefilter", + "git_branch": "RELEASE_3_16", + "git_last_commit": "2138dca", + "git_last_commit_date": "2023-01-19", + "Hash": "456e757607027526632bc38e4d92ff50", + "Requirements": [ + "AnnotationDbi", + "Biobase", + "BiocGenerics", + "annotate", + "survival" + ] + }, "generics": { "Package": "generics", "Version": "0.1.3", @@ -303,6 +621,20 @@ "Hash": "36b4265fb818f6a342bed217549cd896", "Requirements": [] }, + "httr": { + "Package": "httr", + "Version": "1.4.4", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "57557fac46471f0dbbf44705cc6a5c8c", + "Requirements": [ + "R6", + "curl", + "jsonlite", + "mime", + "openssl" + ] + }, "igraph": { "Package": "igraph", "Version": "1.3.5", @@ -324,6 +656,14 @@ "Hash": "0080607b4a1a7b28979aecef976d8bc2", "Requirements": [] }, + "jsonlite": { + "Package": "jsonlite", + "Version": "1.8.4", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "a4269a09a9b865579b2635c77e572374", + "Requirements": [] + }, "labeling": { "Package": "labeling", "Version": "0.4.2", @@ -362,6 +702,27 @@ "rlang" ] }, + "limma": { + "Package": "limma", + "Version": "3.54.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/limma", + "git_branch": "RELEASE_3_16", + "git_last_commit": "1d1fa84", + "git_last_commit_date": "2022-11-01", + "Hash": "8d6904411e61fdbebacaf7ec69ba3e56", + "Requirements": [] + }, + "locfit": { + "Package": "locfit", + "Version": "1.5-9.7", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "08c4156abea85c9b6d4e7798427a887d", + "Requirements": [ + "lattice" + ] + }, "magrittr": { "Package": "magrittr", "Version": "2.0.3", @@ -378,6 +739,17 @@ "Hash": "57af0c3da1f1a50680b186591c3359af", "Requirements": [] }, + "memoise": { + "Package": "memoise", + "Version": "2.0.1", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "e2817ccf4a065c5d9d7f2cfbe7c1d78c", + "Requirements": [ + "cachem", + "rlang" + ] + }, "mgcv": { "Package": "mgcv", "Version": "1.8-41", @@ -389,6 +761,14 @@ "nlme" ] }, + "mime": { + "Package": "mime", + "Version": "0.12", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "18e9c28c1d3ca1560ce30658b22ce104", + "Requirements": [] + }, "mixOmics": { "Package": "mixOmics", "Version": "6.22.0", @@ -437,6 +817,16 @@ "lattice" ] }, + "openssl": { + "Package": "openssl", + "Version": "2.0.5", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "b04c27110bf367b4daa93f34f3d58e75", + "Requirements": [ + "askpass" + ] + }, "patchwork": { "Package": "patchwork", "Version": "1.1.2", @@ -472,6 +862,14 @@ "Hash": "01f28d4278f15c76cddbea05899c5d6f", "Requirements": [] }, + "plogr": { + "Package": "plogr", + "Version": "0.2.0", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "09eb987710984fc2905c7129c7d85e65", + "Requirements": [] + }, "plyr": { "Package": "plyr", "Version": "1.8.8", @@ -482,6 +880,14 @@ "Rcpp" ] }, + "png": { + "Package": "png", + "Version": "0.1-8", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "bd54ba8a0a5faded999a7aab6e46b374", + "Requirements": [] + }, "purrr": { "Package": "purrr", "Version": "1.0.1", @@ -583,6 +989,42 @@ "vctrs" ] }, + "survival": { + "Package": "survival", + "Version": "3.4-0", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "04411ae66ab4659230c067c32966fc20", + "Requirements": [ + "Matrix" + ] + }, + "sva": { + "Package": "sva", + "Version": "3.46.