常见的群体结构的分析方法有admixture分析、系统发生数分析以及主成分分析等。
1、admixture分析
###过滤数据
常用plink软件过滤，在此就不做介绍了，直接开始后续操作。
###dmixture进行群体遗传结构分析（群体数自己决定）
for K in 3 4 5 6 7; do /home/software/admixture_linux-1.3.0/admixture --cv ld.QC.75_noinclude0-502502-geno02-maf03.bed $K | tee log${K}.out; done
###提取CV值：CV error最小的为最佳K值
grep -h CV log*.out
分析结束后生成了自己设定k值的Q文件，用于在R中绘图
R语言绘图
admixture的可视化分为两种
###最佳K值的可视化
ta1 = read.table("ld.QC.75_noinclude0-502502-geno02-maf03.ped.map.3.4.Q") ##用的是最佳K值的那个Q文件
head(ta1)
barplot(t(as.matrix(ta1)),col = rainbow(3),
xlab = "Individual",
ylab = "Ancestry",
border = NA)
####全部K值的可视化(较复杂）
利用表格根据fam文件（三列 1.地区Asia 2.ID名称，与fam文件的一致 3.样本品种）作三列的order.txt并用制表符分隔形式保存
（###可将order.txt文件的第一列地区Asia改成真正的个体名称，这样图中就会显示每个个体名称
###可将order.txt文件中的顺序进行调整则图中的顺序即为order.txt文件的个体顺序）
Session中上传工作目录，需建立一个文件夹（包括Q文件，fam、bed、bim文件，order.txt文件）
##安装软件
install packages（Rcolorbrewer）
##（导入含有Q order.txt bed bid fam的文件夹,修改以下程序中的文件名和K值）
sort.admixture <- function(admix.data){
## sort columns according to the cor
k <- length(admix.data)
n.ind <- nrow(admix.data[[1]])
name.ind <- rownames(admix.data[[1]])
admix.sorted <- list()
if (admix.data[[1]][1,1] > admix.data[[1]][1,2]){
admix.sorted[[1]] <- admix.data[[1]]
}else{
admix.sorted[[1]] <- admix.data[[1]][,c(2,1)]
}
for (i in 1:(k-1)){
admix <- matrix(nrow = n.ind, ncol = (i + 2))
cors <- cor(admix.sorted[[i]], admix.data[[i + 1]])
sorted.loc <- c()
for (j in 1:nrow(cors)){
cor <- cors[j,]
cor[sorted.loc] <- NA
sorted.loc <- c(sorted.loc, which.max(cor))
}
sorted.loc <- c(sorted.loc, which(! 1:ncol(cors) %in% sorted.loc))
cat("n_max = ", sorted.loc, "\n")
admix <- admix.data[[i + 1]][,sorted.loc]
rownames(admix) <- name.ind
admix.sorted[[i + 1]] <- admix
}
return(admix.sorted)
}
sort.iid <- function(k.values, groups){
##k.values <- admix.values[[1]]
##groups <- admix.fam
max.col <- which.max(colSums(k.values))
k.values <- cbind(k.values, groups[match(rownames(k.values), as.character(groups$iid)),])
k.values <- transform(k.values, group = as.factor(k.values$fid))
k.means <- tapply(k.values[,max.col], k.values$group, mean)
k.means <- k.means[order(k.means)]
k.sort <- data.frame(id = names(k.means),
order = order(k.means),
mean = k.means)
k.values$order <- k.sort[match(as.character(k.values$group), k.sort$id), 3]
k.values <- k.values[order(k.values$order, k.values[,max.col]),]
return(rownames(k.values))
}
sort.fid <- function(iid.order, fid.order, fam.table){
new.order <- c()
for (fid in fid.order){
new.order <- c(new.order, which(iid.order %in% fam.table[fam.table$fid == fid, "iid"]))
}
return(iid.order[new.order])
}
read.structure <- function(file, type = "structure"){
if (type == "structure"){
k.values <- read.table(file = file, header = F)
rownames(k.values) <- k.values[,1]
k.values[,1:3] <- NULL
}else{
k.values <- read.table(file = file, header = F)
}
return(k.values)
}
add.black.line <- function(data, groups, nline = 1){
# data <- as.matrix(plot.data[[2]])
# groups <- group.name
# nline <- 3
data <- as.matrix(data)
group.name <- unique(groups)
new.data <- matrix(NA, ncol = ncol(data))
black.data <- matrix(NA, nrow = nline, ncol = ncol(data))
new.name <- c(NA)
for (name in group.name){
new.data <- rbind(new.data, black.data)
new.data <- rbind(new.data, data[which(groups == name),])
new.name <- c(new.name, rep(NA,nline))
new.name <- c(new.name, rownames(data)[which(groups == name)])
}
added.data <- new.data[(nline + 2):nrow(new.data),]
rownames(added.data) <- new.name[(nline + 2):nrow(new.data)]
return(added.data)
}
##============================================================================
##
header <- "ld.QC.75_noinclude0-502502-geno02-maf03" （######plink格式的文件，改为自己使用的文件名）
max.k <- 4
admix.fn <- paste(header, 2:max.k, "Q", sep = ".")
