workflow singlecancer_singlesample_umi{
	String project = "肺癌17基因"
	String bed = "/dataseq/jmdna/database/bed/lung17gene.hg19.liftover.bed"
	String tumor
	String inputDir
	String outputDir
	String need_fusion_test = "yes"
	String need_cnv_test = "yes"
	String need_chemotherapy = "no"
	String ref = "/dataseq/jmdna/database/genome/hg19/hg19.fa"
	String codes_dir = "/dataseq/jmdna/codes/singlecancer_singlesample"
	String accessBed = "/dataseq/jmdna/software/cnvkit-0.9.7/data/access-5k-mappable.hg19.bed"
	String annotateGene = "/dataseq/jmdna/software/cnvkit-0.9.7/data/refFlat.txt"
	String gc_wiggle = "/dataseq/jmdna/codes/pancancer_controlsample/hg19.gc200Base.txt.gz"
	#创建目录
	call create_dir{
		input:
            outputDir=outputDir,
			need_fusion_test=need_fusion_test,
			need_cnv_test=need_cnv_test,
			need_chemotherapy=need_chemotherapy
    }
	call qc{
        input:
            tumor=tumor,
            inputDir=inputDir,
            outputDir=outputDir
    }
    call alignment_bwa{
        input:
            tumor=tumor,
            outputDir=outputDir,
            ref=ref,
            read1=qc.outputFile[0],
            read2=qc.outputFile[1]
    }
    call consensusreads{
        input:
            tumor=tumor,
            outputDir=outputDir,
            ref=ref,
            bam=alignment_bwa.bam
    }		
	call generater_mpileup{
		input:
			tumor=tumor,
			ref=ref,
			bed=bed,
			outputDir=outputDir,
			rmdupBam=consensusreads.outputFile[1]    
	}
	call qc_2{
		input:
			ref=ref,
			bed=bed,
			tumor=tumor,
			outputDir=outputDir,
			codes_dir=codes_dir,
			rmdupBam=consensusreads.outputFile[1]
	}
	call mutation_calling{
        	input:
			codes_dir=codes_dir,
			tumor=tumor,
			outputDir=outputDir,	
			pileup=generater_mpileup.pileup
    }
	
    call annovar{
		input:
			tumor=tumor,
			outputDir=outputDir,
			ref=ref,
			vcf=mutation_calling.vcf,
			rmdupBam=consensusreads.outputFile[1]
    }
	
	if (need_fusion_test=="yes"){
		call fusion{
			input:
				ref=ref,
				codes_dir=codes_dir,
				tumor=tumor,
				outputDir=outputDir,
				rmdupBam=consensusreads.outputFile[1],
				project=project
		}
	}
	
		
#	call chemoTherapy{
#		input:		
#			codes_dir=codes_dir,
#			normal=normal,
#			outputDir=outputDir,
#			ref=ref,
#			project=project,
#			rmdupBam=consensusreads.outputFile[1]
#    }


	if (need_cnv_test=="yes"){
		call cnvkit{
			input:
				tumor=tumor,
				ref=ref,
				bed=bed,
				outputDir=outputDir,
				rmdupBam=consensusreads.outputFile[1],
				accessBed=accessBed,
				codes_dir=codes_dir,
				annotateGene=annotateGene,
				project=project
		}
	}
	
	call dealwithsnvindel{
		input:
			codes_dir=codes_dir,
			project=project,
			outputDir=outputDir,
			tumor=tumor,
			anno=annovar.anno
	}

	call auto_report{
		input:
			codes_dir=codes_dir,
			outputDir=outputDir,
			tumor=tumor,
			snv_result=dealwithsnvindel.snv,
			cnv_result=cnvkit.cnv,
			fusion_result=fusion.fusion
	}
	
	call hotspot{
		input:
        	tumor=tumor,
	    	outputDir=outputDir,
	    	ref=ref,
	    	rmdupBam=consensusreads.outputFile[1],
	    	codes_dir=codes_dir
	}
}
#create project directory
task create_dir{
    String outputDir
	String need_fusion_test
	String need_cnv_test
	String need_chemotherapy
    command <<<
        #创建目录
        if [ ! -d ${outputDir} ];then
            mkdir ${outputDir}
        fi

