VlnPlot画的其实不是原始数据

昨天的推文描述了让小提琴图肚子变大的做法：让你的小提琴肚子大起来?????????????

在此说明：这种不考虑后果，就让肚子大起来的做法是不严谨的。如需使用，建议将原始图和修改图放在一起对比，且在文章中注明。因为我们去掉0之后就改变了本身的数据分布

1.今天聊聊：如何用ggplot代替Vlnplot画图

1.?seurat包中Vlnplot

.libPaths(c( "/home/data/t040413/R/x86_64-pc-linux-gnu-library/4.2",             "/home/data/t040413/R/yll/usr/local/lib/R/site-library",               "/home/data/refdir/Rlib/", "/usr/local/lib/R/library"))?sessionInfo()getwd()?library(Seurat)pbmc=readRDS("~/gzh/featureplot_dotplot_vlnplot/pbmc3k_final.rds")VlnPlot(pbmc,features = 'CD4',pt.size = 1)?

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2.?使用ggplot完全复现Vlnplot

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p=VlnPlot(pbmc,features = 'CD4',slot = 'data')head(p$data)ggplot(p$data  ,aes(ident,CD4,fill=ident)) +   geom_violin(scale = "width",  trim = T )  +   geom_jitter( )+ theme_bw()+RotatedAxis()?

如果不喜欢网格，使用theme_classic()主题就可以了，

如果想调节肚子大小，主要是 geom_violin中的scale和adjust两个参数

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2.Vlnplot画图数据是经过“噪音”处理的

我们来观察一下画图所需要的的数据

1.?不知是否有读者发现，上图CD4的值有负数，但是我们看到原始的画图数据是没有负数的，其取值范围是【0.000000，4.311621】，如下图。

data=FetchData(object = pbmc, vars = 'CD4', slot = 'data')head(data)range(data$CD4)

2. 这就说明我们的画图数据和原始数据是不一样的，为什么呢？然后我去查了一下Vlnplot画图的源代码，结果发现：

vlnplot画图时，给数据添加了噪音 (除了加噪音，Vlnplot还可以选择是否log，在此不表)

data <- FetchData(object = object, vars = features, slot = slot)....noise <- rnorm(n = length(x = data[, feature]))/1e+05#rnorm()?函数用于生成正态分布的随机数。#在上述代码中，n 参数指定了噪声的长度，等于 data[, feature]?列的长度。因此，noise 是一个长度为 data[, feature]?列长度的正态分布随机数向量。#正态噪音是一种常用的随机噪音类型。它具有对称性和均匀性，可以模拟真实数据中的噪声。?

使用trim = F 参数，就可以看到小于0的部分。那些数据都是加了噪音的0

p=VlnPlot(pbmc,features = 'CD4',slot = 'data')head(p$data)ggplot(p$data  ,aes(ident,CD4,fill=ident)) +   geom_violin(scale = "width",  trim = F ,width=1              , adjust = 0.6)  +    # after_stat(density)( kernel="gaussian") +  #"rectangular" # geom_boxplot(width = 0.2, fill = "red") +  geom_jitter(alpha=0.02 )+ theme_classic()+RotatedAxis()?

