需要準備的文件是序列信息bed格式文件或者GRanges格式文件
比如有一個bed文件：
$ cat test.bed
chr1 1000010 1000020
chr2 1000021 1000030
chr3 1000031 1000040
我習慣保存為GR格式：
> test = read.table("test.bed")
> gr = toGR(test)
> gr
GRanges object with 3 ranges and 0 metadata columns:
seqnames ranges strand
<Rle> <IRanges> <Rle>
[1] chr1 1000010-1000020 *
[2] chr2 1000021-1000030 *
[3] chr3 1000031-1000040 *
-------
seqinfo: 3 sequences from an unspecified genome; no seqlengths
toGR是我自己編寫的函數，這里就不放出來了。此外還編寫了另外兩個函數，分別為：
-
getSeq，用于得到堿基序列
getSeq <- function(POSs){
suppressPackageStartupMessages(library(BSgenome.Hsapiens.UCSC.hg19))
seqs = c()
for(i in 1:length(POSs)){
POS = POSs[i]
chr = sapply(strsplit(POS, "[-:]"), function(z) z[1])
strat = as.numeric(sapply(strsplit(POS, "[-:]"), function(z) z[2]))
end = as.numeric(sapply(strsplit(POS, "[-:]"), function(z) z[3]))
seqs = c(seqs, as.character(BSgenome.Hsapiens.UCSC.hg19[[chr]][strat:end]))
}
return(unlist(seqs))
}
-
printSeq，用于打印堿基序列
printSeq <- function(Seq, binwidth = 50){
N_line = nchar(Seq) / binwidth
if(N_line > as.integer(N_line)){
N = as.integer(N_line) + 1
} else{
N = as.integer(N_line)
}
bases = strsplit(Seq, "")[[1]]
for(i in 0:(N-1)){
xseq = paste0(bases[(1 + binwidth*i):(binwidth + i*binwidth)], collapse = "")
if(i != (N-1)){
xseq = paste0(bases[(1 + binwidth*i):(binwidth + i*binwidth)], collapse = "")
} else{
xseq = paste0(bases[(1 + binwidth*i):length(bases)], collapse = "")
}
cat(xseq, "\n")
}
}
默認在fasta中每行顯示50個堿基
然后使用前面的GR數據生成fasta文件：
> seqs = getSeq(as.character(gr))
> seqs
[1] "CTCACCCAGGA" "AAATTGAAGA" "AGAAGGAAAC"
> ID = as.character(seqnames(gr))
> ID
[1] "chr1" "chr2" "chr3"
sink("test.fa")
for(i in 1:length(seqs)){
cat(paste0(">", ID[i]), "\n")
printSeq(seqs[i])
}
system("sed 's/ //g' test.fa > tmp.fa")
system("rm test.fa")
system("mv tmp.fa test.fa")
sink()
這樣就生成了fasta文件了：
$ cat test.fa
>chr1
CTCACCCAGGA
>chr2
AAATTGAAGA
>chr3
AGAAGGAAAC
最后還需要創建索引：
system("samtools faidx test.fa")
這樣就完成了~
好家伙，samtools有提取序列的功能，還能指定每行的堿基數，位點格式為：chr:start-end，例如
$ samtools faidx hg19.fa chr1:2066354-2066580
>chr1:2066354-2066580
AGGCAGGGGACAGACGGACCCGGCCTGCGTTGGCCTGGGGTGACTTCACGGCTCCACTGT
CAGCAAGCGGCCGTCCCGTGGTGGATCCTGTCCGCCCTGCGAGGACACCTGGCTCCATCC
ACACCTGGGCCTCTGTCTCCAGCCGCCGAGGCCGTGACACCATGAGGATCATGTGAGGAG
GGGCAGAGAGAGGCCTCCGGGAGGCCGTCATTCCAGCCCTGCCTTCC
還能將想要提取的區域寫入文件中，并傳遞給-r參數。每個區域單獨一行
區域的格式剛好是Granges對象非常容易生成的格式，所以可以寫個GR轉fasta的小函數：
grToFasta <- function(gr, file){
hg19.fa = "hg19.fa"
pos = as.character(gr)
write.table(pos, "tmp.pos", sep = "\n", quote = FALSE, col.names = FALSE, row.names = FALSE)
system(paste("samtools faidx -n 50", hg19.fa, "-r tmp.pos >", file))
system(paste("rm tmp.pos & samtools faidx", file))
}
我直接好家伙，還是調包香~
