从读取表达矩阵到建立seurat对象都在服务器上进行（文件太大）
建立Seurat对象后，save.image，之后转移到电脑上操作。
读取表达矩阵
> rm(list=ls())
> library(readr)
> control.matrix <- read_tsv("control_matrix.tsv")
── Column specification ───────────────────────────────────────────
cols(
.default = col_double(),
X1 = col_character()
)
ℹ Use `spec()` for the full column specifications.
|====================================================| 100% 1068 MB
Warning message:
Missing column names filled in: 'X1' [1]
> F.matrix <- read_tsv("F_matrix.tsv")
── Column specification ───────────────────────────────────────────
cols(
.default = col_double(),
X1 = col_character()
)
ℹ Use `spec()` for the full column specifications.
|=====================================================| 100% 814 MB
Warning message:
Missing column names filled in: 'X1' [1]
> class(control.matrix)
[1] "spec_tbl_df" "tbl_df" "tbl" "data.frame"
> View(control.matrix)
control.matrix
将data.frame转化为matrix，遇到报错
> control.matrix <- as.matrix(control.matrix)
Error: vector memory exhausted (limit reached?)
原因：电脑内存不够
mac解决：打开终端
步骤1：打开终端，
步骤2：
cd〜
touch .Renviron
打开.Renviron
步骤3：将以下内容保存为 .Renviron 的第一行：
< pre> R_MAX_VSIZE = 100Gb
注意：此限制包括物理限制和虚拟内存；因此，在具有16Gb物理内存的计算机上设置_MAX_VSIZE = 16Gb可能不会防止此错误。您可能需要使用此参数，具体取决于计算机的规格
（参考：https://www.it1352.com/1587330.html）
> F.matrix <- as.matrix(F.matrix)
> class(F.matrix)
[1] "matrix" "array"
构建行名为基因名，列名为细胞名的表达矩阵
#处理矩阵
> F.matrix[1:6,1:6]
X1 AAACATCGAAACATCGCGACTGGA AAACATCGAAACATCGGCTAACGA
[1,] "Gnai3" " 1" " 1"
[2,] "Cdc45" " 0" " 0"
[3,] "H19" " 0" " 0"
[4,] "Apoh" " 0" " 0"
[5,] "Narf" " 0" " 0"
[6,] "Cav2" " 0" " 0"
AAACATCGAAACATCGTAGGATGA AAACATCGAACAACCAGCCACATA AAACATCGAACCGAGAACTATGCA
[1,] " 1" " 0" " 1"
[2,] " 0" " 0" " 0"
[3,] " 0" " 0" " 0"
[4,] " 0" " 0" " 0"
[5,] " 0" " 0" " 0"
[6,] " 0" " 0" " 0"
> rownames(F.matrix)=F.matrix[,1]
> F.matrix[1:6,1:6]
> F.matrix <- F.matrix[,2:ncol(F.matrix)]
> F.matrix[1:6,1:6]
AAACATCGAAACATCGCGACTGGA AAACATCGAAACATCGGCTAACGA AAACATCGAAACATCGTAGGATGA
Gnai3 " 1" " 1" " 1"
Cdc45 " 0" " 0" " 0"
H19 " 0" " 0" " 0"
Apoh " 0" " 0" " 0"
Narf " 0" " 0" " 0"
Cav2 " 0" " 0" " 0"
AAACATCGAACAACCAGCCACATA AAACATCGAACCGAGAACTATGCA AAACATCGAACCGAGAGTCTGTCA
Gnai3 " 0" " 1" " 0"
Cdc45 " 0" " 0" " 0"
H19 " 0" " 0" " 0"
Apoh " 0" " 0" " 0"
Narf " 0" " 0" " 0"
Cav2 " 0" " 0" " 0"
#处理control矩阵
> rownames(control.matrix)=control.matrix[,1]
> control.matrix <- control.matrix[,2:ncol(control.matrix)]
保留行名（基因名）和列名（样本名）信息
> dimnames=list(rownames(F.matrix),colnames(F.matrix))
矩阵中的数字为字符串，将其转换为数值。
>F.matrix=matrix(as.numeric(F.matrix),nrow = nrow(F.matrix),dimnames = dimnames)
在列名的barcode前 添加样本信息
colnames(con.matrix)=paste0('con_', colnames(con.matrix))
colnames(F.matrix)=paste0('F_', colnames(F.matrix))
保存矩阵
write.table(control.matrix,file="control.matrix.txt",sep="\t",row.names=T,quote=F)
write.table(F.matrix,file="F.matrix.txt",sep="\t",row.names=T,quote=F)
重新导入
control.matrix=read.table(file="control.matrix.txt",sep="\t")
F.matrix=read.table(file="F.matrix.txt",sep="\t")
方法1: 先将两个矩阵各自构建Seurat对象，再进行merge
con <- CreateSeuratObject(counts = control.matrix)
F <- CreateSeuratObject(counts = F.matrix)
all.matrix1 <- merge(con,F, add.cell.ids=c("con","F"))
#add.cell.ids是为了防止数据集之间的barcodes重叠，给细胞添加ID
#这个方法得到的seurat对象的orig.ident是character，不是直接两个level。但active.ident是正常的两个level。WHY???（目前看没什么影响）
table(Idents(scRNA1)) #查看每个样多少个细胞
con F
21963 17682
方法2:先将两个矩阵合并再构建一个Seurat对象
按行合并矩阵，取并集
stringsAsFactors=F
all.matrix=merge(control.matrix, F.matrix, by = "row.names", all = TRUE)
#Replace NA's with zeros
all.matrix[is.na(all.matrix)] <- 0
#两个矩阵合并的到了data.frame? 为啥？
#将数据框转化为矩阵
all.matrix=as.matrix(all.matrix)
all.matrix[1:6,1:6]
Row.names con_AAACATCGAAACATCGAATGTTGC con_AAACATCGAAACATCGAGCAGGAA
[1,] "0610005C13Rik" " 0" " 0"
[2,] "0610006L08Rik" " 0" " 0"
[3,] "0610009B22Rik" " 0" " 0"
[4,] "0610009E02Rik" " 0" " 0"
[5,] "0610009L18Rik" " 0" " 0"
[6,] "0610009O20Rik" " 0" " 0"
con_AAACATCGAAACATCGCAGCGTTA con_AAACATCGAACAACCAAAGAGATC
[1,] " 0" " 0"
[2,] " 0" " 0"
[3,] " 1" " 0"
[4,] " 0" " 0"
[5,] " 0" " 0"
[6,] " 0" " 0"
con_AAACATCGAACCGAGAACAGCAGA
[1,] " 0"
[2,] " 0"
[3,] " 0"
[4,] " 0"
[5,] " 0"
#!!!为什么会这样
#按上面处理单个矩阵的方法，处理all.matrix
#保存矩阵
write.table(all.matrix,file="all.matrix.txt",sep="\t",row.names=T,quote=F)
建立Seurat对象
scRNA1 <- CreateSeuratObject(counts = all.matrix)
save.image(file = "scRNA1.RData") #保存
使用filezilla传到电脑，使用Rstudio操作