From 188e7dd8a4fd06f461b75c6537c7e17d36c7eaa7 Mon Sep 17 00:00:00 2001
From: chaopower <chao.zhang@jmdna.com>
Date: Wed, 27 Sep 2023 10:47:03 +0800
Subject: [PATCH] =?UTF-8?q?=E8=B0=83=E6=95=B4call=5Fmutation?=
MIME-Version: 1.0
Content-Type: text/plain; charset=UTF-8
Content-Transfer-Encoding: 8bit

---
 README.md             |   6 +-
 pipeline.wdl          | 201 +++++----------------------------------
 wdl/alignment.wdl     | 122 ++++++++++++++++--------
 wdl/call_mutation.wdl | 215 ++++++++++++++++++++++++++++++++++++++++++
 wdl/qc.wdl            | 118 +++++++++++++++++------
 wdl/task.wdl          |  69 +++++---------
 6 files changed, 439 insertions(+), 292 deletions(-)
 create mode 100644 wdl/call_mutation.wdl

diff --git a/README.md b/README.md
index da61ac9..d16519b 100644
--- a/README.md
+++ b/README.md
@@ -2,8 +2,10 @@
 
 ## run script example 
 ```bash
-/home/zhangchao/soft/jdk-17.0.7+7/bin/java -Dconfig.file=/home/zhangchao/project/pipeline/wdl/cromwell.examples.conf \
--jar /home/zhangchao/soft/cromwell-85.jar run  \
+export PATH=/dataseq/product/workflow/software/bin:$PATH
+
+java17 -Dconfig.file=/home/zhangchao/project/pipeline/wdl/cromwell.examples.conf \
+-jar /dataseq/product/workflow/software/cromwell-85.jar run  \
 --inputs /home/zhangchao/project/pipeline/workflow/test/20230814.json \
 /home/zhangchao/project/pipeline/workflow/pipeline.wdl
 ```
diff --git a/pipeline.wdl b/pipeline.wdl
index 89343c7..eed900a 100644
--- a/pipeline.wdl
+++ b/pipeline.wdl
@@ -1,21 +1,23 @@
 import "./wdl/task.wdl" as mytask
 import "./wdl/qc.wdl"
 import "./wdl/alignment.wdl"
-import "./wdl/statistics.wdl"
+import "./wdl/call_mutation.wdl"
 
 workflow pipeline {
 
     String tumor
     String? normal
-    String inputDir
-    String outputDir
-    String cancer
-    String project="650gene"
-    String codesDir="/dataseq/jmdna/codes/pancancer_controlsample"
-    String ref = "/dataseq/jmdna/database/genome/hg19/hg19.fa"
-    String bed = "/dataseq/jmdna/database/bed/650.bed"
+    Boolean umi=false
 
-    String workdir="${outputDir}/${tumor}"
+    String input_dir
+    String output_dir
+
+    String bed
+
+    String codesDir="/home/zhangchao/project/pipeline/workflow/script"
+    String ref = "/dataseq/jmdna/database/genome/hg19/hg19.fa"
+
+    String workdir="${output_dir}"
 
     call mytask.create_dir as create_dir {
         input:
@@ -26,8 +28,9 @@ workflow pipeline {
         input:
             tumor=tumor,
             normal=normal,
-            inputDir=inputDir,
-            outputDir=workdir
+            umi=umi,
+            input_dir=input_dir,
+            output_dir=workdir
     }
 
     call alignment.alignment as alignment {
@@ -41,183 +44,23 @@ workflow pipeline {
             normal_r1=qc.normal_r1,
             normal_r2=qc.normal_r2,
 
+            umi=umi,
+
             ref=ref,
             bed=bed,
-            outputDir=workdir
+            output_dir=workdir
     }
 
-    call statistics.statistics as statistics {
+    call call_mutation.call_mutation as call_mutation {
         input:
             tumor=tumor,
-            tumor_rmdupBam=alignment.tumor_rmdupBam,
-
+            tumor_rmdup_bam=alignment.tumor_rmdup_bam,
             normal=normal,
-            normal_rmdupBam=alignment.normal_rmdupBam,
+            normal_rmdup_bam=alignment.normal_rmdup_bam,
 