当然，大于0部分肯定也有噪音啊，不过这些噪音都特别小，主要还是为了美化图表

3.开放问题

• 1.你认同seurat包中这种加噪音的方式吗？

• 2.你觉得这种方式是数据造假吗:让你的小提琴肚子大起来

最后，特别鸣谢Jimmy老师

。

大家平时有问题也可以看看生信技能树、单细胞天地、生信菜鸟团公众号。

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源代码详细内容，大家可以看下，部分内容我做了注释：

VlnPlot---->ExIPlot--->SingleExIPlot

> debug(Seurat::  VlnPlot)?> VlnPlot(pbmc3, "CD4",slot = "data")debugging in: VlnPlot(pbmc3, "CD4", slot = "data")?debug: {    if (!is.null(x = split.by) & getOption(x = "Seurat.warn.vlnplot.split",         default = TRUE)) {        message("The default behaviour of split.by has changed.\n",             "Separate violin plots are now plotted side-by-side.\n",             "To restore the old behaviour of a single split violin,\n",             "set split.plot = TRUE.\n      \nThis message will be shown once per session.")        options(Seurat.warn.vlnplot.split = FALSE)    }    return(ExIPlot(object = object, type = ifelse(test = split.plot,         yes = "splitViolin", no = "violin"), features = features,         idents = idents, ncol = ncol, sort = sort, assay = assay,         y.max = y.max, same.y.lims = same.y.lims, adjust = adjust,         pt.size = pt.size, cols = cols, group.by = group.by,         split.by = split.by, log = log, slot = slot, stack = stack,         combine = combine, fill.by = fill.by, flip = flip, add.noise = add.noise,         raster = raster))}Browse[2]> debug: if (!is.null(x = split.by) & getOption(x = "Seurat.warn.vlnplot.split",     default = TRUE)) {    message("The default behaviour of split.by has changed.\n",         "Separate violin plots are now plotted side-by-side.\n",         "To restore the old behaviour of a single split violin,\n",         "set split.plot = TRUE.\n      \nThis message will be shown once per session.")    options(Seurat.warn.vlnplot.split = FALSE)}?Browse[2]> debug: return(ExIPlot(object = object, type = ifelse(test = split.plot,     yes = "splitViolin", no = "violin"), features = features,     idents = idents, ncol = ncol, sort = sort, assay = assay,     y.max = y.max, same.y.lims = same.y.lims, adjust = adjust,     pt.size = pt.size, cols = cols, group.by = group.by, split.by = split.by,     log = log, slot = slot, stack = stack, combine = combine,     fill.by = fill.by, flip = flip, add.noise = add.noise, raster = raster))Browse[2]> ExIPlot?function (object, features, type = "violin", idents = NULL, ncol = NULL,     sort = FALSE, assay = NULL, y.max = NULL, same.y.lims = FALSE,     adjust = 1, cols = NULL, pt.size = 0, group.by = NULL, split.by = NULL,     log = FALSE, slot = "data", stack = FALSE, combine = TRUE,     fill.by = NULL, flip = FALSE, add.noise = TRUE, raster = NULL) {?#在 R 语言中，%||% 运算符称为“或，否则返回”。#它的作用是，如果左边的表达式值不为空，则返回左边的表达式值；#           如果左边的表达式值为空，则返回右边的表达式值。????assay?<-?assay?%||%?DefaultAssay(object?=?object)#首先检查 assay 参数是否为空。如果为空，则将其设置为 DefaultAssay()?函数的返回值。#DefaultAssay()?函数将返回 Seurat 对象的默认测量数据集。????    DefaultAssay(object = object) <- assay    if (isTRUE(x = stack)) {        if (!is.null(x = ncol)) {            warning("'ncol' is ignored with 'stack' is TRUE",                 call. = FALSE, immediate. = TRUE)        }        if (!is.null(x = y.max)) {            warning("'y.max' is ignored when 'stack' is TRUE",                 call. = FALSE, immediate. = TRUE)        }    }    else {????#?这里的ncol目的：根据给定的基因个数来决定最终展示的图形分成几列????        ncol <- ncol %||% ifelse(test = length(x = features) >             9, yes = 4, no = min(length(x = features), 3))    }    #获取画图的数据，极其关键的步骤    data <- FetchData(object = object, vars = features, slot = slot)    pt.size <- pt.size %||% AutoPointSize(data = object)    features <- colnames(x = data)???    #This code snippet ensures that the correct cells are used for generating the violin plot, based on whether specific cell identities have been requested for plotting.    