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FJDpipeline.sh
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FJDpipeline.sh
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#!/bin/bash
#DATE
DATE=`date +%Y-%m-%d`
#······································
#Set the pathways to work for you.
#Create a directory or fix it.
#······································
#Main directory, all data will be stored in this file.
#MDAP=set the patwhay to a principal file which will contain all.
#MDAP='/home/marius/testFJDRP/final_test'
MDAP='/mnt/genetica3/Ionut/PipelineM/pruebas'
#·······································
#Select the directory where fastq files are stored.
#FASTQ directory to look after the .fastq files.
#FQ='/home/marius/genetica3/marius/fastq/NIST70'
FQ='/mnt/genetica3/Ionut/PipelineM/fastqs'
#·······································
#Reference genome directory used as a mini bundle data.
#Where the reference and bundle data is stored
#HG19: where all the references and indexed files will be + bundle data from UCSC.
#HG19='/home/marius/genetica3/marius/hg19bundle'
HG19='/mnt/genetica3/marius/pipeline_practicas_marius/hg19bundle'
#·······································
#Software directory.
#All the software used is stored in this location.
#SFT='/home/marius/software'
SFT='/mnt/genetica3/marius/pipeline_practicas_marius/software'
#Specify where this command is requiered for the script, but gets separated to work.
#GENOTYPE_SCRIPT="${MDAP}/genotype_script.sh"
#Specify the PEDIGREE PATH FOR GENOTYPEGVCFS
#PEDIGREE='home/marius/genetica3/marius/fastq/RP-1773/family_info.txt'
#·······································
#Files generated by the script for each part of the analysis.
#Can change their name or add/remove but then outputs would need redirects.
#IMPORTANT to keep in mind
#If using pathway like ANY=${ANYPATH}/anypath USE DOUBLE QUOTATIONS
#IMPORTANT, or no quotations
#·······································
#Mapped_data: will contain the .SAM files after alignment using BWA.
MD="${MDAP}/mapped_data"
#·······································
#·······································
#Sorted_data: will store the sorted SAM files, after using Picard.
SD="${MDAP}/sorted_data"
#·······································
#·······································
#Dedupped_data: will store the file of the duplicate reads, after using Picard.
DD="${MDAP}/dedupped_data"
#·······································
#·······································
#Recalibrated_base_quality_scores_data: generates a recalibration table based on specified covariates, read_group,reported_quiality_score,machine_cycle and nucleotide_context, after GATK.
RBQSRD="${MDAP}/recalibrated_bqsr_data"
#·······································
#·····································
#Applied_bqsr_data: applying the reacalibration table to the BAM file to continue the analysis based on the READS best selected by GATK.
ABQSRD="${MDAP}/applied_bqsr_data"
#·······································
#·······································
#Plot_recalibration_data: plots the recalibration differences between the first and the second pass of the recalibration.
#First using the APPLIED_RECALIBRATION_BQSR_DATA BAM file, running again BaseRecalibrator and generating the plots with AnalyzeCovariates.
PRD="${MDAP}/plot_recalibration_data"
#·······································
#·······································
#Haplotype_caller_gvcf_data:calling for SNPs and indels via local re-assembly of HAPLOTYPES using HAPLOTYPECALLER by GATK.
HCGVCFD="${MDAP}/haplotype_caller_gvcf_data"
#·······································
#·······································
#If analyzing more than one sample or an family trio, use COMBINE_GVCFS to combine them into a single GVCF.
#If more than a few samples, consider using the alternative tool, GenomicsDBImport(large number of samples).
#Combine_gvcf_data: merge one or more GVCFS into a single g.vcf file
CGVCFD="${MDAP}/combined_gvcf_data"
#·······································
#·······································
#Perform joint genotyping on one or more samples precalled with Haplotype_caller, if one sample,
#straight after haplotypecaller and if more than one use Combine_gvcfs.
#Genotyped_vcf_data: single or single-multisample GVCF as input, output will be a VCF.
GVCFD="${MDAP}/genotyped_vcf_data"
#·······································
#·······································
#HARD_FILTERING, filters for SNPs and filters for INDELs.
#Variant_filtration_vcf_data:hard filtering process to select based on the INFO and FORMAT annotations(QD,MQO,FS,MQ,ReadPosrankSum)
VFVCFD="${MDAP}/variant_filtration_vcf_data"
#·······································
#·······································
#Vep_vcf_annotated_data: annotations added to the CSQ tag in INFO columnd to the VCF format, --vcf argument (change name in VCF_out)
#if TSV will come out with a separated tab value for each annotation in a column. --tab argument for TSV format
VEPVCFAD="${MDAP}/vep_vcf_annotated_data"
#VEP Variables for Variant_effect_predictor, Annotatiting the VCF file.
#VEP DIRECTORY
VEP="${SFT}/variant_effect_predictor/ensembl-vep/vep"
#VEP DIRECTORY for the downloaded files (HUMAN DATABASE FILES,PLUGINS)
VEP_CACHE='/home/marius/.vep'
VEP_CACHE='/mnt/genetica3/marius/pipeline_practicas_marius/software/variant_effect_predictor/.vep'
#VEP_FASTA, reference fasta used by VEP (GRCh37 version)
VEP_FASTA="${VEP_CACHE}/homo_sapiens/93_GRCh37/Homo_sapiens.GRCh37.75.dna.primary_assembly.fa"
#PLUGINS directory for vep.
PLUGIN_DIR="${VEP_CACHE}/Plugins"
#DATABASE directory for vep.
PLUGIN_DBS="${VEP_CACHE}/dbs"
#PLUGINS PATHWAYS FOR VEP to aim after the exact folders and files.