        #创建qc目录
        if [ ! -d ${outputDir}/qc ];then
            mkdir ${outputDir}/qc
        fi

        #创建alignment目录
        if [ ! -d ${outputDir}/alignment ];then
            mkdir ${outputDir}/alignment
        fi

        #创建mutation目录
        if [ ! -d ${outputDir}/mutation ];then
            mkdir ${outputDir}/mutation
        fi

        #创建cnv目录
        if [ ${need_cnv_test} = "yes" ] && [ ! -d ${outputDir}/cnvkit ];then
           mkdir ${outputDir}/cnvkit
        fi

		#创建report目录
        if [ ! -d ${outputDir}/report ];then
            mkdir -p ${outputDir}/report
        fi
		
		#创建chemo目录
        if [ ${need_chemotherapy} = "yes" ] && [ ! -d ${outputDir}/chemo ];then
            mkdir ${outputDir}/chemo
        fi

        #创建fusion目录
        if [ ${need_fusion_test} = "yes" ] && [ ! -d ${outputDir}/fusion ];then
            mkdir ${outputDir}/fusion
        fi
    >>>
	output{
          String newdir = "${outputDir}/report/qc"
      }
}

#generator raw fastq to clean fastq
task qc{
    String tumor
    String inputDir
    String outputDir

    command <<<
        echo processing raw reads with fastp
        fastp -i ${inputDir}/*_${tumor}_*1.fq.gz -o ${outputDir}/qc/${tumor}_clean_R1.fq.gz \
        -I ${inputDir}/*_${tumor}_*2.fq.gz -O ${outputDir}/qc/${tumor}_clean_R2.fq.gz \
        -w 10 \
	--disable_trim_poly_g \
       	--disable_quality_filtering \
        --adapter_sequence AGATCGGAAGAGCACACGTCTGAACTCCAGTCA \
        --adapter_sequence_r2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT \
        -j ${outputDir}/qc/${tumor}.json \
        -h ${outputDir}/qc/${tumor}.html --report_title ${tumor}
	
    >>>

    output{
        Array[String] outputFile = [
        "${outputDir}/qc/${tumor}_clean_R1.fq.gz",
        "${outputDir}/qc/${tumor}_clean_R2.fq.gz",
        "${outputDir}/qc/${tumor}.json",
        "${outputDir}/qc/${tumor}.html"
        ]
    }
}

#alignment clean fastq to reference
task alignment_bwa{
      String tumor
      String ref
      String outputDir
      String read1
      String read2
      command<<<
#fastqtosam
    java -Xmx8G -jar $PICARD FastqToSam \
    FASTQ=${read1} \
    FASTQ2=${read2} \
    OUTPUT=${outputDir}/alignment/unmapped.bam \
    SAMPLE_name=${tumor}


#ExtractUmisFromBam
java -jar /dataseq/jmdna/software/fgbio/target/scala-2.13/fgbio-1.4.0-468a843-SNAPSHOT.jar ExtractUmisFromBam \
    --input=${outputDir}/alignment/unmapped.bam \
    --output=${outputDir}/alignment/unmapped.withUMI.bam \
    --read-structure=1S3M3S+T 1S3M3S+T \
    --single-tag=RX \
    --molecular-index-tags=ZA ZB