fam.fn <- paste(header, "fam", sep = ".")
admix.fam <- read.table(fam.fn, stringsAsFactors = F,
col.names = c("fid", "iid", "pid", "mid", "sex", "pheno"))
admix.values <- lapply(admix.fn, read.table, header = F,
row.names = as.character(admix.fam$iid))
order.fn <- paste(header, "order.txt", sep = ".")
admix.order <- read.table(order.fn, col.names = c("region", "iid", "fid"), stringsAsFactors = F)
id.order <- admix.order$iid
#id.order <- sort.iid(admix.values[[1]], admix.order)
admix.data <- list()
for (i in 1:length(admix.values)){
admix.data[[i]] <- admix.values[[i]][id.order,]
}
species <- as.character(admix.order[,1])
sorted.data <- sort.admixture(admix.data)
## add black line in plot
nline <- 1
plot.data <- list()
group.order <- admix.order[match(id.order, admix.order$iid),3]
for (i in 1:length(sorted.data)){
plot.data[[i]] <- add.black.line(sorted.data[[i]], group.order, nline = nline)
}
## add xlab to plot
plot.id.list <- rownames(plot.data[[1]])
#plot.xlab <- admix.fam[match(x = plot.id.list, table = admix.fam$iid),]
plot.xlab <- admix.order[match(x = plot.id.list, table = admix.order$iid),]
plot.lab <- unique(plot.xlab$fid)
plot.lab <- plot.lab[!is.na(plot.lab)]
plot.at <- c()
start <- 0
for (fid in plot.lab){
xlen <- length(which(plot.xlab$fid == fid))
gap <- start + floor(xlen / 2)
plot.at <- c(plot.at, gap)
start <- start + nline + xlen
}
##=============================================================================
## barplot admixture and structure
library(RColorBrewer)
my.colours <- c(brewer.pal(8, "Dark2"), "mediumblue", "darkred", "coral4",
"purple3", "lawngreen", "dodgerblue1", "paleturquoise3",
"navyblue", "green3", "red1", "cyan",
"orange", "blue", "magenta4", "yellowgreen", "darkorange3",
"grey60", "black")
max.k <- length(plot.data)
n <- dim(plot.data[[1]])[1]
#pdf(file=paste(header, "admix.plot.pdf", sep = "."), width = 16, height = 12)
png(file=paste(header, "admix.plot.png", sep = "."), res=300, width = 2400, height = 2000)
par(mfrow = c(max.k,1), mar=c(0.5,2,0,0), oma=c(6,0,1,0))
par(las=2)
#for (i in 1:(max.k - 1)){
for (i in 1:max.k){
barplot(t(as.matrix(plot.data[[i]])), names.arg = rep(c(""), n),
col = my.colours, border = NA, space = 0, axes = F,
ylab = "")
axis(side = 2, at = 0.5, labels = as.character(i+1), tick = F, hadj = 0)
}
axis(side = 1, at = plot.at, labels = plot.lab, tick = F, lty = 15, cex.axis = 0.5)
#barplot(t(as.matrix(plot.data[[max.k]])), names.arg = rownames(plot.data[[1]]), axes = F,
# col = my.colours, border = NA, space = 0,
# ylab = paste("K=", max.k + 1, sep = ""), cex.axis=0.6(坐标轴字体大小), cex.names=0.6)
dev.off()
2、系统发生树分析
有很多种建树的方法，我们这里介绍下MEGA用NJ法建树和IQ-TREE用ML法建树
过滤的步骤略去
1）MEGA建树
###计算genome
/home/software/plink --bfile XXX-502502-geno02-maf03 --allow-extra-chr --chr-set 26 --genome
编写perl脚本 .pl结尾（Linux下新建.pl文件，直接进入编辑即可，当然，底下这个是现成的，改改可以直接用）
（第一个open里改成自己的genome文件
第二个open里改成自己的fam文件
第三个open里将>后面的改成输出的文件名.meg
在下面的sample_size里，将数量改成自己使用的数量）
#!usr/bin/perl