+            umi=umi,
             ref=ref,
             bed=bed,
-            outputDir=workdir,
-            codesDir=codesDir
-    }
-
-    call mytask.conpair as conpair {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            tumor_rmdupBam=alignment.tumor_rmdupBam,
-            normal_rmdupBam=alignment.normal_rmdupBam,
-            outputDir=workdir,
-            ref=ref
-    }
-
-    call mytask.mutation_calling as mutation_calling {
-        input:
-            name=tumor,
-            tumor_pileup=alignment.tumor_pileup,
-            normal_pileup=alignment.normal_pileup,
-            outputDir=workdir
-    }
-
-    call mytask.annovar as annovar {
-        input:
-            name=tumor,
-            outputDir=workdir,
-            ref=ref,
-            somatic_hc_vcf=mutation_calling.somatic_hc_vcf,
-            germline_vcf=mutation_calling.germline_vcf,
-            loh_hc_vcf=mutation_calling.loh_hc_vcf,
-            rmdupBam=alignment.tumor_rmdupBam
-    }
-
-    call mytask.dealwithsnvindel as dealwithsnvindel {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            somatic_all_anno=annovar.somatic_all_anno,
-            germline_anno=annovar.germline_anno,
-            project=project,
-            outputDir=workdir,
-            cancer=cancer
-    }
-
-    call mytask.hereditary as hereditary {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            outputDir=workdir,
-            project=project,
-            germline_filtered = dealwithsnvindel.germline_filtered
-    }
-
-    call mytask.tmb as tmb {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            outputDir=workdir,
-            somatic_anno=annovar.somatic_anno
-    }
-
-    call mytask.fusion as fusion {
-        input:
-            name=tumor,
-            ref=ref,
-            codesDir=codesDir,
-            outputDir=workdir,
-            rmdupBam=alignment.tumor_rmdupBam,
-            cancer=cancer,
-            project=project,
-            tumor_bamdst_depth=statistics.tumor_bamdst_depth
-    }
-
-    call mytask.cnvkit as cnvkit {
-        input:
-            tumor=tumor,
-            normal=normal,
-
-            tumor_rmdupBam=alignment.tumor_rmdupBam,
-            normal_rmdupBam=alignment.normal_rmdupBam,
-            ref=ref,
-            bed=bed,
-            outputDir=workdir,
-            cancer=cancer,
-            codesDir=codesDir,
-            project=project,
-    }
-
-    call mytask.chemo as chemo {
-        input:
-            codesDir=codesDir,
-            outputDir=workdir,
-            normal=normal,
-            project=project,
-            rmdupBam=alignment.tumor_rmdupBam,
-    }
-
-    call mytask.msi as msi {
-        input:
-            bed=bed,
-            name=tumor,
-            outputDir=workdir,
-            tumor_rmdupBam = alignment.tumor_rmdupBam,
-            normal_rmdupBam =alignment.normal_rmdupBam
-    }
-
-    call mytask.hla as hla {
-        input:
-            inputDir=inputDir,
-            outputDir=workdir,
-            normal=normal,
-    }
-
-    call mytask.neoantigen as neoantigen {
-        input:
-            codesDir=codesDir,
-            outputDir=workdir,
-            name=tumor,
-            somatic_hc_vcf=mutation_calling.somatic_hc_vcf,
-            normal=normal,
-            hla=hla.hla
-    }
-
-    call mytask.mmr as mmr {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            outputDir=workdir,
-            germline_filtered = dealwithsnvindel.germline_filtered
-    }
-
-    call mytask.hrr as hrr {
-        input:
-            codesDir=codesDir,
-            name=tumor,
-            outputDir=workdir,
-            germline_filtered = dealwithsnvindel.germline_filtered
-    }
-
-    call mytask.hotspot as hotspot {
-        input:
-            name=tumor,
-            outputDir=workdir,
-            ref=ref,
-            rmdupBam=alignment.tumor_rmdupBam,
-            codesDir=codesDir,
-    }
-
-    call mytask.auto_report {
-        input:
-            tumor=tumor,
-            normal=normal,
-            outputDir=workdir,
-            codesDir=codesDir,
-            cancer=cancer,
-            cnv_cns=cnvkit.cns,
-            cnv_png=cnvkit.png,
-            fusion_pos=fusion.fusion,
-            snvindel_filtered=dealwithsnvindel.snvindel_filtered,
-            tmb=tmb.tmb,
-            mmr=mmr.mmr,
-            hrr=hrr.hrr,
-            hereditary_pre=hereditary.hereditary_pre
+            output_dir=workdir
     }
 }
diff --git a/wdl/alignment.wdl b/wdl/alignment.wdl
index 304a8ea..8868bed 100644
--- a/wdl/alignment.wdl
+++ b/wdl/alignment.wdl
@@ -4,48 +4,75 @@ task bwa {
     String name
     String read1
     String read2
-    String outputDir
+    String output_dir
     String ref
 