#Loss of Function PLUGIN
LOFTEE="${PLUGIN_DBS}/human_ancestor.fa.gz"
#Loftee folder directoyry (REQUIREMENTS of LoF)
LOFTEE_PATH="${VEP_CACHE}/loftee"
#·······································
#·······································
#dbnSFP plugin, it is a functional prediction and annotation database of all
#potetial non-synonymous single-nucleotide-variants (nsSNVs) in the human genome.
DBNSFP="${PLUGIN_DBS}/dbNSFP_hg19.gz"
#·······································
#·······································
#VCF INPUT
VCF_IN="${VFVCFD}/pre_filtered_ready_to_annotate.vcf"
#·······································
#·······································
#OUTPUT
#Here you can choose from different outputs.
#·······································
#.1:VCF by selecting the --vcf ARGUMENT
#.2:TSV by selecting the --tab ARGUMENT
#.3:DEFAULT --> .TXT + .HTML (just set the name to -o annonated_vcf) and will obtain --> annotated_vcf.txt and annotated_vcf.html
VCF_OUT="${VEPVCFAD}/vep_"$DATE"_annotated.vcf"
#·······································
#·······································
#Also install and set the paths correctly to PERL5LIB by exporting all the commands in libreriasVEP.sh
#selectVariantsData_vcfs, selecting the qualities and the desired FILTERING options.
#SVDVCF='/home/marius/testFJDRP/trioGDBImportJointCallGenotype/selecVariants_data_vcf'
#VTTVCF='/home/marius/testFJDRP/trioGDBImportJointCallGenotype/variantstotable_vcf'
#Last two are still in progress.
#Start pipeline processing.
echo -e '\n\n\n'
echo -e " _____ _ ____ ____ ___ ____ _____ _ ___ _ _ _____ "
echo -e "| ___| | | _ \ | _ \_ _| _ \| ____| | |_ _| \ | | ____|"
echo -e "| |_ _ | | | | | | |_) | || |_) | _| | | | || \| | _| "
echo -e "| _| |_| | |_| | | __/| || __/| |___| |___ | || |\ | |___ "
echo -e "|_| \___/|____/ |_| |___|_| |_____|_____|___|_| \_|_____|"
echo -e " "
echo -e " ____ _____ _ ____ _____ ____ "
echo -e "/ ___|_ _|/ \ | _ \_ _/ ___| "
echo -e "\___ \ | | / _ \ | |_) || | \___ \ "
echo -e " ___) || |/ ___ \| _ < | | ___) |"
echo -e "|____/ |_/_/ \_\_| \_\|_| |____/ "
echo -e " "
echo -e '\n\n\n\n\n\n'
echo ············································································································
echo -e " ___ _ _ ____ _______ _____ _ _ ____ "
echo -e "|_ _| \ | | _ \| ____\ \/ /_ _| \ | |/ ___|"
echo -e " | || \| | | | | _| \ / | || \| | | _ "
echo -e " | || |\ | |_| | |___ / \ | || |\ | |_| |"
echo -e "|___|_| \_|____/|_____/_/\_\___|_| \_|\____|"
echo -e " "
echo -e " ____ _____ _____ _____ ____ _____ _ _ ____ _____ "
echo -e "| _ \| ____| ___| ____| _ \| ____| \ | |/ ___| ____|"
echo -e "| |_) | _| | |_ | _| | |_) | _| | \| | | | _| "
echo -e "| _ <| |___| _| | |___| _ <| |___| |\ | |___| |___ "
echo -e "|_| \_\_____|_| |_____|_| \_\_____|_| \_|\____|_____|"
echo -e " "
echo -e " _____ ___ _ _____ ____ ______ ___ "
echo -e "| ___|_ _| | | ____/ ___| | __ ) \ / / \ "
echo -e "| |_ | || | | _| \___ \ | _ \\ \ /\ / / _ \ "
echo -e "| _| | || |___| |___ ___) | | |_) |\ V V / ___ \ "
echo -e "|_| |___|_____|_____|____/ |____/ \_/\_/_/ \_\ "
echo -e '\n \tINDEXING REFERENCE FILES (BWA)\n'
echo ··············································································································
#Start BWA INDEX.
#Stores .dict and .fai files in HG19 directory.
echo 'Starts BWA INDEX'
echo 'Starts BWA INDEX' >> registerFile
$SFT/bwa/./bwa index $HG19/ucsc.hg19.fasta
echo -e '\nBWA INDEX DONE' ; paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nBWA INDEX DONE' >> registerFile
#REMOVE PREVIOUS UCSC.HG19.FASTA.FAI and UCSC.HG19.DICT file
rm $HG19/ucsc.hg19.fasta.fai $HG19/ucsc.hg19.dict
#Creating .FAI in HG19.
echo 'Create .FAI file, using samtools faidx'
echo 'Create .FAI file, using samtools faidx' >> registerFile
$SFT/samtools/./samtools faidx $HG19/ucsc.hg19.fasta -o $HG19/ucsc.hg19.fai
echo -e '\nucsc.hg19.fasta.FAI DONE'
echo -e '\nucsc.hg19.fasta.FAI DONE' >> registerFile
#Creating .DICT in HG19.
