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
尝试自己调，一晚上也就调到这个程度：
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：
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$gene，slot = "data")
还是很难看，间接证明Seurat包开发者的可视化功力非同一般。但是没有报warning，而且可以看到消失的基因都出现了。
试试直接给scale.data赋值：
pbmc@assays$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。
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(╯‵□′)╯︵┻━┻