     command <<<
-        if [ ! -d ${outputDir}/alignment ];then
-        mkdir ${outputDir}/alignment
+        if [ ! -d ${output_dir}/alignment ];then
+        mkdir ${output_dir}/alignment
         fi
 
         bwa mem -R '@RG\tID:group_n\tLB:library_n\tPL:BGI\tPU:unit1\tSM:${name}' -M -t 10 ${ref} ${read1} ${read2} | \
-        samtools view -@ 10 -bh  -o - | samtools sort -@ 10  -o ${outputDir}/alignment/${name}.sorted.bam
-        samtools index ${outputDir}/alignment/${name}.sorted.bam
+        samtools view -@ 10 -bh  -o - | samtools sort -@ 10  -o ${output_dir}/alignment/${name}.sorted.bam
+        samtools index ${output_dir}/alignment/${name}.sorted.bam
 
     >>>
 
     output {
-        String sortedBam = "${outputDir}/alignment/${name}.sorted.bam"
+        String sorted_bam = "${output_dir}/alignment/${name}.sorted.bam"
     }
 }
 
 #remove PCR duplicates
 
-task markduplicates {
+task markduplicates_genecore {
     String name
     String ref
-    String sortedBam
-    String outputDir
+    String sorted_bam
+    String output_dir
 
     command <<<
-        if [ ! -d ${outputDir}/alignment ];then
-        mkdir ${outputDir}/alignment
+        if [ ! -d ${output_dir}/alignment ];then
+        mkdir ${output_dir}/alignment
         fi
+
+        gencore -i ${sorted_bam} \
+        -o ${output_dir}/alignment/${name}.rmdup.bam \
+        -r ${ref} \
+        -u UMI \
+        -j ${output_dir}/alignment/${name}_rmdup.json \
+        -h ${output_dir}/alignment/${name}_rmdup.html
+
+        samtools index ${output_dir}/alignment/${name}.rmdup.bam
+    >>>
+
+    output {
+        String rmdup_bam = "${output_dir}/alignment/${name}.rmdup.bam"
+    }
+}
+
+task markduplicates_picard {
+    String name
+    String ref
+    String sorted_bam
+    String output_dir
+
+    command <<<
+        if [ ! -d ${output_dir}/alignment ];then
+        mkdir ${output_dir}/alignment
+        fi
+
         java -XX:+UseParallelGC -XX:ParallelGCThreads=2 -Xmx12G  -jar $PICARD MarkDuplicates \
-        I=${sortedBam} \
-        O=${outputDir}/alignment/${name}.rmdup.bam  \
+        I=${sorted_bam} \
+        O=${output_dir}/alignment/${name}.rmdup.bam  \
         CREATE_INDEX=true \
-        M=${outputDir}/alignment/${name}.rmdup.metrics.txt  \
+        M=${output_dir}/alignment/${name}.rmdup.metrics.txt  \
         R=${ref}
 
     >>>
 
     output {
-        String rmdupBam = "${outputDir}/alignment/${name}.rmdup.bam"
+        String rmdup_bam = "${output_dir}/alignment/${name}.rmdup.bam"
     }
 }
 
@@ -56,16 +83,16 @@ task generater_mpileup {
     String rmdupBam
     String ref
     String bed
-    String outputDir
+    String output_dir
 
     command <<<
 
-        samtools mpileup -Bq 20 -Q 20  -f ${ref} -l ${bed} ${rmdupBam} -o ${outputDir}/alignment/${name}.pileup
+        samtools mpileup -Bq 20 -Q 20  -f ${ref} -l ${bed} ${rmdupBam} -o ${output_dir}/alignment/${name}.pileup
 
     >>>
 
     output {
-        String pileup = "${outputDir}/alignment/${name}.pileup"
+        String pileup = "${output_dir}/alignment/${name}.pileup"
     }
 }
 
@@ -79,9 +106,11 @@ workflow alignment {
     String? normal_r1
     String? normal_r2
 
+    Boolean umi
+
     String ref
     String bed
-    String outputDir
+    String output_dir
 
     scatter(name in [tumor, normal]) {
         if (defined(name)) {
@@ -89,36 +118,51 @@ workflow alignment {
                 input:
                     name=name,
                     ref=ref,
-                    outputDir=outputDir,
+                    output_dir=output_dir,
                     read1=if name==tumor then tumor_r1 else normal_r1,
                     read2=if name==tumor then tumor_r2 else normal_r2
 