if (is.null(x = idents)) {        cells <- colnames(x = object)    }    else {?    #Idents(object = object)() 括号用于调用函数。如果函数只有一个参数，则通常可以省略括号。    #但是，在某些情况下，括号仍然是必要的。        cells <- names(x = Idents(object = object)[Idents(object = object) %in%             idents])    }??#这个地方不太好理解，不知道对不对~???#在 R 语言中，[ 运算符用于访问矩阵或列表中的元素。#在 data[cells, , drop = FALSE] 中，第一个参数是 data 矩阵。第二个参数是 cells 向量，它指定要访问的元素的索引。#第三个参数是 drop = FALSE，它指定将结果保留为矩阵    ?    data <- data[cells, , drop = FALSE]    idents <- if (is.null(x = group.by)) {        Idents(object = object)[cells]    }    else {        object[[group.by, drop = TRUE]][cells]    }    if (!is.factor(x = idents)) {        idents <- factor(x = idents)    }    if (is.null(x = split.by)) {        split <- NULL    }    else {        split <- object[[split.by, drop = TRUE]][cells]        if (!is.factor(x = split)) {            split <- factor(x = split)        }        if (is.null(x = cols)) {            cols <- hue_pal()(length(x = levels(x = idents)))            cols <- Interleave(cols, InvertHex(hexadecimal = cols))        }        else if (length(x = cols) == 1 && cols == "interaction") {            split <- interaction(idents, split)            cols <- hue_pal()(length(x = levels(x = idents)))        }        else {            cols <- Col2Hex(cols)        }        if (length(x = cols) < length(x = levels(x = split))) {            cols <- Interleave(cols, InvertHex(hexadecimal = cols))        }        cols <- rep_len(x = cols, length.out = length(x = levels(x = split)))        names(x = cols) <- levels(x = split)        if ((length(x = cols) > 2) & (type == "splitViolin")) {            warning("Split violin is only supported for <3 groups, using multi-violin.")            type <- "violin"        }    }    if (same.y.lims && is.null(x = y.max)) {        y.max <- max(data)    }?    if (isTRUE(x = stack)) {        return(MultiExIPlot(type = type, data = data, idents = idents,             split = split, sort = sort, same.y.lims = same.y.lims,             adjust = adjust, cols = cols, pt.size = pt.size,             log = log, fill.by = fill.by, add.noise = add.noise,             flip = flip))    }?    plots <- lapply(X = features, FUN = function(x) {        return(SingleExIPlot(type = type, data = data[, x, drop = FALSE],             idents = idents, split = split, sort = sort, y.max = y.max,             adjust = adjust, cols = cols, pt.size = pt.size,             log = log, add.noise = add.noise, raster = raster))    })    label.fxn <- switch(EXPR = type, violin = if (stack) {        xlab    } else {        ylab    }, splitViolin = if (stack) {        xlab    } else {        ylab    }, ridge = xlab, stop("Unknown ExIPlot type ", type, call. = FALSE))?    for (i in 1:length(x = plots)) {        key <- paste0(unlist(x = strsplit(x = features[i], split = "_"))[1],             "_")        obj <- names(x = which(x = Key(object = object) == key))        if (length(x = obj) == 1) {            if (inherits(x = object[[obj]], what = "DimReduc")) {                plots[[i]] <- plots[[i]] + label.fxn(label = "Embeddings Value")            }            else if (inherits(x = object[[obj]], what = "Assay")) {                next            }            else {                warning("Unknown object type ", class(x = object),                   immediate. = TRUE, call. = FALSE)                plots[[i]] <- plots[[i]] + label.fxn(label = NULL)            }        }        else if (!features[i] %in% rownames(x = object)) {            plots[[i]] <- plots[[i]] + label.fxn(label = NULL)        }    }    if (combine) {        plots <- wrap_plots(plots, ncol = ncol)        if (length(x = features) > 1) {            plots <- plots & NoLegend()        }    }    return(plots)}<bytecode: 0x565001d8f568><environment: namespace:Seurat>########################################################################################3333??Browse[2]> SingleExIPlotfunction (data, idents, split = NULL, type = "violin", sort = FALSE,     y.max = NULL, adjust = 1, pt.size = 0, cols = NULL, seed.use = 42,     log = FALSE, add.noise = TRUE, raster = NULL) {    if (!is.null(x = raster) && isTRUE(x = raster)) {        if (!PackageCheck("ggrastr", error = FALSE)) {            stop("Please install ggrastr from CRAN to enable rasterization.")        }    }    if (PackageCheck("ggrastr", error = FALSE)) {        if ((nrow(x = data) > 1e+05) & !isFALSE(raster)) {            message("Rasterizing points since number of points exceeds 100,000.",                 "\nTo disable this behavior set `raster=FALSE`")            raster <- TRUE        }    }    if (!is.null(x = seed.use)) {        set.seed(seed = seed.use)    }??#在 R 语言中，||?运算符称为“或，否则返回”。它的作用是，如果左边的表达式值为真，则返回左边的表达式值；如果左边的表达式值为假，则返回右边的表达式值。?#在 if (!is.data.frame(x = data) || ncol(x = data) != 1) { 中，|| 运算符用于检查 data 对象是否是数据框并且是否只有一个列。#如果 data 对象不是数据框，则 is.data.frame(x = data) 表达式值为假，|| 运算符返回右边的表达式值 ncol(x = data) != 1。ncol(x = data) != 1 表达式值也为假，因此 if 语句的条件不满足，stop() 函数会被调用，导致程序中止。??    if (!is.data.frame(x = data) || ncol(x = data) != 1) {        stop("'SingleExIPlot requires a data frame with 1 column")    }    feature <- colnames(x = data)    data$ident <- idents    if ((is.character(x = sort) && nchar(x = sort) > 0) || sort) {        data$ident <- factor(x = data$ident, levels = names(x = rev(x = sort(x = tapply(X = data[,             feature], INDEX = data$ident, FUN = mean), decreasing = grepl(pattern = paste0("^",             tolower(x = sort)), x = "decreasing")))))    }    if (log) {        noise <- rnorm(n = length(x = data[, feature]))/200        data[, feature] <- data[, feature] + 1    }?#rnorm()?函数用于生成正态分布的随机数。#在上述代码中，n 参数指定了噪声的长度，等于 data[, feature]?列的长度。因此，noise 是一个长度为 data[, feature]?列长度的正态分布随机数向量。?#正态噪音是一种常用的随机噪音类型。它具有对称性和均匀性，可以模拟真实数据中的噪声。    else {        noise <- rnorm(n = length(x = data[, feature]))/1e+05    }??    if (!add.noise) {        noise <- noise * 0    }    if (all(data[, feature] == data[, feature][1])) {        warning(paste0("All cells have the same value of ", feature,             "."))    }    else {        data[, feature] <- data[, feature] + noise    }??    axis.label <- "Expression Level"    y.max <- y.max %||% max(data[, feature][is.finite(x = data[,         feature])])    if (type == "violin" && !is.null(x = split)) {        data$split <- split        vln.geom <- geom_violin        fill <- "split"    }?    else if (type == "splitViolin" && !is.null(x = split)) {        data$split <- split        vln.geom <- geom_split_violin        fill <- "split"        type <- "violin"    }    else {        vln.geom <- geom_violin        fill <- "ident"    }???    ##################################################################3#switch(EXPR = type, ...): It uses a switch statement to execute different code blocks based on the type argument, determining the plot type to create.#根据type执行不同的代码块'''    2. Violin Plot Configuration:?If type == "violin":Sets x = "ident", y = feature, xlab = "Identity", ylab = axis.label.Creates violin plot elements using geom_violin or geom_split_violin based on split.Optionally adds jittered points for individual cell visualization.Applies log scaling if log = TRUE.3. Ridge Plot Configuration:?If type == "ridge":Sets x = feature, y = "ident", xlab = axis.label, ylab = "Identity".Creates ridge plot elements using geom_density_ridges.Optionally adds jittered points.Applies log scaling if log = TRUE.'''???    