echo 'Create .DICT file, using picardtools CreateSequnceDictionary'
echo 'Create .DICT file, using picardtools CreateSequnceDictionary' >> registerFile
java -jar $SFT/picard/build/libs/picard.jar CreateSequenceDictionary \
R=$HG19/ucsc.hg19.fasta \
O=$HG19/ucsc.hg19.dict
echo -e '\nucsc.hg19.DICT DONE'
echo -e '\nucsc.hg19.DICT DONE'>> registerFile
echo ············································································································
echo -e " __ __ _ ____ ____ ___ _ _ ____ __ _____ _____ _ _ "
echo -e "| \/ | / \ | _ \| _ \_ _| \ | |/ ___| \ \ / /_ _|_ _| | | |"
echo -e "| |\/| | / _ \ | |_) | |_) | || \| | | _ \ \ /\ / / | | | | | |_| |"
echo -e "| | | |/ ___ \| __/| __/| || |\ | |_| | \ V V / | | | | | _ |"
echo -e "|_| |_/_/ \_\_| |_| |___|_| \_|\____| \_/\_/ |___| |_| |_| |_|"
echo -e " "
echo -e " ______ ___ "
echo -e "| __ ) \ / / \ "
echo -e "| _ \\ \ /\ / / _ \ "
echo -e "| |_) |\ V V / ___ \ "
echo -e "|____/ \_/\_/_/ \_\ "
echo -e '\n \tMAPPING (BWA)\n'
echo ············································································································
#mapping fastq files to reference_genome after BWA INDEX
#reference genome is: UCSC.HG19.FASTA
mkdir $MDAP/mapped_data
echo 'mkdir mapped_data' >> registerFile
#Run BWA mem -t 12
#-t threads
#-P search for Pair mate if not mapped properly, if it found a better hit, skips it.
#PREVIOUS HEADER $SFT/bwa/./bwa mem -t 12 -R @RG\tID:\tPL:illumina\tSM:Analysis_$i $HG19/ucsc.hg19.fasta \
for i in $@
do
echo -e '\nBuilding the header for '$i' ongoing...\n'
# #build header
header=$(zcat $FQ/$i*1.fastq.gz | head -n 1)
echo $header
id=$(echo $header | head -n 1 | cut -f 1-4 -d':' | sed 's/@//' | sed 's/:/_/g')
echo $id
sm=$(echo $header | head -n 1 | grep -Eo '[ATGCN]+$')
echo $sm
echo -e "\nThis is how the new header looks\n"
echo '@RG\tID:'$id'\tSM:'$i'\tLB:'$id'_'$sm'\tSM:'$id'_'$m'\tPL:ILLUMINA'
echo -e '\nHEADER --> DONE\n'
echo -e '\n\nStart BWA MEM for '$i' sample'
echo 'Start BWA MEM for '$i' sample'>> registerFile
echo ············································································································
$SFT/bwa/./bwa mem -v 3 -t 12 -R '@RG\tID:'$id'\tSM:'$i'\tLB:'$id'_'$sm'\tPL:ILLUMINA' \
$HG19/ucsc.hg19.fasta \
$FQ/$i*1.fastq.gz \
$FQ/$i*2.fastq.gz > $MD/mapped_$i.sam
echo -e '\nBWA MEM '$i' DONE' ; paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nBWA MEM '$i' DONE' >> registerFile
#Unzip all the files before continuing the process.
echo 'Unzip mapped sams.'
echo 'Unzip mapped sams.' >> registerFile
#If unzipped not necessary
gunzip -k $MD/mapped_$i.sam.gz
echo 'Gunzip completed.'
echo 'Gunzip completed.' >> registerFile
#Comprobar si el archivo esta en formato zip o no.
done
#
#
echo ···········································································································
echo -e " ____ ___ ____ _____ ___ _ _ ____ ____ _ __ __ "
echo -e "/ ___| / _ \| _ \_ _|_ _| \ | |/ ___| / ___| / \ | \/ |"
echo -e "\___ \| | | | |_) || | | || \| | | _ \___ \ / _ \ | |\/| |"
echo -e " ___) | |_| | _ < | | | || |\ | |_| | ___) / ___ \| | | |"
echo -e "|____/ \___/|_| \_\|_| |___|_| \_|\____| |____/_/ \_\_| |_|"
echo -e '\n \tSORTING SAM (PICARD)\n'
echo ···········································································································
mkdir $MDAP/sorted_data
echo 'mkdir sorted_data'>> registerFile
#Sorting the mapped data.
for i in $@
do
#SORTING THE SAM FILE.
echo 'Run picard SortSam '$i''
echo 'Run picard SortSam '$i''>> registerFile
java -jar $SFT/picard/build/libs/picard.jar SortSam I=$MD/mapped_$i.sam \
O=$SD/sorted$i.bam \
SORT_ORDER=coordinate
echo -e '\nPicard SortSam '$i' DONE' ; paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nPicard SortSam '$i' DONE' >> registerFile
done
echo ············································································································
echo -e " __ __ _ ____ _ _____ _ _ ____ "
echo -e "| \/ | / \ | _ \| |/ /_ _| \ | |/ ___|"
echo -e "| |\/| | / _ \ | |_) | ' / | || \| | | _ "
echo -e "| | | |/ ___ \| _ <| . \ | || |\ | |_| |"
echo -e "|_| |_/_/ \_\_| \_\_|\_\___|_| \_|\____|"
echo -e " "
echo -e " ____ _ _ ____ _ ___ ____ _ _____ _____ ____ "
echo -e "| _ \| | | | _ \| | |_ _/ ___| / \|_ _| ____/ ___| "
echo -e "| | | | | | | |_) | | | | | / _ \ | | | _| \___ \ "
echo -e "| |_| | |_| | __/| |___ | | |___ / ___ \| | | |___ ___) |"
echo -e "|____/ \___/|_| |_____|___\____/_/ \_\_| |_____|____/ "
echo -e " "
echo -e " ____ ___ ____ _ ____ ____ "
echo -e "| _ \_ _/ ___| / \ | _ \| _ \ "
echo -e "| |_) | | | / _ \ | |_) | | | |"
echo -e "| __/| | |___ / ___ \| _ <| |_| |"
echo -e "|_| |___\____/_/ \_\_| \_\____/ "
echo -e " "
echo -e '\n \tMARKING DUPLICATES (PICARD)\n'
echo ············································································································
#Selecting the duplicates reads from the mapped and sorted reads.