#align reads
java -Xmx4G -jar $PICARD SamToFastq \
        I=${outputDir}/alignment/unmapped.withUMI.bam \
        F=/dev/stdout \
        INTERLEAVE=true \
        | bwa mem -p -t 10 ${ref} /dev/stdin \
        | java -Xmx4G -jar $PICARD MergeBamAlignment \
        UNMAPPED=${outputDir}/alignment/unmapped.withUMI.bam \
        ALIGNED=/dev/stdin \
        O=${outputDir}/alignment/${tumor}.mapped.bam \
        R=${ref} \
        SO=coordinate \
        ALIGNER_PROPER_PAIR_FLAGS=True \
        MAX_GAPS=-1 \
        ORIENTATIONS=FR \
        VALIDATION_STRINGENCY=SILENT \
        CREATE_INDEX=True

rm ${outputDir}/alignment/unmapped.bam ${outputDir}/alignment/unmapped.withUMI.bam 
	>>>

      output{
          String bam = "${outputDir}/alignment/${tumor}.mapped.bam"
      }
}

#group by umi and call consensusreads 
task consensusreads{
    String tumor
    String ref
    String outputDir
    String bam

    command<<<
#GroupReadsByUmi
java -Xmx4g -jar /dataseq/jmdna/software/fgbio/target/scala-2.13/fgbio-1.4.0-468a843-SNAPSHOT.jar GroupReadsByUmi \
        --input=${bam} \
        --output=${outputDir}/alignment/${tumor}.grouped.bam \
        --strategy=paired \
        --edits=1 \
        --min-map-q=20 \
		--allow-inter-contig=true

#generate consensus reads
java -Xmx4g -jar /dataseq/jmdna/software/fgbio/target/scala-2.13/fgbio-1.4.0-468a843-SNAPSHOT.jar CallDuplexConsensusReads \
        --input=${outputDir}/alignment/${tumor}.grouped.bam \
        --output=${outputDir}/alignment/${tumor}.consensus.unmapped.bam \
        --error-rate-pre-umi=45 \
        --error-rate-post-umi=30 \
        --min-input-base-quality=20 \
        --min-reads=1 0 0 \
	--threads 20

#remap consensusreads
java -Xmx4G -jar $PICARD SamToFastq \
        I=${outputDir}/alignment/${tumor}.consensus.unmapped.bam \
        F=/dev/stdout \
        INTERLEAVE=true \
        | bwa mem -p -t 10 ${ref} /dev/stdin \
        | java -Xmx4G -jar $PICARD MergeBamAlignment \
        UNMAPPED=${outputDir}/alignment/${tumor}.consensus.unmapped.bam \
        ALIGNED=/dev/stdin \
        O=${outputDir}/alignment/${tumor}.consensus.mapped.bam \
        R=${ref} \
        SO=coordinate \
        ALIGNER_PROPER_PAIR_FLAGS=True \
        MAX_GAPS=-1 \
        ORIENTATIONS=FR \
        VALIDATION_STRINGENCY=SILENT \
        CREATE_INDEX=True    

rm ${outputDir}/alignment/${tumor}.consensus.unmapped.bam
    >>>

    output{
	Array[String] outputFile = [              
	"${outputDir}/alignment/${tumor}.grouped.bam",
	"${outputDir}/alignment/${tumor}.consensus.mapped.bam"
	]
    }	
}

# generater mpileup file
task generater_mpileup{
    String tumor
    String ref
    String bed
    String outputDir
	String rmdupBam

    command<<<

        samtools mpileup -Bq 20 -Q 20  -f ${ref} -l ${bed} \
        ${rmdupBam} -o ${outputDir}/alignment/${tumor}.pileup

    >>>

    output{
        String pileup = "${outputDir}/alignment/${tumor}.pileup"
    }
}

task mutation_calling{
	String codes_dir
	String tumor
	String pileup
	String outputDir
	
	command<<<
	
		java -jar  $VARSCAN mpileup2cns ${pileup} \
        --min-var-freq 0.002 --min-avg-qual 20 --output-vcf 1 --variants  --p-value 0.99 --min-reads2 3 --strand-filter 1 >${outputDir}/mutation/${tumor}.snp.indel.vcf

