一、原始數(shù)據(jù)比對到參考基因組
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bwa ：Version: 0.7.17-r1188
bwa mem -t 10 -R @RG\tID:pc-y2006018-2\tSM:pc-y2006018-2\tLB:Illumina /data3/ref_genome/pco/shajiepu.fasta /data3/Pugionium_cornutum_population/pc-y2006018-2/pc-y2006018-2_1.fq.gz /data3/Pugionium_cornutum_population/pc-y2006018-2/pc-y2006018-2_2.fq.gz
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picard ：v3.11 sortsam
java -Xmx6G -jar /home/jpf/biosoft/picard/picard.jar SortSam I=results/01bwa_mem/pc-y2006018-2.01.bam O=results/02bam_sort/pc-y2006018-2.02.bam SORT_ORDER=coordinate
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picard ：v3.11 MarkDuplicates
java -Xmx40G -jar /home/jpf/biosoft/picard/picard.jar MarkDuplicates I=results/02bam_sort/pc-y2006018-2.02.bam O=results/03bam_picard_markdu/pc-y2006018-2.03.bam M=results/03bam_picard_markdu/pc-y2006018-2.03.bam.metrics REMOVE_DUPLICATES=true OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500
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picard ：v3.11 sortsam
java -Xmx10G -jar /home/jpf/biosoft/picard/picard.jar SortSam SORT_ORDER=coordinate I=results/03bam_picard_markdu/pc-y2006018-2.03.bam O=results/04create_MD5_index/pc-y2006018-2.04.bam COMPRESSION_LEVEL=2 CREATE_MD5_FILE=true CREATE_INDEX=true
二、使用Delly、Manta和smoove進(jìn)行call sv
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Delly ：v1.2.6
/home/jpf/biosoft/Delly/delly_v1.2.6_linux_x86_64bit call -o /data3/call_sv/01Delly_call/pc-y2006018-2.delly.bcf -g /data3/ref_genome/pco/shajiepu.fasta /data3/call_sv/pco_snakemake/results/04create_MD5_index/pc-y2006018-2.04.bam &> /data3/call_sv/01Delly_call/pc-y2006018-2.delly.log
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Manta ：v1.6.0
Manta分為兩步
第一步構(gòu)建初始文件：
/home/jpf/miniconda3/envs/manta_env/bin/configManta.py --bam /data3/call_sv/pco_snakemake/results/04create_MD5_index/pc-y2006018-2.04.bam --referenceFasta /data3/ref_genome/pco/shajiepu.fasta --runDir /data3/call_sv/02Manta_call/pc-y2006018-2 &> /data3/call_sv/02Manta_call/pc-y2006018-2.manta.config.log
第二步運行：
/home/jpf/miniconda3/envs/manta_env/bin/python /data3/call_sv/02Manta_call/pc-y2006018-2/runWorkflow.py &> /data3/call_sv/02Manta_call/pc-y2006018-2/runWorkflow.py.log
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smoove ： v0.2.8
/home/jpf/biosoft/smoove/smoove call --outdir /data3/call_sv/03smoove_call/pc-y2006018-2 --name pc-y2006018-2 --fasta /data3/ref_genome/pco/shajiepu.fasta --genotype /data3/call_sv/pco_snakemake/results/04create_MD5_index/pc-y2006018-2.04.bam &> /data3/call_sv/03smoove_call/pc-y2006018-2.smoove.log
三、使用survivor合并三個軟件的結(jié)果
svrvivor ：v1.0.6
先將每個個體三個軟件結(jié)果合并
/home/jpf/biosoft/SURVIVOR-1.0.6/Debug/SURVIVOR merge /data3/call_sv/04survivor_merge/merge_indiv/pc-y2006018-2.threepath.txt 1 2 1 1 1 50 /data3/call_sv/04survivor_merge/merge_indiv/pc-y2006018-2.three_merge.vcf
再將所有個體合并，最終得到all.indiv.vcf
/home/jpf/biosoft/SURVIVOR-1.0.6/Debug/SURVIVOR merge /data3/call_sv/04survivor_merge/all.indiv.vcf.txt 1 2 1 1 1 50 /data3/call_sv/04survivor_merge/all.indiv.vcf
四、fst計算
vcftools ：v0.1.16
vcftools --vcf /data3/call_sv/04survivor_merge/all.indiv.vcf --weir-fst-pop /data3/call_sv/05fst/1-population.txt --weir-fst-pop /data3/call_sv/05fst/2-population.txt --out /data3/call_sv/05fst/p1_p2_window --fst-window-size 10000 --fst-window-step 2500
#窗口大小為1kb，步長為2.5kb
后續(xù)提取前fst5%區(qū)域的基因進(jìn)行富集分析