# define array of input and output files
open (AAA,"ld.QC.0-1-cattle-290-chr1-29-snp-indel.filter.pass-502502-geno01-maf00.genome.genome") || die "can't open AAA"; ##用自己上一步的genome文件
open (BBB,"290-cattle-breed.fam.txt") || die "can't open BBB"; ##使用自己的fam文件
open (CCC,">4-22-lzx_Dis.meg"); ##输出文件名在>后面改
my @aa=<AAA>;
my @bb=<BBB>;
$sample_size=290; ### 个体数目，改成自己用的数目
print CCC "#mega\n!Title: $sample_size pigs;\n!Format DataType=Distance DataFormat=UpperRight NTaxa=$sample_size;\n\n";
foreach ($num1=0;$num1<=$#bb;$num1++){
chomp $bb[$num1];
@arraynum1=split(/\s+/,$bb[$num1]);
print CCC "#$arraynum1[1]\n"; ##个体的ID名称
}
print CCC "\n";
@array=();
foreach ($num2=1;$num2<=$#aa;$num2++){
chomp $aa[$num2];
@arraynum1=split(/\s+/,$aa[$num2]);
push(@array,1-$arraynum1[12]);
}
@array2=(0);
$i=$sample_size;
while ($i>0){
push(@array2,$array2[$#array2]+$i);
$i=$i-1;
}
print "@array2";
for ($i=($sample_size-1); $i>=0; $i=$i-1){
print CCC " " x ($sample_size-($i+1));
for ($j=$array2[$sample_size-$i-1]; $j<=$array2[$sample_size-$i]-1; $j++){
print CCC "$array[$j] ";
}
print CCC "\n";
}
close AAA;
close BBB;
close CCC;
#######生成.meg文件利用MEGA软件可视化
2)IQ-TREE建树
#####转成map ped
/home/software/plink --allow-extra-chr --chr-set 27 -bfile ld.QC.xll_noinclude0.recode-502502-geno02-maf03 --recode --out ld.QC.xll_noinclude0.recode-502502-geno02-maf03
#####转成fa
nohup python /home/hmy/ped2fa.py ld.QC.xll_noinclude0.recode-502502-geno02-maf03.ped ld.QC.xll_noinclude0.recode-502502-geno02-maf03.fa &
打开fa文件修改个体ID，当然也可以不改
#####过滤I成N
sed -i "s/I/N/g" ld.QC.xll_noinclude0.recode-502502-geno02-maf03.fa
#####mafft比对
####多序列比对：是指把多条（3 条或以上）有系统进化关系的蛋白质或核酸序列进行比对，尽可能地把相同的碱基或氨基酸残基排在同一列上。
###这样做的意义是，对齐的碱基或氨基酸残基在进化上是同源的，即来自共同祖先（common ancestor）。
nohup mafft --auto ld.QC.xll_noinclude0.recode-502502-geno02-maf03.fa > ld.xll_noinclude0.recode-502502-geno02-maf03_aligned.fa &
#####Iqtree（构建进化树）
nohup /home/software/iqtree-1.6.12-Linux/bin/iqtree -s ld.QC.75_xiugai_noinclude0-502502-geno02-maf03.fa -m TEST -st DNA -bb 1000 -nt AUTO &
#####itol画树（网页搜索）
用bionj文件
3、主成分分析
（gcta）（gcta用的是数字染色体，若不是，注意替换下）
###make germ
nohup /home/software/gcta_1.92.3beta3/gcta64 --bfile ld.QC.75_noinclude0-502502-geno02-maf03 --make-grm --autosome-num 26 --out ld.QC.75_noinclude0-502502-geno02-maf03.gcta &
# autosome表示只选出常染色体来运行
###pca
nohup /home/software/gcta_1.92.3beta3/gcta64 --grm ld.QC.75_noinclude0-502502-geno02-maf03.gcta --pca 5 --out ld.QC.75_noinclude0-502502-geno02-maf03.gcta.out &
########输入的文件就是.gcta,这个文件不是上一步的生成文件，pca后面跟的是要做几个主成分的比较
R绘图
################
a=read.table("pca.eigenvec",header=F)
head(a)
dim(a)
b=read.table("pca.txt",header=F) ######pca.txt为四列的文件，（1列为品种，2列和4列为个体ID，3列为排序数字）
head(b)
library("ggplot2")
qplot(a[,3],a[,4],col=b[,1])
Breed=b[,1]
p = ggplot(data = a ,
aes(x = a[,3],
y = a[,4], ############x = a[,3], y = a[,4]代表第3列和第4列比较也就是pc1与pc2比较
group = Breed,
shape = Breed,
color = Breed)
)+geom_point(size=2) +scale_shape_manual(values = seq(0,75))
p + labs(x = "pc1", y = "pc2") #############修改x轴和y轴的坐标名称
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