             }
-            call markduplicates {
-                input:
-                    name=name,
-                    ref=ref,
-                    outputDir=outputDir,
-                    sortedBam=bwa.sortedBam
+            if (name==tumor) {
+                if (umi) {
+                    call markduplicates_genecore as tumor_markduplicates_genecore {
+                        input:
+                            name=name,
+                            ref=ref,
+                            output_dir=output_dir,
+                            sorted_bam=bwa.sorted_bam,
+                    }
+                }
+                if (!umi) {
+                    call markduplicates_picard as tumor_markduplicates_picard {
+                        input:
+                            name=name,
+                            ref=ref,
+                            output_dir=output_dir,
+                            sorted_bam=bwa.sorted_bam,
+                    }
+                }
             }
-            call generater_mpileup {
-                input:
-                    name=name,
-                    ref=ref,
-                    outputDir=outputDir,
-                    bed=bed,
-                    rmdupBam=markduplicates.rmdupBam
+
+            if (name==select_first([normal, 'None'])) {
+                call markduplicates_picard as normal_markduplicates_picard {
+                    input:
+                        name=name,
+                        ref=ref,
+                        output_dir=output_dir,
+                        sorted_bam=bwa.sorted_bam,
+                }
             }
 
         }
     }
 
     output {
-        String tumor_sortedBam = "${outputDir}/alignment/${tumor}.sorted.bam"
-        String tumor_rmdupBam = "${outputDir}/alignment/${tumor}.rmdup.bam"
-        String tumor_pileup = "${outputDir}/alignment/${tumor}.pileup"
-        String normal_sortedBam = "${outputDir}/alignment/${normal}.sorted.bam"
-        String normal_rmdupBam = "${outputDir}/alignment/${normal}.rmdup.bam"
-        String normal_pileup = "${outputDir}/alignment/${normal}.pileup"
+        String tumor_sorted_bam = "${output_dir}/alignment/${tumor}.sorted.bam"
+        String tumor_rmdup_bam = "${output_dir}/alignment/${tumor}.rmdup.bam"
+        String tumor_pileup = "${output_dir}/alignment/${tumor}.pileup"
+        String normal_sorted_bam = "${output_dir}/alignment/${normal}.sorted.bam"
+        String normal_rmdup_bam = "${output_dir}/alignment/${normal}.rmdup.bam"
+        String normal_pileup = "${output_dir}/alignment/${normal}.pileup"
     }
 }
\ No newline at end of file
diff --git a/wdl/call_mutation.wdl b/wdl/call_mutation.wdl
new file mode 100644
index 0000000..41efa3f
--- /dev/null
+++ b/wdl/call_mutation.wdl
@@ -0,0 +1,215 @@
+task mutation_calling_umi {
+    String name
+    String output_dir
+    String rmdup_bam
+    String ref
+    String bed
+    command <<<
+
+        if [ ! -d ${output_dir}/mutation ];then
+        mkdir ${output_dir}/mutation
+        fi
+
+
+        #1条call
+        # 这个情况是reads数目只有1，但是如果去掉了这个reads数导致数据量减少很多
+        # -r 3 是指有3条这样样的reads支撑
+        # -f 是指频率 以2条方式的call出来的变异频率可以比1条的方式更可信
+
+        java -jar /dataseq/jmdna/software/VarDict-1.8.3/lib/VarDict-1.8.3.jar \
+        -G ${ref} \
+        -f 0.001 \
+        -N ${name} \
+        -b ${rmdup_bam} \
+        -UN -Q 20 -m 3 -r 3 -th 10 -c 1 -S 2 -E 3 -g 4 ${bed} \
+        | /dataseq/jmdna/software/VarDict-1.8.3/bin/teststrandbias.R \
+        | /dataseq/jmdna/software/VarDict-1.8.3/bin/var2vcf_valid.pl \
+        -N ${name} -E -f 0.001 > ${output_dir}/mutation/${name}.1r.snp.indel.vcf
+
+        #提取>=2条矫正的序列
+        python3 /home/zhangchao/project/pipeline/control/script/fetch_bam.py ${output_dir}/alignment/${name}.rmdup.bam ${output_dir}/alignment/${name}.2r.rmdup.bam
+        samtools index ${output_dir}/alignment/${name}.2r.rmdup.bam
+
+        # 保证 1r call mut umi family 里面有2条reads
+        #2条矫正的call
+        java -jar /dataseq/jmdna/software/VarDict-1.8.3/lib/VarDict-1.8.3.jar -G ${ref} \
+        -f 0.0001 -N ${name}_2r -b ${output_dir}/alignment/${name}.2r.rmdup.bam \
+        -UN -Q 20 -m 3 -r 1 -th 10 -c 1 -S 2 -E 3 -g 4 ${bed} | /dataseq/jmdna/software/VarDict-1.8.3/bin/teststrandbias.R \
+        | /dataseq/jmdna/software/VarDict-1.8.3/bin/var2vcf_valid.pl -N ${name} -E -f 0.001 >${output_dir}/mutation/${name}.2r.snp.indel.vcf
+
+        #merge突变，以1条方式call的>0.01的突变+两条方式的对一条方式的低频区域（AF<0.01）进行矫正。
+        perl  /home/zhangchao/project/pipeline/control/script/1r_plus_2r.pl \
+        ${output_dir}/mutation/${name}.1r.snp.indel.vcf \
+        ${output_dir}/mutation/${name}.2r.snp.indel.vcf \
+        ${output_dir}/mutation/${name}.snp.indel.vcf
+
+        table_annovar.pl \
+        ${output_dir}/mutation/${name}.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,cytoBand \
+        -argument '-splicing_threshold 2 -hgvs',,,,,,,,,, \
+        --intronhgvs 50 \
+        -operation g,f,f,f,f,f,f,f,f,f,r \
+        --outfile ${output_dir}/mutation/${name}.snp.indel.anno
+
+    >>>
+
+    output {
+        String vcf = "${output_dir}/mutation/${name}.filter.flag.snp.indel.vcf"