mkdir $MDAP/dedupped_data
echo 'mkdir dedupped_data'>> registerFile
for i in $@
do
#Mark duplicates PICARD
echo 'Start picard MarkDuplicates '$i' '
echo 'Start picard MarkDuplicates '$i' '>>registerFile
java -jar $SFT/picard/build/libs/picard.jar MarkDuplicates \
I=$SD/sorted$i.bam \
O=$DD/dedupped_$i.bam \
M=$DD/marked_dup_metrics_$i.txt \
REMOVE_DUPLICATES=true \
AS=SortOrder
echo -e '\n PICARD MarkDuplicates '$i' DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\n PICARD MarkDuplicates '$i' DONE' >> registerFile
done
echo ············································································································
echo -e " ____ _____ ____ _ _ ____ ____ _____ ____ ____ _ __ __ "
echo -e "| _ \| ____| _ \| | | | _ \| _ \| ____| _ \ | __ ) / \ | \/ |"
echo -e "| | | | _| | | | | | | | |_) | |_) | _| | | | | | _ \ / _ \ | |\/| |"
echo -e "| |_| | |___| |_| | |_| | __/| __/| |___| |_| | | |_) / ___ \| | | |"
echo -e "|____/|_____|____/ \___/|_| |_| |_____|____/ |____/_/ \_\_| |_|"
echo -e " "
echo -e " ___ _ _ ____ _______ __ ____ _ ___ _____ ___ _ _____ "
echo -e "|_ _| \ | | _ \| ____\ \/ / | __ ) / \ |_ _| | ___|_ _| | | ____| "
echo -e " | || \| | | | | _| \ / | _ \ / _ \ | | | |_ | || | | _| "
echo -e " | || |\ | |_| | |___ / \ | |_) / ___ \ | | | _| | || |___| |___ _ "
echo -e "|___|_| \_|____/|_____/_/\_\ |____/_/ \_\___| |_| |___|_____|_____(_)"
echo -e " "
echo -e '\n \t Dedupped BAM index (BAI) file. \n'
echo ············································································································
#Create a .BAI file to compare original vs removed duplicates reads.
for i in $@
do
# #Indexing the BAM files.
# #Generating the .BAI files from the DEDUPPED (markedDuplicates from the original SAM/BAM file).
echo 'Indexing '$i' BAM files'
echo 'Indexing '$i' BAM files' >> registerFile
java -jar $SFT/picard/build/libs/picard.jar BuildBamIndex \
I=$DD/dedupped_$i.bam \
O=$DD/dedupped_$i.bai
echo -e '\n PICARD BuildBamIndex '$i' DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\n PICARD BuildBamIndex '$i' DONE' >> registerFile
done
echo ············································································································
echo -e " ____ ___ ____ ____ "
echo -e "| __ ) / _ \/ ___|| _ \ "
echo -e "| _ \| | | \___ \| |_) | "
echo -e "| |_) | |_| |___) | _ < "
echo -e "|____/ \__\_\____/|_| \_\ "
echo -e " "
echo -e " ____ _____ ____ _ _ ___ ____ ____ _ _____ ___ ___ _ _ "
echo -e "| _ \| ____/ ___| / \ | | |_ _| __ )| _ \ / \|_ _|_ _/ _ \| \ | | "
echo -e "| |_) | _|| | / _ \ | | | || _ \| |_) | / _ \ | | | | | | | \| | "
echo -e "| _ <| |__| |___ / ___ \| |___ | || |_) | _ < / ___ \| | | | |_| | |\ | "
echo -e "|_| \_\_____\____/_/ \_\_____|___|____/|_| \_\/_/ \_\_| |___\___/|_| \_| "
echo -e " "
echo -e " ____ _ _____ _ "
echo -e "| _ \ / \|_ _|/ \ "
echo -e "| | | |/ _ \ | | / _ \ "
echo -e "| |_| / ___ \| |/ ___ \ "
echo -e "|____/_/ \_\_/_/ \_\ "
echo -e '\n \tBQSR (GATK)\n'
echo -e '\t -.1 Recalibration data table\n'
echo -e '\t -.2 Recalibration data table \n'
echo ············································································································
#Recalibrating the reads using base quality score reads.
mkdir $MDAP/recalibrated_bqsr_data
echo 'mkdir recalibrated_bqsr_data' >> registerFile
for i in $@
do
# #GATK BaseRecalibration first table
# #BaseRecalibration + table
echo 'Starts GATK '$1' Recalibrator'
echo 'Starts GATK '$1' Recalibrator' >> registerFile
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar BaseRecalibrator \
-R $HG19/ucsc.hg19.fasta \
-I $DD/dedupped_$i.bam \
--known-sites $HG19/dbsnp_138.hg19.vcf.gz \
--known-sites $HG19/1000G_phase1.indels.hg19.sites.vcf.gz \
--known-sites $HG19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz \
-O $RBQSRD/before_recalibrated_bqsr_data_$i.recal.table
# --bqsr 1st_racalibration.table
echo -e '\n GATK BaseRecalibrator '$i' DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\n GATK BaseRecalibrator '$i' DONE' >>registerFile
done
echo ············································································································
echo -e " _ ____ ____ _ __ _____ _ _ ____ ____ ___ ____ ____ "
echo -e " / \ | _ \| _ \| | \ \ / /_ _| \ | |/ ___| | __ ) / _ \/ ___|| _ \ "
echo -e " / _ \ | |_) | |_) | | \ V / | || \| | | _ | _ \| | | \___ \| |_) | "
echo -e " / ___ \| __/| __/| |___| | | || |\ | |_| | | |_) | |_| |___) | _ < "
echo -e "/_/ \_\_| |_| |_____|_| |___|_| \_|\____| |____/ \__\_\____/|_| \_\ "
echo -e " "
echo -e "__ _____ _____ _ _ ____ _ _____ _ __ "
echo -e "\ \ / /_ _|_ _| | | | / ___| / \|_ _| |/ / "
echo -e " \ \ /\ / / | | | | | |_| | | | _ / _ \ | | | ' / "
echo -e " \ V V / | | | | | _ | | |_| |/ ___ \| | | . \ "
echo -e " \_/\_/ |___| |_| |_| |_| \____/_/ \_\_| |_|\_\ "
echo -e " "
echo -e '\n \tApplying BQSR GATK\n'
echo ············································································································
#Applying the recalibration table to the bam file to continue the analysis.