switch(EXPR = type, violin = {        x <- "ident"        y <- paste0("`", feature, "`")        xlab <- "Identity"        ylab <- axis.label??    #geom <- list(...): This line creates a list named geom to store geometric elements that will be added to the ggplot object.??        geom <- list(vln.geom(scale = "width", adjust = adjust,             trim = TRUE), theme(axis.text.x = element_text(angle = 45,             hjust = 1)))?#vlnplot调整图形胖瘦的核心参数?geom#scale = "width": Scales the violins based on the number of observations in each group, ensuring equal widths.#adjust = adjust: Adjusts the bandwidth of the kernel density estimation used to create the violin shapes. This value likely comes from a user-specified argument.#trim = TRUE: Trims the violins at the extremes to limit the influence of outliers.            ???        if (is.null(x = split)) {            if (isTRUE(x = raster)) {                jitter <- ggrastr::rasterize(geom_jitter(height = 0,                   size = pt.size, show.legend = FALSE))            } else {                jitter <- geom_jitter(height = 0, size = pt.size,                   show.legend = FALSE)            }        } else {            if (isTRUE(x = raster)) {                jitter <- ggrastr::rasterize(geom_jitter(position = position_jitterdodge(jitter.width = 0.4,                   dodge.width = 0.9), size = pt.size, show.legend = FALSE))            } else {                jitter <- geom_jitter(position = position_jitterdodge(jitter.width = 0.4,                   dodge.width = 0.9), size = pt.size, show.legend = FALSE)            }        }        log.scale <- scale_y_log10()        axis.scale <- ylim?    }, ridge = {        x <- paste0("`", feature, "`")        y <- "ident"        xlab <- axis.label        ylab <- "Identity"        geom <- list(geom_density_ridges(scale = 4), theme_ridges(),             scale_y_discrete(expand = c(0.01, 0)), scale_x_continuous(expand = c(0,                 0)))        jitter <- geom_jitter(width = 0, size = pt.size, show.legend = FALSE)        log.scale <- scale_x_log10()        axis.scale <- function(...) {            invisible(x = NULL)        }    }, stop("Unknown plot type: ", type))??    plot <- ggplot(data = data, mapping = aes_string(x = x, y = y,         fill = fill)[c(2, 3, 1)]) + labs(x = xlab, y = ylab,         title = feature, fill = NULL) + theme_cowplot() +         theme(plot.title = element_text(hjust = 0.5))?    plot <- do.call(what = "+", args = list(plot, geom))?    plot <- plot + if (log) {        log.scale    }    else {        axis.scale(min(data[, feature]), y.max)    }    if (pt.size > 0) {        plot <- plot + jitter    }?    if (!is.null(x = cols)) {        if (!is.null(x = split)) {            idents <- unique(x = as.vector(x = data$ident))            splits <- unique(x = as.vector(x = data$split))            labels <- if (length(x = splits) == 2) {                splits            }            else {                unlist(x = lapply(X = idents, FUN = function(pattern,                   x) {                  x.mod <- gsub(pattern = paste0(pattern, "."),                     replacement = paste0(pattern, ": "), x = x,                     fixed = TRUE)                  x.keep <- grep(pattern = ": ", x = x.mod, fixed = TRUE)                  x.return <- x.mod[x.keep]                  names(x = x.return) <- x[x.keep]                  return(x.return)                }, x = unique(x = as.vector(x = data$split))))            }            if (is.null(x = names(x = labels))) {                names(x = labels) <- labels            }        }        else {            labels <- levels(x = droplevels(data$ident))        }        plot <- plot + scale_fill_manual(values = cols, labels = labels)    }    return(plot)}<bytecode: 0x564ff95dec80><environment: namespace:Seurat>?