	>>>
	
	output{
        String vcf = "${outputDir}/mutation/${tumor}.snp.indel.vcf"        
    }
}

# hotspot
task hotspot{
	String tumor
	String outputDir
	String ref
	String rmdupBam
	String codes_dir
	command<<<
	
	mkdir -p ${outputDir}/mutation/hotspot
	samtools mpileup -Bq 20 -Q 20  -f ${ref} -l ${codes_dir}/hotspot.bed ${rmdupBam} -o ${outputDir}/mutation/hotspot/${tumor}.hotspot.pileup
	java -jar $VARSCAN mpileup2cns ${outputDir}/mutation/hotspot/${tumor}.hotspot.pileup --min-var-freq 0.001 --min-avg-qual 20 --output-vcf 1 --variants --p-value 0.99 --min-reads2 2 --strand-filter 0 >${outputDir}/mutation/hotspot/${tumor}.hotspot.L.snp.indel.vcf
	java -jar $VARSCAN mpileup2cns ${outputDir}/mutation/hotspot/${tumor}.hotspot.pileup --min-var-freq 0.002 --min-avg-qual 20 --output-vcf 1 --variants --p-value 0.99 --min-reads2 3 --strand-filter 1 >${outputDir}/mutation/hotspot/${tumor}.hotspot.H.snp.indel.vcf
	perl ${codes_dir}/hotspot.hvl.pl ${outputDir} ${tumor} 

	if [ -e "${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.vcf" ]; then
		table_annovar.pl \
		${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.vcf \
		/dataseq/jmdna/software/annovar/humandb/ \
		-buildver hg19 -nastring . -vcfinput -remove -otherinfo \
		-protocol refGene,avsnp150,cosmic91,clinvar_20220320,1000g2015aug_all,1000g2015aug_eas,esp6500siv2_all,exac03nontcga,gnomad_genome,dbnsfp35c \
		-operation g,f,f,f,f,f,f,f,f,f \
		--outfile ${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.anno


#		java -jar /dataseq/jmdna/software/GenomeAnalysisTK.3.7.jar -T VariantAnnotator \
#		-R ${ref} \
#		-I ${outputDir}/alignment/${tumor}.rmdup.bam \
#		-V ${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.vcf \
#		-o ${outputDir}/mutation/hotspot/${tumor}.hotspot.TandemRepeatAnnotator.vcf \
#		--annotation TandemRepeatAnnotator

#		grep -v "^##" ${outputDir}/mutation/hotspot/${tumor}.hotspot.TandemRepeatAnnotator.vcf | cut -f8| paste ${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.anno.hg19_multianno.txt  -  >${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.Somatic.annoall.hg19_multianno.txt		
	cp ${outputDir}/mutation/hotspot/${tumor}.hotspot.snp.indel.anno.hg19_multianno.txt ${outputDir}/report/${tumor}.hotspot.snp.indel.anno.hg19_multianno.txt

	fi
	
	>>>
	output{
				String  hotspot = "${outputDir}/mutation/hotspot/${tumor}.hotspot.H.snp.indel.vcf"
	}
}

task annovar{
	String tumor
	String outputDir
	String ref
    String vcf
	String rmdupBam
    command<<<

        table_annovar.pl \
        ${outputDir}/mutation/${tumor}.snp.indel.vcf \
        /dataseq/jmdna/software/annovar/humandb/ \
        -buildver hg19 -nastring . -vcfinput -remove -otherinfo \
        -protocol refGene,avsnp150,cosmic91,clinvar_20220320,1000g2015aug_all,1000g2015aug_eas,esp6500siv2_all,exac03nontcga,gnomad_genome,dbnsfp35c \
        -argument '-splicing_threshold 2 -hgvs',,,,,,,,, \
        --intronhgvs 50 \
        -operation g,f,f,f,f,f,f,f,f,f \
	--outfile ${outputDir}/mutation/${tumor}.snp.indel.annoall


