前言

小編最近看到一個文章，該文章從同義突變位點中提取4dtv用來做後續分析，原文見 Genomic Consequences of Long-Term Population Decline in Brown Eared Pheasant ,所以很是好奇是怎麼實現的，但文章沒有說明怎麼實現的，還好通過百度最終找到了破解方法，這裡主要參考了大神的部落格，部落格原連結見從SnpEff註釋得到的VCF中過濾4DTV位點

安裝環境

（1）java #該軟體是Java編寫，小編這裡用的java-1.8.0

（2）snpEff #上邊提到的博主基於該軟體編寫的提取4DTV指令碼

（3）python3以及pysam模組

pysam模組

pip3 install pysam -i https://pypi.tuna.tsinghua.edu.cn/simple

snpEff軟體安裝

wget  https://nchc.dl.sourceforge.net/project/snpeff/snpEff_v4_5covid19_core.zip
unzip snpEff_v4_5covid19_core.zip #解壓安裝
cd snpEff ; chmod 755 *jar #進入目錄，可以看到兩個jar執行程式，分別是SnpSift和snpEff，給執行許可權

snpEff軟體配置

該軟體是一個很強大的軟體，自帶有很多基因組的資料庫，小編在此不再介紹，有興趣可以上官網檢視，

mkdir -p data/apple
ascp -i ~/asperaweb_id_dsa.openssh  -QTr -l200m  anonftp@ftp.ncbi.nlm.nih.gov:genomes/all/GCF/002/114/115/GCF_002114115.1_ASM211411v1/GCF_002114115.1_ASM211411v1_genomic.fna.gz ./  #ascp用法見小編關於該工具的部落格
ascp -i ~/asperaweb_id_dsa.openssh  -QTr -l200m  anonftp@ftp.ncbi.nlm.nih.gov:genomes/all/GCF/002/114/115/GCF_002114115.1_ASM211411v1/GCF_002114115.1_ASM211411v1_genomic.gff.gz ./

基因組和gff註釋檔案下載下來後，解壓並命名為sequences.fa和genes.gff，這裡命名錯誤後續會報錯

cd ../../
vim snpEff.config,修改配置檔案，找到Databases & Genomes，修改如下，注意命名需要嚴格參照小編的來，apple是上邊建立在data資料庫裡的檔名，字尾必須有.genome

# Databases & Genomes
#Malus_domestica
apple.genome    : Malus_domestica
apple.reference : https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/002/114/115/GCF_002114115.1_ASM211411v1/GCF_002114115.1_ASM211411v1_genomic.fna.gz \     # Genome sequence
                 https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/002/114/115/GCF_002114115.1_ASM211411v1/GCF_002114115.1_ASM211411v1_genomic.gff.gz \     # gff3  

 

snpEff自由資料庫建立

這裡需要較長時間，最好是在後臺nohup執行,尤其是比較大的基因組

java -jar snpEff.jar build -gff3 -v apple 

提取4DTV位點

java -jar snpEff.jar  ann apple apple.vcf.gz >apple.snpEff.vcf
python3 calc_4dTv_in_eff_vcf.py snpEff.vcf 4dTv.snpEff.vcf genome.sequences.fa

上邊的apple apple.vcf.gz為最原始的輸入vcf檔案，4dTv.snpEff.vcf既為最終需要的4DTV輸出vcf檔案，這裡最核心的為 calc_4dTv_in_eff_vcf.py 指令碼，該指令碼引自 從SnpEff註釋得到的VCF中過濾4DTV位點
，指令碼程式碼如下：

#!/usr/bin/env python3

from sys import argv
from pysam import VariantFile
from pysam import FastaFile

file_in = argv[1]
file_out = argv[2]
fafile = argv[3]

codon = set(["TC", "CT", "CC", "CG", "AC", "GT", "GC", "GG"])
rev_dict = dict(A='T',T='A', C='G', G='C')

bcf_in = VariantFile(file_in)
bcf_out = VariantFile(file_out, "w", header = bcf_in.header)
fa_in = FastaFile(fafile)

for rec in bcf_in.fetch():
    # no annotation available
    if len(rec.info.items()) == 0:
        continue
    ann = rec.info['ANN']
    info = rec.info['ANN'][0].split('|')
    # only use synonymouse variants
    if info[1] != "synonymous_variant":
        continue
    # only the 3rd position can be 4dTv
    if int(info[9][2:-3]) % 3 != 0:
        continue

    # determine the strand by the REF column and mutation
    # if the ref is not same as the mutation site
    if rec.ref == info[9][-3]:
        pre = fa_in.fetch(rec.chrom, rec.pos-3, rec.pos-1)
        pre = pre.upper()
    else:
        tmp = fa_in.fetch(rec.chrom, rec.pos, rec.pos+2)
        tmp = tmp.upper()
        pre = rev_dict[tmp[1]] + rev_dict[tmp[0]]
    if pre not in codon:
        continue
    bcf_out.write(rec)

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