Variant Calling Pipeline using GATK4

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Identifying genomic variants using next-generation sequencing data, such as single nucleotide polymorphisms (SNPs) and DNA insertions and deletions (indels), is an important aspect of scientific discovery. This pipeline was created to help researchers reliably and quickly discover and annotate sequence variants. The pipeline is based on the Broad Institute's recommended best practices for variant discovery analysis and uses the Genome Analysis Toolkit 4 (GATK4) to perform variant calling. SnpEff is employed to annotate and predict the effects of variants after SNPs have been detected. This pipeline is designed for calling variants in clonal samples, or samples from a single person. In these samples, the frequency of variations is estimated to be 1 (for haploids or homozygous diploids) or 0.5 (for heterozygous diploids).

Code Snippets

    """
    bwa mem \
	-K 100000000 \
	-v 3 \
	-t ${task.cpus} \
	-Y \
	-R \"${readGroup}\" \
	$ref \
	${reads[0]} \
	${reads[1]} \
	> ${pair_id}_aligned_reads.sam
    """

NextFlow BWA From line 48 of master/main.nf

    """
    mkdir -p ${params.tmpdir}/${workflow.runName}/${pair_id}
    gatk --java-options "-Djava.io.tmpdir=${params.tmpdir}/${workflow.runName}/${pair_id}" \
	 MarkDuplicatesSpark \
	-I $aligned_reads \
	-M ${pair_id}_dedup_metrics.txt \
	-O ${pair_id}_sorted_dedup.bam 
    rm -r ${params.tmpdir}/${workflow.runName}/${pair_id}
    """ 

NextFlow gatk From line 82 of master/main.nf

    """
    java -jar \$PICARD_JAR \
        CollectAlignmentSummaryMetrics \
	R=${params.ref} \
        I=${sorted_dedup_reads} \
	O=${pair_id}_alignment_metrics.txt
    java -jar \$PICARD_JAR \
        CollectInsertSizeMetrics \
        INPUT=${sorted_dedup_reads} \
	OUTPUT=${pair_id}_insert_metrics.txt \
        HISTOGRAM_FILE=${pair_id}_insert_size_histogram.pdf 
    samtools depth -a ${sorted_dedup_reads} > ${pair_id}_depth_out.txt
    """

NextFlow SAMtools From line 111 of master/main.nf

    """
    gatk HaplotypeCaller \
	-R $ref \
	-I $input_bam \
	-O ${pair_id}_raw_variants_${round}.vcf \
    """

NextFlow gatk From line 147 of master/main.nf

    """
    gatk SelectVariants \
	-R $ref \
	-V $raw_variants \
	-select-type SNP \
	-O ${pair_id}_raw_snps_${round}.vcf
    gatk SelectVariants \
        -R $ref \
        -V $raw_variants \
        -select-type INDEL \
        -O ${pair_id}_raw_indels_${round}.vcf
    """

NextFlow gatk From line 173 of master/main.nf

    """
    gatk VariantFiltration \
	-R $ref \
	-V $raw_snps \
	-O ${pair_id}_filtered_snps_${round}.vcf \
	-filter-name "QD_filter" -filter "QD < 2.0" \
	-filter-name "FS_filter" -filter "FS > 60.0" \
	-filter-name "MQ_filter" -filter "MQ < 40.0" \
	-filter-name "SOR_filter" -filter "SOR > 4.0" \
	-filter-name "MQRankSum_filter" -filter "MQRankSum < -12.5" \
	-filter-name "ReadPosRankSum_filter" -filter "ReadPosRankSum < -8.0"
    """

NextFlow gatk From line 203 of master/main.nf

    """
    gatk VariantFiltration \
        -R $ref \
        -V $raw_indels \
        -O ${pair_id}_filtered_indels_${round}.vcf \
	-filter-name "QD_filter" -filter "QD < 2.0" \
	-filter-name "FS_filter" -filter "FS > 200.0" \
	-filter-name "SOR_filter" -filter "SOR > 10.0"
    """

NextFlow gatk From line 233 of master/main.nf

    """
    echo "New Round: " $new_round
    gatk SelectVariants \
	--exclude-filtered \
	-V $filtered_snps \
	-O ${pair_id}_bqsr_snps.vcf
    gatk SelectVariants \
        --exclude-filtered \
        -V $filtered_indels \
        -O ${pair_id}_bqsr_indels.vcf
    gatk BaseRecalibrator \
	-R $ref \
	-I $input_bam \
	--known-sites ${pair_id}_bqsr_snps.vcf \
	--known-sites ${pair_id}_bqsr_indels.vcf \
	-O ${pair_id}_recal_data.table
    gatk ApplyBQSR \
        -R $ref \
        -I $input_bam \
        -bqsr ${pair_id}_recal_data.table \
        -O ${pair_id}_recal.bam
    gatk BaseRecalibrator \
        -R $ref \
	-I ${pair_id}_recal.bam \
        --known-sites ${pair_id}_bqsr_snps.vcf \
	--known-sites ${pair_id}_bqsr_indels.vcf \
	-O ${pair_id}_post_recal_data.table
    """	

NextFlow gatk From line 287 of master/main.nf

    """
    gatk AnalyzeCovariates \
	-before $recal_table \
	-after $post_recal_table \
	-plots ${pair_id}_recalibration_plots.pdf
    """

NextFlow gatk From line 329 of master/main.nf

    """
    java -jar \$SNPEFF_JAR -v \
	-dataDir $params.snpeff_data \
	$params.snpeff_db \
	$filtered_snps > ${pair_id}_filtered_snps.ann.vcf
    """

NextFlow From line 351 of master/main.nf

"""
parse_metrics.sh ${pair_id} > ${pair_id}_report.csv
"""

NextFlow From line 382 of master/main.nf

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Created: 1yr ago

Updated: 1yr ago

Maitainers: public

URL: https://gencore.bio.nyu.edu/variant-calling-pipeline-gatk4/

Name: variant-calling-pipeline-gatk4

Version: 1

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License: GNU General Public License v3.0

Keywords:

Indel detection SNP annotation Variant calling gatk BWA SAMtools DNA

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