+    }
+}
+
+task mutation_calling_tissue {
+    String name
+    String bed
+    String ref
+    String output_dir
+    String rmdup_bam
+
+    command <<<
+        if [ ! -d ${output_dir}/mutation ];then
+        mkdir ${output_dir}/mutation
+        fi
+
+        # vardict
+        java -jar /dataseq/jmdna/software/VarDict-1.8.3/lib/VarDict-1.8.3.jar \
+        -G ${ref} \
+        -f 0.01 \
+        -N ${name} \
+        -b ${rmdup_bam} \
+        -UN \
+        -Q 20 \
+        -m 3 \
+        -r 3 \
+        -th 10 \
+        -c 1 -S 2 -E 3 -g 4 ${bed} |/dataseq/jmdna/software/VarDict-1.8.3/bin/teststrandbias.R \
+        |/dataseq/jmdna/software/VarDict-1.8.3/bin/var2vcf_valid.pl -N ${name} -E -f 0.01 >${output_dir}/mutation/${name}.snp.indel.vcf
+
+        table_annovar.pl \
+        ${output_dir}/mutation/${name}.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',,,,,,,,, \
+        -operation g,f,f,f,f,f,f,f,f,f \
+        --intronhgvs 50 \
+        --outfile ${output_dir}/mutation/${name}.snp.indel.anno
+
+    >>>
+
+    output {
+        String vcf = "${output_dir}/mutation/${name}.snp.indel.vcf"
+    }
+}
+
+task mutation_calling_tissue_control {
+    String name
+    String bed
+    String ref
+    String output_dir
+    String tumor_rmdup_bam
+    String normal_rmdup_bam
+
+    command <<<
+        if [ ! -d ${output_dir}/mutation ];then
+        mkdir ${output_dir}/mutation
+        fi
+
+        java -jar /dataseq/jmdna/software/VarDict-1.8.3/lib/VarDict-1.8.3.jar \
+        -G ${ref} \
+        -f 0.01 \
+        -N ${name} \
+        -b "${tumor_rmdup_bam}|${normal_rmdup_bam}" \
+        -UN -Q 20 -m 3 -r 3 -th 20 -c 1 -S 2 -E 3 -g 4 ${bed} | /dataseq/jmdna/software/VarDict-1.8.3/bin/testsomatic.R \
+        | /dataseq/jmdna/software/VarDict-1.8.3/bin/var2vcf_paired.pl -N ${name} -f 0.01 > ${output_dir}/mutation/${name}.snp.indel.vcf
+
+        table_annovar.pl \
+        ${output_dir}/mutation/${name}.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',,,,,,,,, \
+        -operation g,f,f,f,f,f,f,f,f,f \
+        --intronhgvs 50 \
+        --outfile ${output_dir}/mutation/${name}.snp.indel.anno
+
+    >>>
+
+    output {
+        String vcf = "${output_dir}/mutation/${name}.snp.indel.vcf"
+    }
+}
+
+workflow call_mutation {
+
+    String tumor
+    String tumor_rmdup_bam
+    String? normal
+    String? normal_rmdup_bam
+    Boolean umi
+    String output_dir
+    String ref
+    String bed
+
+    scatter(name in [tumor, normal]) {
+        if (defined(name)) {
+            if (name==tumor) {
+                if (umi) {
+                    call mutation_calling_umi as tumor_mutation_calling_umi {
+                        input:
+                            name=name,
+                            output_dir=output_dir,
+                            ref=ref,
+                            bed=bed,
+                            rmdup_bam=tumor_rmdup_bam
+                    }
+                }
+                if (!umi) {
+                    # 单样本模式，normal没有定义
+                    if (name==select_first([normal, tumor])) {
+                        call mutation_calling_tissue as tumor_mutation_calling_tissue {
+                            input:
+                                name=name,
+                                output_dir=output_dir,
+                                ref=ref,
+                                bed=bed,
+                                rmdup_bam=normal_rmdup_bam
+                        }
+                    }
+                    # 双样本模式，normal有定义
+                    if (name!=select_first([normal, tumor])) {
+                        call mutation_calling_tissue_control as tumor_mutation_calling_tissue_control {
+                            input:
+                                name=name,
+                                output_dir=output_dir,
+                                ref=ref,
+                                bed=bed,
+                                tumor_rmdup_bam=tumor_rmdup_bam,
+                                normal_rmdup_bam=normal_rmdup_bam
+                        }
+                    }
+                }
+            }
+            if (name==select_first([normal, 'None'])) {
+                if (umi) {
+                    call mutation_calling_tissue as normal_mutation_calling_tissue {
+                        input:
+                            name=name,
+                            output_dir=output_dir,
+                            ref=ref,
+                            bed=bed,
+                            rmdup_bam=normal_rmdup_bam
+                    }
+                }
+
+            }
+
+        }
+    }
+
+    output {
+        String somatic_vcf = "${output_dir}/mutation/${tumor}.snp.indel.vcf"
+        String somatic_nc_vcf = "${output_dir}/mutation/${normal}.snp.indel.vcf"
+    }
+}
\ No newline at end of file
diff --git a/wdl/qc.wdl b/wdl/qc.wdl
index 60d6930..fccd4bf 100644
--- a/wdl/qc.wdl
+++ b/wdl/qc.wdl
@@ -1,31 +1,71 @@
 task runqc {
     String name
-    String inputDir
-    String outputDir
+    String input_dir
+    String output_dir
 
