【佳學(xué)基因檢測(cè)】如何從基因組序列文件中獲取特定基因的全部序列、編碼序列、啟動(dòng)子序列?
一、從基因組序列文件獲取特定基因序列需要參照基因組序列和注釋文件
1. 從NCBI數(shù)據(jù)庫(kù)下載人類基因組參照基因組數(shù)據(jù)文件。https://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/annotation/GRCh38_latest/refseq_identifiers/GRCh38_latest_genomic.fna.gz,下載后的文件格式是FASTA文件格式。文件的存儲(chǔ)為:GCF_000001405.35_GRCh38.p9_genomic.fna, 查看前5行的內(nèi)容:
head -5 /media/jiaxue/0B8B16F90B8B16F9/reference/GCF_000001405.35_GRCh38.p9_genomic.fna
顯示結(jié)果為:
2. 從NCBI下載人類基因組注釋文件,下載地址為:https://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/annotation/GRCh38_latest/refseq_identifiers/GRCh38_latest_genomic.gff.gz, 存儲(chǔ)為:GRCh38_latest_genomic.gff.gz,將文件解壓為GRCh38_latest_genomic.gff基因注釋文件為
GTF
格式,共有9列,看前9列信息(第三列包含了不同的元件注釋)
cut -f 1-9
/media/jiaxue/0B8B16F90B8B16F9/reference/GRCh38_latest_genomic.gff | head
head -15 /media/jiaxue/0B8B16F90B8B16F9/reference/GRCh38_latest_genomic.gff 顯示內(nèi)容為:
3. 基因組注釋文件信息內(nèi)容的解釋
二、參照基因組基因信息提取軟件介紹gffread
這里用到了gffread
, 運(yùn)行如下命令,安裝gffread。
conda install -c bioconda gffread
運(yùn)行 gffread -h 查看軟件是否安裝成功。
提取轉(zhuǎn)錄本序列、CDS和蛋白序列
gffre
ad -h
可以參考所有可用參數(shù),如果有特殊情況需要考慮的,還需配合其它參數(shù)使用。
1.獲取轉(zhuǎn)錄本序列
轉(zhuǎn)到注釋文件所在的文件夾: cd /media/jiaxue/0B8B16F90B8B16F9/reference/
gffread GRCh38_latest_genomic.gff -g GRCh38_latest_genomic.fna -w jiaxue.transcripts.fa
輸入基因組文件和注釋文件需要匹配,否則會(huì)終止。輸入匹配的文件后顯示了如下記錄:
內(nèi)容如下:
head GRCh38.transcripts.fa
>rna-NR_046018.2
2.獲取CDS序列
# 獲取CDS序列
gffread GRCh38_latest_genomic.gff -g GRCh38_latest_genomic.fna -x jiaxue.CDS.fa
內(nèi)容如下
head -150 jiaxue.CDS.fa
>rna-NM_001005484.2
3.獲取蛋白序列
# 獲取蛋白序列
gffread GRCh38_latest_genomic.gff -g GRCh38_latest_genomic.fna -y jiaxue.protein.fa
采用如下命令顯示內(nèi)容蛋白質(zhì)序列: head -150 jiaxue.protein.fa
>rna-NM_001005484.2
MKKVTAEAISWNESTSETNNSMVTEFIFLGLSDSQELQTFLFMLFFVFYGGIVFGNLLIVITVVSDSHLH
SPMYFLLANLSLIDLSLSSVTAPKMITDFFSQRKVISFKGCLVQIFLLHFFGGSEMVILIAMGFDRYIAI
CKPLHYTTIMCGNACVGIMAVTWGIGFLHSVSQLAFAVHLLFCGPNEVDSFYCDLPRVIKLACTDTYRLD
IMVIANSGVLTVCSFVLLIISYTIILMTIQHRPLDKSSKALSTLTAHITVVLLFFGPCVFIYAWPFPIKS
LDKFLAVFYSVITPLLNPIIYTLRNKDMKTAIRQLRKWDAHSSVKF
>rna-XM_047436352.1
解析GTF文件的結(jié)構(gòu)
針對(duì)本GTF,對(duì)于gene
元件,基因名字 (Gene symbol
)在第14列。
head -n 1 GRCh38.gtf | sed 's/"/ /g' | tr ' ' '
' | sed = | sed 'N;s/
/ /'
1 chr20
2 ensembl_havana
3 gene
4 87250
5 97094
6 .