mkdir $MDAP/applied_bqsr_data
echo 'mkdir applied_bqsr_data' >> registerFile
for i in $@
do
# #ApplyBQSR
echo 'Starts picard '$i' ApplyBQSR'
echo 'Starts picard '$i' ApplyBQSR' >> registerFile
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar ApplyBQSR \
-R $HG19/ucsc.hg19.fasta \
-I $DD/dedupped_$i.bam \
--bqsr $RBQSRD/before_recalibrated_bqsr_data_$i.recal.table \
-O $ABQSRD/applied_bqsr_data_$i.bam
echo -e '\nGATK ApplyBQSR '$i' DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nGATK ApplyBQSR '$i' DONE' >>registerFile
done
echo ············································································································
echo -e " _ _ _ _ _ __ ____________ "
echo -e " / \ | \ | | / \ | | \ \ / /__ / ____|"
echo -e " / _ \ | \| | / _ \ | | \ V / / /| _| "
echo -e " / ___ \| |\ |/ ___ \| |___| | / /_| |___ "
echo -e "/_/ \_\_| \_/_/ \_\_____|_| /____|_____|"
echo -e " ____ _____ ___ ____ ___ _ _____ _____ ____ _____ ___ ____ "
echo -e " / ___/ _ \ \ / / \ | _ \|_ _| / \|_ _| ____/ ___| | ___/ _ \| _ \ "
echo -e "| | | | | \ \ / / _ \ | |_) || | / _ \ | | | _| \___ \ | |_ | | | | |_) | "
echo -e "| |__| |_| |\ V / ___ \| _ < | | / ___ \| | | |___ ___) | | _|| |_| | _ < "
echo -e " \____\___/ \_/_/ \_\_| \_\___/_/ \_\_| |_____|____/ |_| \___/|_| \_\ "
echo -e " "
echo -e " ____ ____ _____ ____ _ ___ _____ ____ "
echo -e "| _ \| _ \| ___| | _ \| | / _ \_ _/ ___| "
echo -e "| |_) | | | | |_ | |_) | | | | | || | \___ \ "
echo -e "| __/| |_| | _| | __/| |__| |_| || | ___) | "
echo -e "|_| |____/|_| |_| |_____\___/ |_| |____/ "
echo -e " "
echo -e " ____ ___ __ __ ____ _ ____ _ _____ ___ ___ _ _ ___ _____ "
echo -e " / ___/ _ \| \/ | _ \ / \ | _ \ / \|_ _|_ _/ _ \| \ | | / _ \| ___| "
echo -e "| | | | | | |\/| | |_) / _ \ | |_) | / _ \ | | | | | | | \| | | | | | |_ "
echo -e "| |__| |_| | | | | __/ ___ \| _ < / ___ \| | | | |_| | |\ | | |_| | _| "
echo -e " \____\___/|_| |_|_| /_/ \_\_| \_\/_/ \_\_| |___\___/|_| \_| \___/|_| "
echo -e " "
echo -e " _____ _ _ _____ "
echo -e "|_ _| | | | ____| "
echo -e " | | | |_| | _| "
echo -e " | | | _ | |___ "
echo -e " |_| |_| |_|_____| "
echo -e " "
echo -e " ____ _____ ____ _ _ ___ ____ ____ _ _____ ___ ___ _ _ "
echo -e "| _ \| ____/ ___| / \ | | |_ _| __ )| _ \ / \|_ _|_ _/ _ \| \ | | "
echo -e "| |_) | _|| | / _ \ | | | || _ \| |_) | / _ \ | | | | | | | \| | "
echo -e "| _ <| |__| |___ / ___ \| |___ | || |_) | _ < / ___ \| | | | |_| | |\ | "
echo -e "|_| \_\_____\____/_/ \_\_____|___|____/|_| \_\/_/ \_\_| |___\___/|_| \_| "
echo -e " "
echo -e " ____ _ _____ _ ____ _ _____ _ __ "
echo -e "| _ \ / \|_ _|/ \ / ___| / \|_ _| |/ / "
echo -e "| | | |/ _ \ | | / _ \ | | _ / _ \ | | | ' / "
echo -e "| |_| / ___ \| |/ ___ \ | |_| |/ ___ \| | | . \ "
echo -e "|____/_/ \_\_/_/ \_\ \____/_/ \_\_| |_|\_\ "
echo -e " "
echo -e '\n \tAnalyze Covariates for PDF plots comparation of the Recalibration GATK\n'
echo -e "Comparisson among the bias of the sequencer errors"
echo ············································································································
mkdir $MDAP/plot_recalibration_data
echo 'mkdir plot_recalibration_data' >> registerFile
echo 'Starts GATK Second Recalibration'
echo 'Starts GATK Second Recalibration' >> registerFile
#GATK BaseRecalibration second table for next step AnalyzeCovariates.
#Generates the second pass table.