#		java -jar /dataseq/jmdna/software/GenomeAnalysisTK.3.7.jar -T VariantAnnotator \
#		-R ${ref} \
#		-I ${rmdupBam} \
#		-V ${vcf} \
#		-o ${outputDir}/mutation/${tumor}.TandemRepeatAnnotator.vcf \
#		--annotation TandemRepeatAnnotator

#	grep -v "^##" ${outputDir}/mutation/${tumor}.TandemRepeatAnnotator.vcf | cut -f8| paste ${outputDir}/mutation/${tumor}.snp.indel.hg19_multianno.txt  -  >${outputDir}/mutation/${tumor}.snp.indel.annoall.hg19_multianno.txt

    >>>
	output{
        String anno = "${outputDir}/mutation/${tumor}.snp.indel.annoall.hg19_multianno.txt"
    }
}

task qc_2{
	String ref
	String bed
	String tumor
	String outputDir
	String codes_dir
	#for task chain
	String rmdupBam
	
	command <<<
		mkdir ${outputDir}/qc/group_bamdst ${outputDir}/qc/consensus_bamdst
        bamdst -p ${bed} -o ${outputDir}/qc/group_bamdst ${outputDir}/alignment/${tumor}.mapped.bam
		bamdst -p ${bed} -o ${outputDir}/qc/consensus_bamdst ${outputDir}/alignment/${tumor}.consensus.mapped.bam
	
		java -Xmx4g -jar /dataseq/jmdna/software/fgbio/target/scala-2.13/fgbio-1.4.0-468a843-SNAPSHOT.jar CollectDuplexSeqMetrics \
                -i ${outputDir}/alignment/${tumor}.grouped.bam \
                -o ${outputDir}/qc/${tumor}_umi_qc \
                -d ${tumor} \
                -u true
		
		samtools flagstat -@ 10 ${outputDir}/alignment/${tumor}.mapped.bam >${outputDir}/qc/${tumor}.flagstat
		samtools stats --reference ${ref} -t ${bed} -@ 10 ${outputDir}/alignment/${tumor}.consensus.mapped.bam > ${outputDir}/alignment/${tumor}.stat
		Rscript ${codes_dir}/qc_umi.r ${outputDir} ${tumor}
		Rscript ${codes_dir}/InsertAndDepthStat.R ${outputDir}/qc/${tumor}_InsertAndDepthStat ${outputDir}/qc/group_bamdst/insertsize.plot ${outputDir}/qc/group_bamdst/depth_distribution.plot
#		mv ${outputDir}/qc/${tumor}_qcstat.txt ${outputDir}/report/qc/${tumor}_qcstat.txt
	>>>		
}

task fusion{
	String ref
	String codes_dir
	String tumor
	String outputDir
	#for task chain
	String rmdupBam
	String project

	command<<<
	
		# Extract the discordant paired-end alignments.
		samtools view -b -F 1294 ${rmdupBam} > ${outputDir}/fusion/${tumor}.discordants.bam

		# Extract the split-read alignments
		samtools view -h ${rmdupBam} \
		| /dataseq/jmdna/software/lumpy-sv/scripts/extractSplitReads_BwaMem -i stdin \
		| samtools view -Sb - \
		> ${outputDir}/fusion/${tumor}.splitters.bam

		lumpyexpress \
		-B ${rmdupBam} \
		-S ${outputDir}/fusion/${tumor}.splitters.bam \
		-D ${outputDir}/fusion/${tumor}.discordants.bam \
		-o ${outputDir}/fusion/${tumor}.fusion.vcf

		perl ${codes_dir}/fusion.filter.pl ${outputDir}/fusion/${tumor}.fusion.vcf ${outputDir}/fusion/${tumor}.fusion.filter.vcf

		svtyper \
		-B ${rmdupBam} \
		-i ${outputDir}/fusion/${tumor}.fusion.filter.vcf \
		-T ${ref} \
		-o ${outputDir}/fusion/${tumor}.fusion.gt.vcf

