Seurat::DoHeatmap 的谜之报错

最近在学习Seurat教程（Seurat版本3.0.2），发现运行到倒数第二步做热图展示marker genes时总是报错

主要问题：

DoHeatmap(pbmc, features = top10$gene) + NoLegend()

运行结果：

Warning message:
In DoHeatmap(pbmc, features = top10$gene) :
The following features were omitted as they were not found in the scale.data slot for the RNA assay: NEAT1, SLC25A39, AC090498.1, CTSS, DNAJB1, HSP90AA1, PRF1, NKG7, KLRD1, CD8B, DUSP4, CD8A, CCL5, CD27, GZMK, CD69, TNFAIP3, ZFP36L2, IL7R

图片倒是生成了，但是数了一下基因数，确实少了很多，尤其是Cluster 0 和1,都没几个剩下的了

1565194413212.png

尝试自己调，一晚上也就调到这个程度：

1565194545049.png

好丑，还是继续研究下包里的代码吧/(ㄒoㄒ)/~~

DoHeatMap

看了一下代码，里面有这样一句：

  if (any(!features %in% possible.features)) {
    bad.features <- features[!features %in% possible.features]
    features <- features[features %in% possible.features]
    if (length(x = features) == 0) {
      stop("No requested features found in the ", 
           slot, " slot for the ", assay, " assay.")
    }
    warning("The following features were omitted as they were not found in the ", 
            slot, " slot for the ", assay, " assay: ", 
            paste(bad.features, collapse = ", "))

这个bad.features来自features和possible.feature的比对，之后判断若features内元素个数不为零则warning："The following features were omitted as they were not found in the ······“

function (object, features = NULL, cells = NULL, group.by = "ident", 
          group.bar = TRUE, disp.min = -2.5, disp.max = NULL, slot = "scale.data", 
          assay = NULL, label = TRUE, size = 5.5, hjust = 0, angle = 45, 
          raster = TRUE, draw.lines = TRUE, lines.width = NULL, group.bar.height = 0.02, 
          combine = TRUE) 

往回找，原来DoHeatMap（）有个默认参数slot = "scale.data"，自动用scale.data的数据画图。
看一下这个Seurat对象的结构：

1565195235734.png

哈哈，发现问题了，原来counts （应该是raw read count）和data 都是20647行，每行对应一个基因，但scale.data只有2000行。

> pbmc <- ScaleData(
+   object = pbmc,
+   do.scale = TRUE,
+   do.center = FALSE,
+   vars.to.regress = c("percent.mt"))

重新ScaleData()一下，发现数据没有任何改变····

算了，试试用data作图：

DoHeatmap(pbmc, features = top10$gene，slot = "data") 

1565195494082.png

还是很难看，间接证明Seurat包开发者的可视化功力非同一般。但是没有报warning，而且可以看到消失的基因都出现了。

试试直接给scale.data赋值：

pbmc@assays$RNA@scale.data <- scale(pbmc@assays$RNA@data, scale = TRUE)
DoHeatmap(pbmc, features = top10$gene,size = 0.5,slot = "scale.data") + NoLegend()

这次也没有报warning，配色还是有点难看，大概是scale参数设置的不同，想完全复现可能还得查ScaleData的代码了。不过基本上能用了。增加了height，避免文字挤在一起，其实最好是保存成ggplot的对象，拼图的时候再改。

1565195816712.png

总结：

产生这个问题的主要原因是ScaleData()指令生成scale.data（封装在Seurat对象里）中基因数减少。

因为Seurat对象封装了好几层，并且对S4对象的操作也不太熟悉，所以一开始不太容易发现原因。

另外，操作多个Seurat对象时注意偶尔rm（）+gc（）清空内存，我24G内存都好几次99%内存占用(╯‵□′)╯︵┻━┻