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/sva", + "git_branch": "RELEASE_3_16", + "git_last_commit": "4aac49c", + "git_last_commit_date": "2022-11-01", + "Hash": "cff27a16344227cf1b74703774380618", + "Requirements": [ + "BiocParallel", + "edgeR", + "genefilter", + "limma", + "matrixStats", + "mgcv" + ] + }, + "sys": { + "Package": "sys", + "Version": "3.4.1", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "34c16f1ef796057bfa06d3f4ff818a5d", + "Requirements": [] + }, "tibble": { "Package": "tibble", "Version": "3.1.8", @@ -670,6 +1112,25 @@ "Repository": "CRAN", "Hash": "c0e49a9760983e81e55cdd9be92e7182", "Requirements": [] + }, + "xtable": { + "Package": "xtable", + "Version": "1.8-4", + "Source": "Repository", + "Repository": "CRAN", + "Hash": "b8acdf8af494d9ec19ccb2481a9b11c2", + "Requirements": [] + }, + "zlibbioc": { + "Package": "zlibbioc", + "Version": "1.44.0", + "Source": "Bioconductor", + "git_url": "https://git.bioconductor.org/packages/zlibbioc", + "git_branch": "RELEASE_3_16", + "git_last_commit": "d39f0b0", + "git_last_commit_date": "2022-11-01", + "Hash": "6f578730acbdfc7ce2ca49df29bb5352", + "Requirements": [] } } } -- GitLab From 9e99c7187cfeb691e0751bc89368e5261d410201 Mon Sep 17 00:00:00 2001 From: DURANTE ARTHUR <arthur.durante@inrae.fr> Date: Wed, 25 Jan 2023 10:15:53 +0100 Subject: [PATCH 4/8] Update README.md --- README.md | 58 ------------------------------------------------------- 1 file changed, 58 deletions(-) diff --git a/README.md b/README.md index 9fb94034..2049d2b9 100644 --- a/README.md +++ b/README.md @@ -1,59 +1 @@ # pigheat_transcriptome - - - - -## Collaborate with your team - -- [ ] [Invite team members and collaborators](https://docs.gitlab.com/ee/user/project/members/) -- [ ] [Create a new merge request](https://docs.gitlab.com/ee/user/project/merge_requests/creating_merge_requests.html) -- [ ] [Automatically close issues from merge requests](https://docs.gitlab.com/ee/user/project/issues/managing_issues.html#closing-issues-automatically) -- [ ] [Enable merge request approvals](https://docs.gitlab.com/ee/user/project/merge_requests/approvals/) -- [ ] [Automatically merge when pipeline succeeds](https://docs.gitlab.com/ee/user/project/merge_requests/merge_when_pipeline_succeeds.html) - -# Editing this README - -When you're ready to make this README your own, just edit this file and use the handy template below (or feel free to structure it however you want - this is just a starting point!). Thank you to [makeareadme.com](https://www.makeareadme.com/) for this template. - -## Suggestions for a good README -Every project is different, so consider which of these sections apply to yours. The sections used in the template are suggestions for most open source projects. Also keep in mind that while a README can be too long and detailed, too long is better than too short. If you think your README is too long, consider utilizing another form of documentation rather than cutting out information. - -## Name -Choose a self-explaining name for your project. - -## Description -Let people know what your project can do specifically. Provide context and add a link to any reference visitors might be unfamiliar with. A list of Features or a Background subsection can also be added here. If there are alternatives to your project, this is a good place to list differentiating factors. - -## Badges -On some READMEs, you may see small images that convey metadata, such as whether or not all the tests are passing for the project. You can use Shields to add some to your README. Many services also have instructions for adding a badge. - -## Visuals -Depending on what you are making, it can be a good idea to include screenshots or even a video (you'll frequently see GIFs rather than actual videos). Tools like ttygif can help, but check out Asciinema for a more sophisticated method. - -## Installation -Within a particular ecosystem, there may be a common way of installing things, such as using Yarn, NuGet, or