7 +
8 .
9 gene_id
10 ENSG00000178591
11 ; gene_version
12 6
13 ; gene_name
14 DEFB125
15 ; gene_source
16 ensembl_havana
17 ; gene_biotype
18 protein_coding
19 ;
針對(duì)本GTF,對(duì)于transcript
元件,基因名字 (Gene symbol
)在第18列。
sed -n '2p' GRCh38.gtf | sed 's/"/ /g' | tr ' ' '
' | sed = | sed 'N;s/
/ /'
1 chr20
2 havana
3 transcript
4 87250
5 97094
6 .
7 +
8 .
9 gene_id
10 ENSG00000178591
11 ; gene_version
12 6
13 ; transcript_id
14 ENST00000608838
15 ; transcript_version
16 1
17 ; gene_name
18 DEFB125
19 ; gene_source
20 ensembl_havana
21 ; gene_biotype
22 protein_coding
23 ; transcript_name
24 DEFB125-202
25 ; transcript_source
26 havana
27 ; transcript_biotype
28 processed_transcript
29 ; transcript_support_level
30 2
31 ;
這個(gè)查看信息在哪一列是很常用的檢查文件結(jié)構(gòu)提取對(duì)應(yīng)信息的方式,簡(jiǎn)化為一個(gè)腳本checkCol.sh
檢查某個(gè)文件的指定行(默認(rèn)為先進(jìn)行)
checkCol.sh -f GRCh38.gtf
1 chr20
2 ensembl_havana
3 gene
4 87250
5 97094
6 .
7 +
8 .
9 gene_id "ENSG00000178591"; gene_version "6"; gene_name "DEFB125"; gene_source "ensembl_havana"; gene_biotype "protein_coding";
檢查標(biāo)準(zhǔn)輸入的先進(jìn)行
sed 's/"/ /g' GRCh38.gtf | checkCol.sh -f -
1 chr20
2 ensembl_havana
3 gene
4 87250
5 97094
6 .
7 +
8 .
9 gene_id
10 ENSG00000178591
11 ; gene_version
12 6
13 ; gene_name
14 DEFB125
15 ; gene_source
16 ensembl_havana
17 ; gene_biotype
18 protein_coding
19 ;
提取基因啟動(dòng)子序列
首先確定啟動(dòng)子區(qū)域,這里定義轉(zhuǎn)錄起始位點(diǎn)上游1000 bp
和下游500 bp
為啟動(dòng)子區(qū)域。
sed 's/"/ /g' GRCh38.gtf | awk 'BEGIN{OFS=FS=" "}{if($3=="gene") {if($7=="+") {start=$4-1000; end=$4+500;} else {if($7=="-") start=$5-500; end=$5+1000; } if(start<0) start=0; print $1,start,end,$14,$10,$7;}}' >GRCh38.promoter.bed
啟動(dòng)子區(qū)域如下 (這個(gè)bed文件也可以用于ChIP-seq類型的數(shù)據(jù)分析確定peak是否在啟動(dòng)子區(qū)域)
head GRCh38.promoter.bed
chr20 86250 87750 DEFB125 ENSG00000178591 +
chr20 141369 142869 DEFB126 ENSG00000125788 +
chr20 156470 157970 DEFB127 ENSG00000088782 +
chr20 189181 190681 DEFB128 ENSG00000185982 -
chr20 226258 227758 DEFB129 ENSG00000125903 +
chr20 256736 258236 DEFB132 ENSG00000186458 +
chr20 266186 267686 AL034548.1 ENSG00000272874 +
chr20 290278 291778 C20orf96 ENSG00000196476 -
chr20 295968 297468 ZCCHC3 ENSG00000247315 +
chr20 347724 349224 NRSN2-AS1 ENSG00000225377 -
然后提取序列。這里用到了bedtools
工具,官方有提供編譯好的二進(jìn)制文件,下載下來(lái)即可使用。
# -name: 輸出基因名字(bed文件的第四列)
# -s: 考慮到正反鏈(對(duì)于啟動(dòng)子區(qū)域,是否考慮鏈的信息關(guān)系不太大)
bedtools getfasta -name -s -fi GRCh38.fa -bed GRCh38.promoter.bed >GRCh38.promoter.fa