     command <<<
-        echo "###### ${name} fastp beginning at: $(date) ######"
+        # echo "###### ${name} fastp beginning at: $(date) ######"
 
-        if [ ! -d ${outputDir}/qc ];then
-        mkdir ${outputDir}/qc
+        if [ ! -d ${output_dir}/qc ];then
+        mkdir ${output_dir}/qc
         fi
 
-        fastp -i ${inputDir}/*_${name}_*1.fq.gz -o ${outputDir}/qc/${name}_clean_R1.fq.gz \
-        -I ${inputDir}/*_${name}_*2.fq.gz -O ${outputDir}/qc/${name}_clean_R2.fq.gz \
+        fastp \
+        -i ${input_dir}/*_${name}_*1.fq.gz \
+        -o ${output_dir}/qc/${name}_clean_R1.fq.gz \
+        -I ${input_dir}/*_${name}_*2.fq.gz \
+        -O ${output_dir}/qc/${name}_clean_R2.fq.gz \
         -w 10 \
+        -e 20 \
         --correction \
         --overlap_len_require 10 \
-        -j ${outputDir}/qc/${name}.json \
-        -h ${outputDir}/qc/${name}.html \
-        --report_title $name \
-        -e 20
+        -j ${output_dir}/qc/${name}.json \
+        -h ${output_dir}/qc/${name}.html \
+        --report_title ${name}
 
