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1.reseq [2017/12/15 15:36] – removed hyjeong1.reseq [2021/03/17 13:09] (current) – external edit 127.0.0.1
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 +  ######################### 
 +  #   2017 C&K Workshop   # 
 +  #   Resequencing Code   # 
 +  #        2017. 11       # 
 +  ######################### 
 +   
 +  ########Resequencing ########### 
 +   
 +  cd /home/workshop/1.WGS_practice/Genome_analysis 
 +   
 +  ### FASTQC 
 +   
 +  fastqc --nogroup paired_end_sep1.fq paired_end_sep2.fq 
 +   
 +  ### TRIMMOMATIC 
 +   
 +  java -jar /home/Program/Trimmomatic-0.33/trimmomatic-0.33.jar \ 
 +  PE paired_end_sep1.fq paired_end_sep2.fq paired_end_1P.fastq.gz paired_end_1U.fastq.gz \ 
 +  paired_end_2P.fastq.gz paired_end_2U.fastq.gz \ 
 +  ILLUMINACLIP:/home/Program/Trimmomatic-0.33/adapters/total-TruSeq-PE.fa:2:30:10 \ 
 +  TRAILING:20 MINLEN:75 
 +   
 +   
 +  ### REFERENCE INDEXING 
 +   
 +  /home/Program/bowtie2-2.2.5/bowtie2-build Reference.fa Reference 
 +   
 +  ### READ MAPPING 
 +   
 +  /home/Program/bowtie2-2.2.5/bowtie2 \ 
 +  -x Reference \ 
 +  -1 paired_end_1P.fastq.gz -2 paired_end_2P.fastq.gz \ 
 +  -S paired_end.sam \ 
 +  2> /home/workshop/1.WGS_practice/Genome_analysis/paired_end.bowtie2stat 
 +   
 +  ### ADD_RG  
 +   
 +  java -jar /home/Program/picard-tools-1.138/picard.jar AddOrReplaceReadGroups \ 
 +  INPUT=paired_end.sam \ 
 +  OUTPUT=paired_end_RG.bam \ 
 +  SORT_ORDER=coordinate RGID=paired_end RGLB=paired_end RGPL=illumina RGPU=non RGSM=paired_end \ 
 +  VALIDATION_STRINGENCY=LENIENT 
 +   
 +  ### REMOVE_DU 
 +   
 +  java -jar /home/Program/picard-tools-1.138/picard.jar MarkDuplicates \ 
 +  INPUT=paired_end_RG.bam \ 
 +  OUTPUT=paired_end_RG_DU.bam \ 
 +  METRICS_FILE=paired_end_RG_DU.metrics REMOVE_DUPLICATES=true ASSUME_SORTED=true \ 
 +  VALIDATION_STRINGENCY=LENIENT  
 +   
 +  ### FIXMATE 
 +   
 +  java -jar /home/Program/picard-tools-1.138/picard.jar FixMateInformation \ 
 +  INPUT=paired_end_RG_DU.bam \ 
 +  OUTPUT=paired_end_RG_DU_FIX.bam \ 
 +  SORT_ORDER=coordinate \ 
 +  VALIDATION_STRINGENCY=LENIENT 
 +   
 +  ### INDEXING 
 +   
 +  /home/Program/samtools-1.2/samtools faidx Reference.fa 
 +   
 +  /home/Program/samtools-1.2/samtools index paired_end_RG_DU_FIX.bam 
 +   
 +  ### REALIGN 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T RealignerTargetCreator \ 
 +  -R Reference.fa \ 
 +  -I paired_end_RG_DU_FIX.bam \ 
 +  -o paired_end_RG_DU_FIX_Realign.intervals  
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T IndelRealigner \ 
 +  -R Reference.fa \ 
 +  -I paired_end_RG_DU_FIX.bam \ 
 +  -o paired_end_RG_DU_FIX_Realign.bam \ 
 +  -targetIntervals paired_end_RG_DU_FIX_Realign.intervals 
 +   
 +  ### BQ_RECAL 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T BaseRecalibrator \ 