#instead of second table, we use the BAM created by ApplyBQSR to regenrate a new TABLE for plot.
for i in $@
do
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar BaseRecalibrator \
-I $ABQSRD/applied_bqsr_data_$i.bam \
-R $HG19/ucsc.hg19.fasta \
--known-sites $HG19/dbsnp_138.hg19.vcf.gz \
--known-sites $HG19/1000G_phase1.indels.hg19.sites.vcf.gz \
--known-sites $HG19/Mills_and_1000G_gold_standard.indels.hg19.sites.vcf.gz \
-O $PRD/after_recalibrated_bqsr_data_$i.recal.table
#second bqsr table for comparation.
done
echo 'Second recalibration GATK table --> DONE'
#Full generating the Plots of the recalibration tables using AnalyzeCovariates and saving a csv copy.
#Analyze the tables.
echo -e '\n Generating Plots, pdf and csv files'
for i in $@
do
echo -e '\nGenerating the files for '$i' starting...'
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar AnalyzeCovariates \
-before $RBQSRD/before_recalibrated_bqsr_data_$i.recal.table \
-after $PRD/after_recalibrated_bqsr_data_$i.recal.table \
-csv $PRD/BQSR_$i.csv \
-plots $PRD/AnalyzeCovariates_bqsr_$i.pdf
echo '\nPlot and CSV file for '$i' is DONE!'
done
echo -e '\n Plots files generated --> DONE'
#Obtaining an CSV and PDF file of the comparrisson between first and second pass of the recalibration applied to the bam fi$
echo ············································································································
echo -e " _ _ _ ____ _ ___ _______ ______ _____ "
echo -e "| | | | / \ | _ \| | / _ \_ _\ \ / / _ \| ____|"
echo -e "| |_| | / _ \ | |_) | | | | | || | \ V /| |_) | _| "
echo -e "| _ |/ ___ \| __/| |__| |_| || | | | | __/| |___ "
echo -e "|_| |_/_/ \_\_| |_____\___/ |_| |_| |_| |_____|"
echo -e " "
echo -e " ____ _ _ _ _____ ____ "
echo -e " / ___| / \ | | | | | ____| _ \ "
echo -e "| | / _ \ | | | | | _| | |_) | "
echo -e "| |___ / ___ \| |___| |___| |___| _ < "
echo -e " \____/_/ \_\_____|_____|_____|_| \_\ "
echo -e " "
echo -e '\n \tHAPLOTYPE CALLER GATK\n'
echo ············································································································
#Ready to call for Variants.
mkdir $MDAP/haplotype_caller_gvcf_data
echo 'mkdir haplotype_caller_gvcf_data' >> registerFile
for i in $@
do
#HaplotypeCaller for each sample for later joint genotyping.
echo -e '\nGATK HaplotypeCallerGVCF for '$i' STARTS'
echo -e '\nGATK HaplotypeCallerGVCF for '$i' STARTS'>> registerFile
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar HaplotypeCaller \
-R $HG19/ucsc.hg19.fasta \
-I $ABQSRD/applied_bqsr_data_$i.bam \
-ERC GVCF \
-bamout $HCGVCFD/HC_bamout_$i.bam \
-O $HCGVCFD/HC_data_$i.g.vcf \
-G StandardAnnotation \
-G AS_StandardAnnotation \
-G StandardHCAnnotation \
-A FisherStrand -A StrandOddsRatio -A RMSMappingQuality -A MappingQualityRankSumTest -A ReadPosRankSumTest -A DepthPerSampleHC -A BaseQualityRankSumTest -A ExcessHet -A StrandArtifact \
--annotate-with-num-discovered-alleles=true
echo -e '\nGATK HaplotypeCallerGVCF ERC GVCF for '$i' DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nGATK HaplotypeCallerGVCF ERC GVCF for '$i' DONE' >> registerFile
done
#If more than one input the pipeline will continue the Joint analysis.
if [ $# -gt 1 ]
then
mkdir $MDAP/combined_gvcf_data
echo 'mkdir combined_gvcf_data'>> registerFile
# #Obtenido en GCVF pasamos al Joint Genotyping on one or more samples called with HC.
#
echo -e '\nUsing GATK COMBINEGVCFs for merging GVCFs'
echo -e '\nUsing GATK COMBINEGVCFs for merging GVCFs'>> registerFile
echo -e " ____ ___ __ __ ____ ___ _ _ _____ ______ ______ _____ ____ "
echo -e " / ___/ _ \| \/ | __ )_ _| \ | | ____| / ___\ \ / / ___| ___/ ___| "
echo -e " | | | | | | |\/| | _ \| || \| | _| | | _ \ \ / / | | |_ \___ \ "
echo -e " | |__| |_| | | | | |_) | || |\ | |___ | |_| | \ V /| |___| _| ___) | "
echo -e " \____\___/|_| |_|____/___|_| \_|_____| \____| \_/ \____|_| |____/ "
