################################ Section 1:FASTQ to BAM ###############################

## mapping

#bwa mem -t 32 -M /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta H11_CCACTC_L007_R1_001.fastq H11_CCACTC_L007_R2_001.fastq > mem.sam

################################## Section 2:Calling Variants #################################

## add read groups

#java -Xmx2g -jar /leofs/noncode/softwares/picard-tools-1.112/AddOrReplaceReadGroups.jar

#       INPUT=mem.sam OUTPUT=addrg_mem.bam

#       SORT_ORDER=coordinate

#       RGID=group1 RGLB=lib1 RGPL=illumina RGPU=unit1 RGSM=sample1

## dedupping

#java -Xmx2g -jar /leofs/noncode/softwares/picard-tools-1.112/MarkDuplicates.jar

#       INPUT=addrg_mem.bam OUTPUT=dedup_mem.bam

#       METRICS_FILE=metrics.mem.txt READ_NAME_REGEX=".*?([0-9]+):([0-9]+):([0-9]+)$"

## validate bam formate

#java -Xmx2g -jar /leofs/noncode/softwares/picard-tools-1.112/ValidateSamFile.jar

#       INPUT=dedup_mem.bam OUTPUT=validate_mem.txt

## build index form bam file

#java -Xmx4g -jar /leofs/noncode/softwares/picard-tools-1.112/BuildBamIndex.jar

#       INPUT=dedup_mem.bam

## Local Realignment around Indels (~6h)

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T RealignerTargetCreator

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I dedup_mem.bam

#       -known /leofs/noncode/xcl/references/human/hg19/1000G_phase1.indels.hg19.sites.vcf

#       -o target_interval.list

#(If this step goes wrong,it may be the reference FASTA not sorted as chr1..chrY..chrM.Just run again,it maybe do well);

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T IndelRealigner

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I dedup_mem.bam

#       -targetIntervals target_interval.list

#       -known /leofs/noncode/xcl/references/human/hg19/1000G_phase1.indels.hg19.sites.vcf

#       -o realigned_mem.bam

## Base quality scores recalibration (BQSR)

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T BaseRecalibrator

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I realigned_mem.bam

#       -knownSites /leofs/noncode/xcl/references/human/hg19/dbsnp_138.hg19.vcf

#       -knownSites /leofs/noncode/xcl/references/human/hg19/1000G_phase1.indels.hg19.sites.vcf

#       -o recal_data_mem.table

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T BaseRecalibrator

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I realigned_mem.bam

#       -knownSites /leofs/noncode/xcl/references/human/hg19/dbsnp_138.hg19.vcf

#       -knownSites /leofs/noncode/xcl/references/human/hg19/1000G_phase1.indels.hg19.sites.vcf

#       -BQSR recal_data_mem.table

#       -o post_recal_data_mem.table

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T AnalyzeCovariates

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -before recal_data_mem.table

#       -after post_recal_data_mem.table

#       -plots recalibration_plots.pdf

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T PrintReads

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I realigned_mem.bam

#       -BQSR recal_data_mem.table

#       -o recal_mem.bam

## Variant Discovery

#  Calling Variants (~6h)

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T HaplotypeCaller

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -I recal_mem.bam

#       --dbsnp /leofs/noncode/xcl/references/human/hg19/dbsnp_138.hg19.vcf

#       --emitRefConfidence GVCF

#       --variant_index_type LINEAR

#       --variant_index_parameter 128000

#       -o gvcf_mem.vcf

#       -nct 16

#(If this step goes wrong with the ERROR MESSAGE:"There was a failure because you did not provide enough memory to run this program.  See the -Xmx JVM argument to adjust the maximum heap size provided to Java".You could change the parameter "-nct 16" to "-nct 2" or lower.)

# Joint Genotyping

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T GenotypeGVCFs

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       --variant gvcf_mem.vcf

#       -o raw.snps.indels.vcf

#       -nt 16

############################################ Section 3:Filtering Variants###########################

# Variant Filtering

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T SelectVariants

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -V gvcf_mem.vcf

#       -selectType SNP

#       -o raw_snps.vcf

#       -nt 16

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T VariantFiltration

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -V raw_snps.vcf

#       --filterExpression "QD < 2.0 || FS > 60.0 || MQ < 40.0 || HaplotypeScore > 13.0 ||MappingQualityRankSum < -12.5||     ReadPosRankSum < -8.0"

#       --filterName "my_snp_filter"

#       -o filtered_snps.vcf

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T SelectVariants

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -V gvcf_mem.vcf

#       -selectType INDEL

#       -o raw_indels.vcf

#       -nt 16

#

#java -Xmx4g -jar /leofs/noncode/softwares/gatk/GenomeAnalysisTK.jar

#       -T VariantFiltration

#       -R /leofs/noncode/reference/gatk/genome/hg19/ucsc.hg19.fasta

#       -V raw_indels.vcf

#       --filterExpression "QD < 2.0 || FS > 200.0 || MQ < 40.0 || ReadPosRankSum < -20.0"

#       --filterName "my_snp_filter"

#       -o filtered_indels.vcf

pipeline_gatk.txt