 +  -R Reference.fa \ 
 +  -I paired_end_RG_DU_FIX_Realign.bam \ 
 +  -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov ContextCovariate \ 
 +  -knownSites Reference.vcf \ 
 +  -o paired_end_RECAL.grp 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T PrintReads \ 
 +  -R Reference.fa \ 
 +  -I paired_end_RG_DU_FIX_Realign.bam \ 
 +  -BQSR paired_end_RECAL.grp \ 
 +  -o paired_end_RECAL.bam 
 +   
 +  ### INDEXING 
 +   
 +  /home/Program/samtools-1.2/samtools index paired_end_RECAL.bam 
 +   
 +  ### IGV 
 +  java -Xmx1500m -jar /home/Program/IGV_2.3.80/igv.jar 
 +   
 +   
 +   
 +  ############ VARIANT CALLING ######### 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar -T UnifiedGenotyper \ 
 +  -R Reference.fa \ 
 +  -I paired_end_RECAL.bam \ 
 +  -o sample01.raw.vcf \ 
 +  -glm BOTH 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar \ 
 +  -R Reference.fa \ 
 +  -T SelectVariants \ 
 +  --variant sample01.raw.vcf \ 
 +  -o snp_sample01.raw.vcf \ 
 +  -selectType SNP 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar \ 
 +  -R Reference.fa \ 
 +  -T SelectVariants \ 
 +  --variant sample01.raw.vcf \ 
 +  -o indel_sample01.raw.vcf \ 
 +  -selectType INDEL 
 +   
 +  ### VARIANT FILTERING 
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar \ 
 +  -R Reference.fa \ 
 +  -T VariantFiltration \ 
 +  --variant indel_sample01.raw.vcf \ 
 +  -o filtered_indel_sample01.vcf \ 
 +  --filterExpression "MQ0 >= 4 && ((MQ0 / (1.0*DP)) > 0.1)" --filterName "HARD_TO_VALIDATE" \ 
 +  --filterExpression "QUAL < 30" --filterName "QualFilter" \ 
 +  --filterExpression "QD < 5.0" --filterName "QD5" \ 
 +  --filterExpression "FS > 200.0" --filterName "FS200"  
 +   
 +  java -jar /home/Program/GenomeAnalysisTK-3.4-46/GenomeAnalysisTK.jar \ 
 +  -R Reference.fa \ 
 +  -T VariantFiltration \ 
 +  --variant snp_sample01.raw.vcf \ 
 +  -o filtered_snp_sample01.vcf \ 
 +  --clusterSize 3 --clusterWindowSize 10 \ 
 +  --mask filtered_indel_sample01.vcf --maskName "InDel" \ 
 +  --filterExpression "MQ0 >= 4 && ((MQ0 / (1.0*DP)) > 0.1)" --filterName "HARD_TO_VALIDATE" \ 
 +  --filterExpression "QUAL < 30" --filterName "QualFilter" \ 
 +  --filterExpression "FS > 200.0 " --filterName "FS200" 
 +   
 +   
 +  ### VARIANT ANNOTATION 
 +  #java -jar /home/Program/snpEff/snpEff.jar databases | more 
 +  #java -jar /home/Program/snpEff/snpEff.jar -download GRCh38.82 
 +   
 +  java -jar /home/Program/snpEff/snpEff.jar Human3 filtered_snp_sample01.vcf -s filtered_snp_sample01.ann.html >filtered_snp_sample01.ann.vcf 
 +  java -jar /home/Program/snpEff/snpEff.jar Human3 filtered_indel_sample01.vcf -s filtered_indel_sample01.ann.html >filtered_indel_sample01.ann.vcf 
 +   
 +   
 +  ### IGV 
 +  java -Xmx1500m -jar /home/Program/IGV_2.3.80/igv.jar 
 +   
 +  ### END ### 
 +  
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