序列信息如下:
head GRCh38.promoter.fa | cut -c 1-60
>DEFB125::chr20:86250-87750(+)
ATAATTTGAAGTGAGGTAATGTGATTCCTCTAGTTTTGTTCTTTTTGCTTAGGATGGCTT
>DEFB126::chr20:141369-142869(+)
AATATTCAAGAGAATGCCAAGAAAGCTACAAGAACAAATAGCAGGTCAGTCGTTGCCTGG
>DEFB127::chr20:156470-157970(+)
ATATCCGTCACCTCAAACATTTATCATTTGTATTGGGAACATTCAAAATCCTCTCTTCTA
>DEFB128::chr20:189181-190681(-)
AAAAAAGAAAAAGAACTCCAAGTCTAATAAGACCAGAGACCTGCCCTTTATGGGTCTGCA
>DEFB129::chr20:226258-227758(+)
GAGTGGAAGGTGGGAGGAGGGAGAGGATGAGGAAAAATAACTAATGGACACTAGGCTTAA
如果不想要坐標(biāo)信息,可對(duì)序列名字做一下簡(jiǎn)化
cut -d ':' -f 1 GRCh38.promoter.fa >GRCh38.promoter.simplename.fa
head GRCh38.promoter.simplename.fa | cut -c 1-60
>DEFB125
ATAATTTGAAGTGAGGTAATGTGATTCCTCTAGTTTTGTTCTTTTTGCTTAGGATGGCTT
>DEFB126
AATATTCAAGAGAATGCCAAGAAAGCTACAAGAACAAATAGCAGGTCAGTCGTTGCCTGG
>DEFB127
ATATCCGTCACCTCAAACATTTATCATTTGTATTGGGAACATTCAAAATCCTCTCTTCTA
>DEFB128
AAAAAAGAAAAAGAACTCCAAGTCTAATAAGACCAGAGACCTGCCCTTTATGGGTCTGCA
>DEFB129
GAGTGGAAGGTGGGAGGAGGGAGAGGATGAGGAAAAATAACTAATGGACACTAGGCTTAA
提取基因序列
提取基因序列的操作也類似于提取啟動(dòng)子序列。這里要注意GFF文件的序列位置是從1
開(kāi)始,而bed
文件的位置是從0
開(kāi)始,前閉后開(kāi),所以要對(duì)序列的起始位置進(jìn)行-1的操作。
type="gene"
sed 's/"/ /g' GRCh38.gtf | awk -v type="${type}" 'BEGIN{OFS=FS=" "}{if($3==type) {print $1,$4-1,$5,$14,".",$7}}' >GRCh38.gene.bed
head GRCh38.gene.bed
chr20 87249 97094 DEFB125 . +
chr20 142368 145751 DEFB126 . +
chr20 157469 159163 DEFB127 . +
chr20 187852 189681 DEFB128 . -
chr20 227257 229886 DEFB129 . +
chr20 257735 261096 DEFB132 . +
提取基因序列
bedtools getfasta -name -s -fi GRCh38.fa -bed GRCh38.gene.bed >GRCh38.gene.fa
# 查看序列
head GRCh38.gene.fa | cut -c 1-60
>DEFB125::chr20:87249-97094(+)
ACAGGAATTCATATCGGGGTGATCACTCAGAAGAAAAGGTGAATACCGGATGTTGTAAGC
>DEFB126::chr20:142368-145751(+)
GCCATACACTTCAGCAGAGTTTGCAACTTCTCTTCTAAGTCTTTATCCTTCCCCCAAGGC
>DEFB127::chr20:157469-159163(+)
CTCTGAGGAAGGTAGCATAGTGTGCAGTTCACTGGACCAAAAGCTTTGGCTGCACCTCTT
>DEFB128::chr20:187852-189681(-)
GGCACACAGACCACTGGACAAAGTTCTGCTGCCTCTTTCTCTTGGGAAGTCTGTAAATAT
提取非編碼RNA的序列
在GTF文件中有轉(zhuǎn)錄本類型的注釋,包含下面這些注釋類型
ntisense_RNA
lincRNA
miRNA
misc_RNA
processed_pseudogene
processed_transcript
protein_coding
rRNA
scaRNA
sense_intronic
sense_overlapping
snoRNA
snRNA
TEC
transcribed_processed_pseudogene
transcribed_unitary_pseudogene
transcribed_unprocessed_pseudogene
unitary_pseudogene
unprocessed_pseudogene
我們只篩選lincRNA
grep 'transcript_biotype "lincRNA"' GRCh38.gtf >GRCh38.lincRNA.gtf