Homebrew. However, consider the possibility that whoever is reading your README is a novice and would like more guidance. Listing specific steps helps remove ambiguity and gets people to using your project as quickly as possible. If it only runs in a specific context like a particular programming language version or operating system or has dependencies that have to be installed manually, also add a Requirements subsection. - -## Usage -Use examples liberally, and show the expected output if you can. It's helpful to have inline the smallest example of usage that you can demonstrate, while providing links to more sophisticated examples if they are too long to reasonably include in the README. - -## Support -Tell people where they can go to for help. It can be any combination of an issue tracker, a chat room, an email address, etc. - -## Roadmap -If you have ideas for releases in the future, it is a good idea to list them in the README. - -## Contributing -State if you are open to contributions and what your requirements are for accepting them. - -For people who want to make changes to your project, it's helpful to have some documentation on how to get started. Perhaps there is a script that they should run or some environment variables that they need to set. Make these steps explicit. These instructions could also be useful to your future self. - -You can also document commands to lint the code or run tests. These steps help to ensure high code quality and reduce the likelihood that the changes inadvertently break something. Having instructions for running tests is especially helpful if it requires external setup, such as starting a Selenium server for testing in a browser. - -## Authors and acknowledgment -Show your appreciation to those who have contributed to the project. - -## License -For open source projects, say how it is licensed. - -## Project status -If you have run out of energy or time for your project, put a note at the top of the README saying that development has slowed down or stopped completely. Someone may choose to fork your project or volunteer to step in as a maintainer or owner, allowing your project to keep going. You can also make an explicit request for maintainers. -- GitLab From ff3e62ccd17c36c894b4f6d4c31aa8bca4f937af Mon Sep 17 00:00:00 2001 From: DURANTE ARTHUR <arthur.durante@inrae.fr> Date: Wed, 25 Jan 2023 13:45:29 +0100 Subject: [PATCH 5/8] Update README.md --- README.md | 1 + 1 file changed, 1 insertion(+) diff --git a/README.md b/README.md index 2049d2b9..3e4e333c 100644 --- a/README.md +++ b/README.md @@ -1 +1,2 @@ # pigheat_transcriptome +*** -- GitLab From 8513cc6556b4ba3645444bfbf5af2b7d15790fe1 Mon Sep 17 00:00:00 2001 From: DURANTE ARTHUR <arthur.durante@inrae.fr> Date: Wed, 25 Jan 2023 13:57:41 +0100 Subject: [PATCH 6/8] Update README.md --- README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/README.md b/README.md index 3e4e333c..a278afc9 100644 --- a/README.md +++ b/README.md @@ -1,2 +1,2 @@ # pigheat_transcriptome -*** +this repertory contains as of today the scripts and graphics for the analysis of the Crossbred Large white and Creole pig transcriptome used for the Pig Heat project. The files and data are based on the [previous work](https://forgemia.inra.fr/genepi/analyses/pig-heat-scripts) on the project. The existing files were processed and complemented during Arthur Durante's internship in the GenEpi team from January to July 2023 under the supervision of Guillaume Devailly. -- GitLab From a498cbd600a32a0674bac689f9eba406b582f861 Mon Sep 17 00:00:00 2001 From: Arthur Durante <arthur.durante@inrae.fr> Date: Wed, 25 Jan 2023 15:00:49 +0100 Subject: [PATCH 7/8] ComBat normalisation --- 01_metadata_cleaning.R | 7 +- 02_transcriptome_normalisation.R | 86 +++++++++++++----- .../PCA_batch_after_normalisation.png | Bin 31382 -> 40841 bytes .../PCA_batch_after_normalisation_bande.png | Bin 0 -> 71740 bytes .../PCA_batch_after_normalisation_group.png | Bin 0 -> 33797 bytes .../PCA_batch_after_normalisation_sex.png | Bin 0 -> 30531 bytes .../PCA_batch_before_normalisation.png | Bin 31109 -> 40134 bytes .../boxplot_selset_after_normalisation.png | Bin 121155 -> 121163 bytes 8 files changed, 68 insertions(+), 25 deletions(-) create mode 100644 plot/transcriptome_normalisation/PCA_batch_after_normalisation_bande.png create mode 100644 plot/transcriptome_normalisation/PCA_batch_after_normalisation_group.png create mode 100644 plot/transcriptome_normalisation/PCA_batch_after_normalisation_sex.png diff --git a/01_metadata_cleaning.R b/01_metadata_cleaning.R index 95f5a414..6d592a3f 100644 --- a/01_metadata_cleaning.R +++ b/01_metadata_cleaning.R @@ -1,3 +1,4 @@ -# loading transcrptomic data obtained by Get-Trix -library(data.table) -transcriptome <- fread("data/raw/MA0050_All_All_pData.csv") +# Transcriptome metadata has been assembled by this script: +# https://forgemia.inra.fr/genepi/analyses/pig-heat-scripts/-/blob/master/6.9_clean_transcripto_md.R +# and reused as is. + diff --git a/02_transcriptome_normalisation.R b/02_transcriptome_normalisation.R index 3d58b87a..5137beee 100644 --- a/02_transcriptome_normalisation.R +++ b/02_transcriptome_normalisation.R @@ -130,7 +130,8 @@ png( plotscale <- 3 plotIndiv( pca_nona, - group = samples$Manip, + group = samples$Manip, + ellipse = TRUE, legend = TRUE, legend.title = "Batch", ind.names = FALSE, title = "pca from raw data", @@ -144,42 +145,83 @@ plotIndiv( ) dev.off() -## normalisation & metadata ------- +# ComBat batch removal ------- +# checking the coordination of names +table(colnames(transcriptomes_nona)==samples$X) +#TRUE : 718 +table(colnames(transcriptomes_nona)==metadata$trix_name) +#TRUE : 718 model_4_pca <- model.matrix(~ sex + cond, data = metadata) normalised_set <- ComBat(transcriptomes_nona, batch = samples$Manip, mod = model_4_pca) -fwrite(normalised_set, file = "data/raw/normalised_transcriptomic_data.csv") +fwrite( + data.table( + probe_name = probes$ProbeName, + normalised_set + ), + file = "data/processed/transcriptomes_combat_normalised.tsv", sep = "\t" +) -## pca & boxplotfrom normalised data ------------- +## boxplot from normalised data ----- + +png( + "plot/transcriptome_normalisation/boxplot_selset_after_normalisation.png", + width = 2500, height = 800, pointsize = 16 +) +expboxplot(normalised_set, colors = my_colours[samples$Manip], title = "after ComBat") +dev.off() + +## pca from normalised data ------------- pca_norm <- pca(t(normalised_set), ncomp = 4, center = TRUE, scale = TRUE) +my_plot_pca <- function(pca, group) { + plotscale <- 3 + plotIndiv( + pca, + group = group, + ellipse = TRUE, + legend = TRUE, legend.title = "Batch", + ind.names = FALSE, + title = "pca from normalised data", + size.title = rel(1) * plotscale, + size.subtitle = rel(1.5) * plotscale, + size.xlabel = rel(1) * plotscale, + size.ylabel = rel(1) * plotscale, + size.axis = rel(0.8) * plotscale, + size.legend = rel(1) * plotscale, + size.legend.title = rel(1.1) * plotscale + ) +} + png( "plot/transcriptome_normalisation/PCA_batch_after_normalisation.png", width = 800, height = 800 ) -plotscale <- 3 -plotIndiv( - pca_norm, - group = samples$Manip, - legend = TRUE, legend.title = "Batch", - ind.names = FALSE, - title = "pca from normalised data", - size.title = rel(1) * plotscale, - size.subtitle = rel(1.5) * plotscale, - size.xlabel = rel(1) * plotscale, - size.ylabel = rel(1) * plotscale, - size.axis = rel(0.8) * plotscale, - size.legend = rel(1) * plotscale, - size.legend.title = rel(1.1) * plotscale +my_plot_pca(pca_norm, samples$Manip) +dev.off() + +#containing experimental data + +png( + "plot/transcriptome_normalisation/PCA_batch_after_normalisation_group.png", + width = 800, height = 800 ) +my_plot_pca(pca_norm,metadata$cond) dev.off() png( - "plot/transcriptome_normalisation/boxplot_selset_after_normalisation.png", - width = 2500, height = 800, pointsize = 16 + "plot/transcriptome_normalisation/PCA_batch_after_normalisation_sex.png", + width = 800, height = 800 +) +my_plot_pca(pca_norm,metadata$sex) +dev.off() + +png( + "plot/transcriptome_normalisation/PCA_batch_after_normalisation_bande.png", + width = 1200, height = 800 ) -expboxplot(normalised_set, colors = my_colours[samples$Manip], title = "normalised data") +my_plot_pca(pca_norm,metadata$bande) dev.off() # TO DO @@ -188,7 +230,7 @@ dev.off() # faire tourner sva::ComBat en retirant le batch, sur un model sex + cond (done) # faire les plots ACP et boxplot sur les données normalisés (pca-done; boxplot-done) # exporter les données normalisés avec fwrite (done) -# commencer le Readme +# commencer le Readme (wip) diff --git a/plot/transcriptome_normalisation/PCA_batch_after_normalisation.png b/plot/transcriptome_normalisation/PCA_batch_after_normalisation.png index 28376f3cec71f86732122ca5f7e40147901789fe..2457cf876e7ae15903b1484b2494d2f2a69aa16d 100644 GIT binary patch literal 40841 zcmd?Qc|4Tg7dSpM3}c<KX7`MpkO+}w4B3~GUC5AZDYCDFnHu|$wJZ^3UrX6iLK9M! zvX(G*ktKWlM(@w}_xk<wd%b@D|DNZ#ujiTPp5>l<?m73Kd&ONb)nlUPp$CCLOa}To z<{%IiaQX+MqWrR2y<-OgLDa7pTU@5Rf<USukQ+!<6{Jcji6FN|Q2sq`ZcT1(47a8x zw<e{G=EfB8@bD-oC|peLqtsRfxt&%&y(kaF)56V7)s6Dds7j>31O=Ibd5V2{VKgxm zh}OguXhu_@&E)j*F)>Lt@$oV7p_COS$s`PgpC$$V)x|_(q6;w5ZJ2@rOaY}FbNeTv 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------------------ 1 file changed, 18 deletions(-) diff --git a/02_transcriptome_normalisation.R b/02_transcriptome_normalisation.R index 5137beee..d3f05ee7 100644 --- a/02_transcriptome_normalisation.R +++ b/02_transcriptome_normalisation.R @@ -31,7 +31,6 @@ replace_na_by_rowmean <- function(x) { } - # data loading ---------- samples <- fread("data/raw/MA0050_All_All_pData.csv") @@ -120,7 +119,6 @@ ggsave( transcriptomes_nona <- replace_na_by_rowmean(transcriptomes) ## PCA by batch ------------ - pca_nona <- pca(t(transcriptomes_nona), ncomp = 4, center = TRUE, scale = TRUE) png( @@ -163,7 +161,6 @@ fwrite( ) ## boxplot from normalised data ----- - png( "plot/transcriptome_normalisation/boxplot_selset_after_normalisation.png", width = 2500, height = 800, pointsize = 16 @@ -172,7 +169,6 @@ expboxplot(normalised_set, colors = my_colours[samples$Manip], title = "after Co dev.off() ## pca from normalised data ------------- - pca_norm <- pca(t(normalised_set), ncomp = 4, center = TRUE, scale = TRUE) my_plot_pca <- function(pca, group) { @@ -201,8 +197,6 @@ png( my_plot_pca(pca_norm, samples$Manip) dev.off() -#containing experimental data - png( "plot/transcriptome_normalisation/PCA_batch_after_normalisation_group.png", width = 800, height = 800 @@ -223,15 +217,3 @@ png( ) my_plot_pca(pca_norm,metadata$bande) dev.off() - -# TO DO -# installer sva (done) -# charger le fichier trx_metadata_gd avec fread (done) -# faire tourner sva::ComBat en retirant le batch, sur un model sex + cond (done) -# faire les plots ACP et boxplot sur les données normalisés (pca-done; boxplot-done) -# exporter les données normalisés avec fwrite (done) -# commencer le Readme (wip) - - - - -- GitLab