		table_annovar.pl \
		${outputDir}/fusion/${tumor}.fusion.gt.vcf \
		/dataseq/jmdna/software/annovar/humandb/ \
		-buildver hg19 -nastring . -vcfinput -remove -otherinfo \
		-protocol refGene \
		-operation g \
		--outfile ${outputDir}/fusion/${tumor}.fusion
	
		perl ${codes_dir}/fusion.reanno.pl ${outputDir}/qc/consensus_bamdst/depth.tsv.gz ${outputDir} ${tumor}
		perl ${codes_dir}/fusion_targetTherapy.pl ${codes_dir} ${tumor} ${outputDir} ${project}

	>>>
	
	output{
        String fusion = "${outputDir}/fusion/${tumor}.fusion.pos.txt"
    }
}

task chemoTherapy{
        String codes_dir
        String normal
        String outputDir
        String ref
		String project
        #for task chain
		Array[String] rmdupBam
        command <<<
                perl ${codes_dir}/chemo/singlecancer_chemo_2.pl ${codes_dir} ${outputDir} ${normal} ${ref} ${project}
        >>>
}

task cnvkit{
    String ref
    String bed
    String tumor
    String outputDir
    String accessBed
    String annotateGene
	String codes_dir
	String project
    #for task chain
    String rmdupBam

    command <<<

	echo run cnvkit batch to processing cnv calling
        cnvkit.py batch \
	${rmdupBam} \
	-r ${codes_dir}/cnvkit-lung17gene-cfDNA/lung17gene_pool_tumor_reference.cnn \
        --drop-low-coverage  --scatter --diagram --output-dir ${outputDir}/cnvkit

	perl ${codes_dir}/cnv_targetTherapy_umi.pl ${codes_dir} ${tumor} ${outputDir} ${project}

    >>>

	output{
        String cnv = "${outputDir}/cnvkit/${tumor}.cnv.pos.txt"
    }
}

task dealwithsnvindel{
	String codes_dir
	String project
	String outputDir
	String tumor
	String anno

	command <<<
	
	perl ${codes_dir}/pick_mut_splice_promoter.pl ${codes_dir} ${tumor} ${outputDir} ${project}
	perl ${codes_dir}/targetTherapy.pl ${codes_dir} ${tumor} ${outputDir} ${project}
	
	>>>
    output{
        String snv = "${outputDir}/mutation/${tumor}.snvindel.pos.txt"
    }
}

task auto_report{
    String codes_dir
	String outputDir
	String tumor
	String snv_result
	String cnv_result
	String fusion_result
    command <<<
    python ${codes_dir}/sample_post.py -s ${tumor} -o ${outputDir}
    python ${codes_dir}/drug_dedup.py ${outputDir} ${tumor}	
    python ${codes_dir}/report_template/lung_17gene_umi_report.py ${outputDir} ${tumor}
    python ${codes_dir}/qc_check.py ${outputDir} ${tumor} c    
    python3 ${codes_dir}/wdl_check.py -o ${outputDir}

    cp ${outputDir}/fusion/${tumor}.fuison.vus.txt ${outputDir}/report/
    cp ${outputDir}/mutation/${tumor}.snvindel.vus.txt ${outputDir}/report/
    cp ${outputDir}/mutation/${tumor}.target.splicing.txt ${outputDir}/report/
    cp ${outputDir}/qc/${tumor}_qc.txt ${outputDir}/report/
    cp ${outputDir}/qc/qc_fail.txt ${outputDir}/report/
#    cp ${outputDir}/fusion/${tumor}.longindel.pos.txt ${outputDir}/report/
    cp ${outputDir}/mutation/${tumor}.target.splicing.txt ${outputDir}/report/	
	>>>
	output{
        String report = "${outputDir}/report/${tumor}_report.docx"
    }

}