# #Switch to the directory containing the Haplotypecaller generated gvcfs and obtaining a list use CombineGVCFS
# #only works like this or by separated.
cd $HCGVCFD ; ls *.g.vcf > my_list_of_gvcfs_files_to_combine.list
# #List obtained
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar CombineGVCFs \
-R $HG19/ucsc.hg19.fasta \
--variant $HCGVCFD/my_list_of_gvcfs_files_to_combine.list \
-O $CGVCFD/combined.g.vcf
echo ············································································································
echo -e " _ ___ ___ _ _ _____ "
echo -e " | |/ _ \_ _| \ | |_ _|"
echo -e " _ | | | | | || \| | | | "
echo -e "| |_| | |_| | || |\ | | | "
echo -e " \___/ \___/___|_| \_| |_| "
echo -e " "
echo -e " ____ _ _ ___ _______ ______ ___ _ _ ____ "
echo -e " / ___| \ | |/ _ \_ _\ \ / / _ \_ _| \ | |/ ___|"
echo -e "| | _| \| | | | || | \ V /| |_) | || \| | | _ "
echo -e "| |_| | |\ | |_| || | | | | __/| || |\ | |_| |"
echo -e " \____|_| \_|\___/ |_| |_| |_| |___|_| \_|\____|"
echo -e " "
echo -e '\n \tJOINT GENOTYPING (GATK)\n'
echo ············································································································
echo -e '\nGATK COMBINEGVCFs DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nGATK COMBINEGVCFs DONE' >> registerFile
mkdir $MDAP/genotyped_vcf_data
echo 'mkdir genotyped_data_vcf'>> registerFile
#GenotypeGVCFs into final VCF
echo -e '\nUsing GATK GenotypeGVCFs for final VCF'
echo -e " ____ _____ _ _ ___ _______ ______ _____ ____ "
echo -e " / ___| ____| \ | |/ _ \_ _\ \ / / _ \| ____| _ \ "
echo -e " | | _| _| | \| | | | || | \ V /| |_) | _| | | | | "
echo -e " | |_| | |___| |\ | |_| || | | | | __/| |___| |_| | "
echo -e " \____|_____|_| \_|\___/ |_| |_| |_| |_____|____/ "
echo -e " "
echo -e " ____ ___ __ __ ____ ___ _ _ _____ ____ ______ ______ _____ ____ "
echo -e " / ___/ _ \| \/ | __ )_ _| \ | | ____| _ \ / ___\ \ / / ___| ___/ ___| "
echo -e " | | | | | | |\/| | _ \| || \| | _| | | | | | | _ \ \ / / | | |_ \___ \ "
echo -e " | |__| |_| | | | | |_) | || |\ | |___| |_| | | |_| | \ V /| |___| _| ___) | "
echo -e " \____\___/|_| |_|____/___|_| \_|_____|____/ \____| \_/ \____|_| |____/ "
echo -e " "
echo -e " __ __ _ _ _ _____ ___ "
echo -e " | \/ | | | | | |_ _|_ _| "
echo -e " | |\/| | | | | | | | | | "
echo -e " | | | | |_| | |___| | | | "
echo -e " |_| |_|\___/|_____|_| |___| "
echo -e " "
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar GenotypeGVCFs \
-R $HG19/ucsc.hg19.fasta \
-V $CGVCFD/combined.g.vcf \
-O $GVCFD/genotyped_data.vcf
bash $GENOTYPE_SCRIPT
echo -e '\nGATK GenotypeGVCFs DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nGATK GenotypeGVCFs DONE' >> registerFile
#if only one INPUT continue to joint analysis.
else
mkdir $MDAP/genotyped_vcf_data
echo 'mkdir genotyped_data_vcf'>> registerFile
echo -e " ____ _____ _ _ ___ _______ ______ _____ ____ "
echo -e " / ___| ____| \ | |/ _ \_ _\ \ / / _ \| ____| _ \ "
echo -e " | | _| _| | \| | | | || | \ V /| |_) | _| | | | | "
echo -e " | |_| | |___| |\ | |_| || | | | | __/| |___| |_| | "
echo -e " \____|_____|_| \_|\___/ |_| |_| |_| |_____|____/ "
echo -e " "
echo -e " ____ ___ __ __ ____ ___ _ _ _____ ____ ______ ______ _____ ____ "
echo -e " / ___/ _ \| \/ | __ )_ _| \ | | ____| _ \ / ___\ \ / / ___| ___/ ___| "
echo -e " | | | | | | |\/| | _ \| || \| | _| | | | | | | _ \ \ / / | | |_ \___ \ "
echo -e " | |__| |_| | | | | |_) | || |\ | |___| |_| | | |_| | \ V /| |___| _| ___) | "
echo -e " \____\___/|_| |_|____/___|_| \_|_____|____/ \____| \_/ \____|_| |____/ "
echo -e " "
echo -e " ____ ___ _ ___ "
echo -e " / ___| / _ \| | / _ \ "
echo -e " \___ \| | | | | | | | | "
echo -e " ___) | |_| | |__| |_| | "
echo -e " |____/ \___/|_____\___/ "
echo -e " "
#GenotypeGVCFs into final VCF
echo -e '\nUsing GATK GenotypeGVCFs for final VCF'
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar GenotypeGVCFs \
-R $HG19/ucsc.hg19.fasta \
-V $HCGVCFD/HC_data_$1.g.vcf \
-G StandardAnnotation \
-O $GVCFD/genotyped_data.vcf
echo -e '\nGATK GenotypeGVCFs DONE' ;paplay /usr/share/sounds/freedesktop/stereo/complete.oga
echo -e '\nGATK GenotypeGVCFs DONE' >> registerFile
fi
echo ············································································································
echo -e " _ _ _ ____ ____ _____ ___ _ _____ _____ ____ ___ _ _ ____ "
echo -e "| | | | / \ | _ \| _ \ | ___|_ _| | |_ _| ____| _ \|_ _| \ | |/ ___|"
echo -e "| |_| | / _ \ | |_) | | | | | |_ | || | | | | _| | |_) || || \| | | _ "
echo -e "| _ |/ ___ \| _ <| |_| | | _| | || |___| | | |___| _ < | || |\ | |_| |"
echo -e "|_| |_/_/ \_\_| \_\____/ |_| |___|_____|_| |_____|_| \_\___|_| \_|\____|"
echo -e " "
echo -e "\n \tHard filtering if less than 30 samples and doing it in the classical way, selecting variants\n"
echo ············································································································
#HARD FILTERING
#First step extacting the SNP's
#Second step extracting the INDEL's
mkdir $MDAP/variant_filtration_vcf_data
echo "variantfiltration_data_vcf ">> registerFile
#1.Extract the SNP's from the call set.
echo "Extract the SNP's from the call set."
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar SelectVariants \
-R $HG19/ucsc.hg19.fasta \
-V $GVCFD/genotyped_data.vcf \
--select-type-to-include SNP \
-O $VFVCFD/selected_raw_snp.vcf
#Creates the selected_raw_snps vcf containing just the SNP's from the original callset.