-        echo "###### ${name} fastp end at: $(date) ######"
     >>>
     output {
-        String r1 = "${outputDir}/qc/${name}_clean_R1.fq.gz "
-        String r2 = "${outputDir}/qc/${name}_clean_R2.fq.gz "
-        String json = "${outputDir}/qc/${name}.json"
+        String r1 = "${output_dir}/qc/${name}_clean_R1.fq.gz "
+        String r2 = "${output_dir}/qc/${name}_clean_R2.fq.gz "
+        String json = "${output_dir}/qc/${name}.json"
+    }
+
+}
+
+task umiqc {
+    String name
+    String input_dir
+    String output_dir
+
+    command <<<
+        # echo "###### ${name} fastp beginning at: $(date) ######"
+
+        if [ ! -d ${output_dir}/qc ];then
+        mkdir ${output_dir}/qc
+        fi
+
+        fastp -i ${input_dir}/*_${name}_*1.fq.gz -o ${output_dir}/qc/${name}_clean_R1.fq.gz \
+        -I ${input_dir}/*_${name}_*2.fq.gz -O ${output_dir}/qc/${name}_clean_R2.fq.gz \
+        -w 10 \
+        --correction \
+        --overlap_len_require=10 \
+        --umi \
+        --umi_loc=per_read \
+        --umi_len=4 \
+        --umi_prefix=UMI \
+        --umi_skip=3 \
+        --umi_delim \ : \
+        --disable_trim_poly_g \
+        -j ${output_dir}/qc/${name}.json \
+        -h ${output_dir}/qc/${name}.html \
+        --report_title ${name}
+
+    >>>
+
+    output {
+        String r1 = "${output_dir}/qc/${name}_clean_R1.fq.gz "
+        String r2 = "${output_dir}/qc/${name}_clean_R2.fq.gz "
+        String json = "${output_dir}/qc/${name}.json"
     }
 
 }
@@ -34,27 +74,49 @@ workflow qc {
 
     String tumor
     String? normal
-    String inputDir
-    String outputDir
+    Boolean umi
+    String input_dir
+    String output_dir
 
     scatter(name in [tumor, normal]) {
         if (defined(name)) {
-            call runqc {
-                input:
-                    name=name,
-                    inputDir=inputDir,
-                    outputDir=outputDir
+            if (name==tumor) {
+                if (umi) {
+                    call umiqc as run_umi_qc {
+                        input:
+                            name=name,
+                            input_dir=input_dir,
+                            output_dir=output_dir
+                    }
+                }
+                if (!umi) {
+                    call runqc as run_tumor_qc {
+                        input:
+                            name=name,
+                            input_dir=input_dir,
+                            output_dir=output_dir
+                    }
+                }
             }
+            if (name==select_first([normal, 'None'])) {
+                call runqc as run_normal_qc {
+                    input:
+                        name=name,
+                        input_dir=input_dir,
+                        output_dir=output_dir
+                }
+            }
+
         }
     }
 
     output {
-        String tumor_r1 = "${outputDir}/qc/${tumor}_clean_R1.fq.gz "
-        String tumor_r2 = "${outputDir}/qc/${tumor}_clean_R2.fq.gz "
-        String tumor_json = "${outputDir}/qc/${tumor}.json"
-        String normal_r1 = "${outputDir}/qc/${normal}_clean_R1.fq.gz "
-        String normal_r2 = "${outputDir}/qc/${normal}_clean_R2.fq.gz "
-        String normal_json = "${outputDir}/qc/${normal}.json"
+        String tumor_r1 = "${output_dir}/qc/${tumor}_clean_R1.fq.gz "
+        String tumor_r2 = "${output_dir}/qc/${tumor}_clean_R2.fq.gz "
+        String tumor_json = "${output_dir}/qc/${tumor}.json"
+        String normal_r1 = "${output_dir}/qc/${normal}_clean_R1.fq.gz "
+        String normal_r2 = "${output_dir}/qc/${normal}_clean_R2.fq.gz "
+        String normal_json = "${output_dir}/qc/${normal}.json"
     }
 }
 
diff --git a/wdl/task.wdl b/wdl/task.wdl
index 23cbf13..50c742d 100644
--- a/wdl/task.wdl
+++ b/wdl/task.wdl
@@ -3,7 +3,7 @@
 task create_dir {
     String workdir
     command <<<
-        if [ ! -d ${workdir}];then
+        if [ ! -d ${workdir} ];then
         mkdir -p ${workdir}/log
         fi
 
@@ -12,9 +12,10 @@ task create_dir {
 
 task mutation_calling {
     String name
-    String tumor_pileup
-    String normal_pileup
+    String tumor_rmdupBam
+    String normal_rmdupBam
     String outputDir
+    String bed
 
     command <<<
 
@@ -22,47 +23,27 @@ task mutation_calling {
         mkdir ${outputDir}/mutation
         fi
 