gffread GRCh38.lincRNA.gtf -g GRCh38.fa -w GRCh38.lincRNA.fa
head GRCh38.lincRNA.fa | cut -c 1-60
>ENST00000608495
GTCGCACGCGCTGGCCAAACGGGCGCACCAGACACTTTTCAGGGCCCTGCCAAAGACCTC
CTGGCGTCCCAGACACAAGAGATCCAGGCCAAGACTCACACTTCACAAGATACACAGACA
GGAACAGGAAATTCCATGAAACTTCCATTTACCCAATTAGCCGGACTCACTGAGCCCCAG
TCAACCAACTCCTACTAAAATTAAAAAGTAATGTGTGGTATAGATTGGAATAATAGACAT
AAACGATGGGAGGCGGAGAGGGGTGAGGGTTGAAAAATTACCTATTGGGTGCAACATTCA
AATGGGGCACTAGAAGCCCACTCCACCACTATGCAATATATGTATTTGTACCCCGTAAAT
提取一個(gè)個(gè)外顯子序列
獲取外顯子的坐標(biāo)
type="exon"
sed 's/"/ /g' GRCh38.gtf | awk -v type="${type}" 'BEGIN{OFS=FS=" "}{if($3==type) {print $1,$4-1,$5,$14,$20,$7}}' >GRCh38.exon.bed
# 查看文件內(nèi)容
head GRCh38.exon.bed
chr20 87249 87359 ENST00000608838 DEFB125 +
chr20 96004 97094 ENST00000608838 DEFB125 +
chr20 87709 87767 ENST00000382410 DEFB125 +
chr20 96004 96533 ENST00000382410 DEFB125 +
chr20 142368 142686 ENST00000382398 DEFB126 +
chr20 145414 145751 ENST00000382398 DEFB126 +
chr20 142633 142686 ENST00000542572 DEFB126 +
chr20 145414 145488 ENST00000542572 DEFB126 +
chr20 145578 145749 ENST00000542572 DEFB126 +
chr20 157469 157593 ENST00000382388 DEFB127 +
提取序列
# -name: 輸出基因名字(bed文件的第四列)
# -s: 考慮到正反鏈(對(duì)于啟動(dòng)子區(qū)域,是否考慮鏈的信息關(guān)系不太大)
bedtools getfasta -name -s -fi GRCh38.fa -bed GRCh38.exon.bed >GRCh38.exon.fa
# 查看序列信息
head GRCh38.exon.fa | cut -c 1-60
>ENST00000608838::chr20:87249-87359(+)
ACAGGAATTCATATCGGGGTGATCACTCAGAAGAAAAGGTGAATACCGGATGTTGTAAGC
>ENST00000608838::chr20:96004-97094(+)
GTAGCTTTGAACCCCAAAAATGTTGGAAGAATAATGTAGGACATTGCAGAAGACGATGTT
>ENST00000382410::chr20:87709-87767(+)
ATGAATATCCTGATGCTGACCTTCATTATCTGTGGGTTGCTAACTCGGGTGACCAAAG
>ENST00000382410::chr20:96004-96533(+)
GTAGCTTTGAACCCCAAAAATGTTGGAAGAATAATGTAGGACATTGCAGAAGACGATGTT
提取一個(gè)個(gè)內(nèi)含子序列
確定內(nèi)含子區(qū)域
sed 's/"/ /g' GRCh38.gtf | awk 'BEGIN{OFS=FS=" ";oldtr="";}{if($3=="exon") {tr=$14; if(oldtr!=tr) {start=$5; oldtr=tr;} else {print $1,start,$4-1,tr,$20,$7; start=$5;} } }' >GRCh38.intron.bed
# 查看文件內(nèi)容
head GRCh38.intron.bed
chr20 87359 96004 ENST00000608838 DEFB125 +
chr20 87767 96004 ENST00000382410 DEFB125 +
chr20 142686 145414 ENST00000382398 DEFB126 +
chr20 142686 145414 ENST00000542572 DEFB126 +
chr20 145488 145578 ENST00000542572 DEFB126 +
chr20 157593 158773 ENST00000382388 DEFB127 +
chr20 189681 187852 ENST00000334391 DEFB128 -
chr20 227346 229277 ENST00000246105 DEFB129 +
提取序列同上。
(責(zé)任編輯:佳學(xué)基因)