#2.Apply the filters to the SNP's callset.
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar VariantFiltration \
-R $HG19/ucsc.hg19.fasta \
-V $VFVCFD/selected_raw_snp.vcf \
--filter-expression "QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0" \
--filter-name "my_SNP_filter" \
-O $VFVCFD/filtered_SNP_data.vcf
#3. Extract the INDELS from the ORIGINAL call set.
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar SelectVariants \
-R $HG19/ucsc.hg19.fasta \
-V $GVCFD/genotyped_data.vcf \
--select-type-to-include INDEL \
-O $VFVCFD/selected_raw_indels.vcf
#Creates the selected_raw_indels vcf containing just the INDEL's from the original callset.
#4.Apply the filters to the INDEL's callset.
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar VariantFiltration \
-R $HG19/ucsc.hg19.fasta \
-V $VFVCFD/selected_raw_indels.vcf \
--filter-expression "QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0" \
--filter-name "my_INDEL_filter" \
-O $VFVCFD/filtered_INDEL_data.vcf
#Filtered INDELS and SNPS of the original file.
#Combine Variants after using SNPS and INDELS filtering into a single file and get it ready for annotation.
java -jar $SFT/gatk/build/libs/gatk-package-4.0.6.0-22-g9d9484f-SNAPSHOT-local.jar MergeVcfs \
-R $HG19/ucsc.hg19.fasta \
-I $VFVCFD/filtered_SNP_data.vcf \
-I $VFVCFD/filtered_INDEL_data.vcf \
-O $VFVCFD/filtered_INDEL_SNP_data.vcf
echo ············································································································
echo -e "Filtering QUALITY,chrM,chrUn,my_SNP_filter and my_INDEL_filter out of the VCF file"
echo ············································································································
awk 'BEGIN {FS=OFS="\t"}{if ($1 ~/^#/){print $0} else if ($1 ~/^chrM.*/ || $1 ~/^chrUn.*/ || $7 ~/my_SNP_filter/ || $7 ~/my_INDEL_filter/ || $6 < 100) {print ""} else {print $0}}' $VFVCFD/filtered_INDEL_SNP_data.vcf > $VFVCFD/test; gawk 'NF > 0' $VFVCFD/test > $VFVCFD/pre_filtered_ready_to_annotate.vcf
echo ············································································································
echo -e "__ ___ ____ ___ _ _ _ _____ "
echo -e "\ \ / / \ | _ \|_ _| / \ | \ | |_ _|"
echo -e " \ \ / / _ \ | |_) || | / _ \ | \| | | | "
echo -e " \ V / ___ \| _ < | | / ___ \| |\ | | | "
echo -e " \_/_/ \_\_| \_\___/_/ \_\_| \_| |_| "
echo -e " "
echo -e " _ _ _ _ _ ___ _____ _ _____ ___ ___ _ _ "
echo -e " / \ | \ | | \ | |/ _ \_ _|/ \|_ _|_ _/ _ \| \ | |"
echo -e " / _ \ | \| | \| | | | || | / _ \ | | | | | | | \| |"
echo -e " / ___ \| |\ | |\ | |_| || |/ ___ \| | | | |_| | |\ |"
echo -e "/_/ \_\_| \_|_| \_|\___/ |_/_/ \_\_| |___\___/|_| \_|"
echo -e " "
echo -e "\n \tVARIANT ANNOTATION (VEP ENSEMBL)\n"
echo ············································································································
mkdir $MDAP/vep_vcf_annotated_data
echo ············································································································
echo -e '\nFilter by popuplation frequencies\n'
echo ············································································································
#RUN vep
#················································································································
perl $VEP --everything --allele_number --cache --offline --dir_plugins $PLUGIN_DIR \
--dir $VEP_CACHE --v --assembly GRCh37 --fork 12 --fasta $VEP_FASTA \
--force_overwrite --symbol --canonical --sift b --polyphen b --af_1kg --af_gnomad --af_esp --af \
--ccds --protein --uniprot --hgvs --pubmed --biotype --regulatory --numbers --domains \
--gene_phenotype --max_af --variant_class --filter_common --force_overwrite \
--plugin LoF,human_ancestor_fa:${LOFTEE},$LOFTEE_PATH \
--plugin dbNSFP,$DBNSFP,ALL \
--vcf --fields "VARIANT_CLASS,Location,Uploaded_variation,Allele,Gene,Feature,Feature_type,BIOTYPE,SYMBOL,Consequence,CANONICAL,LoF_flags,LoF_filter,LoF,CDS_position,HGVSc,HGVSp,HGVSg,SIFT,PolyPhen,CADD_raw,FATHMM_pred,GERP++_RS,GERP++_NR,SWISSPROT,PUBMED,CLIN_SIG,FILTER,cDNA_position,EUR_AF,gnomAD_exomes_AF,gnomAD_genomes_AF,1000Gp3_AF,1000Gp3_EUR_AF,ExAC_EAS_AF" \
-i $VCF_IN -o $VCF_OUT
echo "Filtering population frequencies and Consequences out of the VCF/TSV file"
awk 'BEGIN{FS=OFS="\t"}{
if ($1 ~/^#/)
{
print $0
}
else if (($30 < 0.0100 || $30 == " ") && ($31 < 0.0100 || $31 == " ") && ($32 < 0.0100 || $32 == " ") && ($33 < 0.0100 || $33 == " ") && ($34 < 0.0100 || $34 == " ") && ($35 < 0.0100 || $35 == " "))
{
print $0
}
}' $VCF_OUT > pre-filtered.vcf
echo -e '\n VEP prefiltered VCF file ready in the MDAP directory'
echo -e 'DONE'