-        java -jar $VARSCAN somatic ${tumor_pileup} ${normal_pileup} \
-        --output-snp ${outputDir}/mutation/${name}.snp.vcf \
-        --output-indel ${outputDir}/mutation/${name}.indel.vcf \
-        --min-var-freq 0.01 \
-        --min-freq-for-hom 0.9 \
-        --somatic-p-value 0.05 \
-        --output-vcf 1 \
-        --min-avg-qual 20  \
-        --min-coverage-normal 10 \
-        --min-coverage-tumor 30 \
-        --min-reads2 3
+        java -jar /dataseq/jmdna/software/VarDict-1.8.3/lib/VarDict-1.8.3.jar \
+        -G /dataseq/jmdna/database/genome/hg19/hg19.fa \
+        -f 0.01 \
+        -N ${name} \
+        -b "${tumor_rmdupBam}|${normal_rmdupBam}" \
+        -UN -Q 20 -m 3 -r 3 -th 20 -c 1 -S 2 -E 3 -g 4 ${bed} | \
+        /dataseq/jmdna/software/VarDict-1.8.3/bin/testsomatic.R | \
+        /dataseq/jmdna/software/VarDict-1.8.3/bin/var2vcf_paired.pl -N ${name} -f 0.01 \
+        > ${outputDir}/mutation/${name}_vardict.snp.indel.vcf
 
-        java -jar $VARSCAN processSomatic \
-        ${outputDir}/mutation/${name}.snp.vcf \
-        --min-tumor-freq 0.01 \
-        --max-normal-freq 0.01 \
-        --p-value 0.05
-
-        java -jar $VARSCAN  processSomatic \
-        ${outputDir}/mutation/${name}.indel.vcf \
-        --min-tumor-freq 0.01 \
-        --max-normal-freq 0.01 \
-        --p-value 0.05
-
-        java -jar $GATK MergeVcfs \
-        -I ${outputDir}/mutation/${name}.snp.Somatic.hc.vcf \
-        -I ${outputDir}/mutation/${name}.indel.Somatic.hc.vcf \
-        -O ${outputDir}/mutation/${name}.snp.indel.Somatic.hc.vcf \
-        -D /dataseq/jmdna/database/genome/hg19/hg19.dict
-
-        java -jar $GATK MergeVcfs \
-        -I ${outputDir}/mutation/${name}.snp.Germline.vcf \
-        -I ${outputDir}/mutation/${name}.indel.Germline.vcf \
-        -O ${outputDir}/mutation/${name}.snp.indel.Germline.vcf \
-        -D /dataseq/jmdna/database/genome/hg19/hg19.dict
-
-        java -jar $GATK MergeVcfs \
-        -I ${outputDir}/mutation/${name}.snp.LOH.hc.vcf \
-        -I ${outputDir}/mutation/${name}.indel.LOH.hc.vcf \
-        -O ${outputDir}/mutation/${name}.snp.indel.LOH.hc.vcf \
-        -D /dataseq/jmdna/database/genome/hg19/hg19.dict
+        vep \
+        --input_file ${outputDir}/mutation/${name}_vardict.snp.indel.vcf \
+        --output_file ${outputDir}/mutation/${name}_vardict_vep.snp.indel.vcf \
+        --format vcf \
+        --vcf \
+        --symbol \
+        --terms SO \
+        --hgvs \--fasta /dataseq/jmdna/database/genome/hg19/hg19.fa \
+        --offline --cache --dir_cache /home/software/.vep \
+        --pick \
+        --force_overwrite
 
     >>>
 
@@ -580,7 +561,7 @@ task auto_report {
         perl /home/jm001/test_duantao/database_update/codes/682/indication.pl ${outputDir} ${cancer}
         python3 ${codesDir}/drug_dedup.py ${outputDir} ${tumor}
         perl ${codesDir}/file_format_change.pl ${outputDir} ${tumor}
-        python3 ${codesDir}/682gene_tissue_control_report.py ${outputDir} ${tumor} ${normal} ${cancer}
+        python3 ${codesDir}/report_template/682gene_tissue_control_report.py ${outputDir} ${tumor} ${normal} ${cancer}
 
         ln -s  ${cnv_cns} ${outputDir}/report/
         ln -s  ${cnv_png} ${outputDir}/report/