Variant Calling in NA12878 was performed using the GATK best practices workflow.

Fastq files were obtained from the following link https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR5665260 To download the files I installed SRAtoolkit on the server and used the fasterq dump command:

fasterq-dump SRR5665260

Reference genome was downloaded from the following link: https://console.cloud.google.com/storage/browser/genomics-public-data/resources/broad/hg38/v0;tab=objects?prefix=&forceOnObjectsSortingFiltering=false The fasta file, .fai file, .dict, dbsnp file, known indel sites were also downloaded.

GTF file was downloaded from Gencode: https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_39/gencode.v39.annotation.gtf.gz

Input

SRR5665260_1.fastq,
SRR5665260_2.fastq

Command

fastp -i SRR5665260_1.fastq -I SRR5665260_2.fastq -o SRR5665260_1.fastp.fastq -O SRR5665260_2.fastp.fastq --detect_adapter_for_pe 

Output

SRR5665260_1.fastp.fastq,
SRR5665260_2.fastp.fastq,
fastp.html,
fastp.json

Input

resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta,# reference genome
gencode_v39_annotation.gtf #gtf file

Command

STAR --runThreadN 6 --runMode genomeGenerate --genomeDir reference_genome_hg38_index/ --genomeFastaFiles reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta --sjdbGTFfile gtf_files/gencode_v39_annotation.gtf --sjdbOverhang 143

Output

genome Dir

Input

genomeDir,
SRR5665260_1.fastp.fastq,
SRR5665260_2.fastp.fastq

Command

nohup STAR --runThreadN 6 --genomeDir reference_genome_hg38_index/ --readFilesIn SRR5665260_1.fastp.fastq SRR5665260_2.fastp.fastq --sjdbOverhang 143 --twopassMode Basic --outSAMtype BAM SortedByCoordinate --outFileNamePrefix NA12878_RNA & 

Output

NA12878_RNAAligned.sortedByCoord.out.bam #bam file sorted by coordinate

Input

NA12878_RNAAligned.sortedByCoord.out.bam

Command

gatk MarkDuplicates --INPUT NA12878_RNAAligned.sortedByCoord.out.bam --OUTPUT NA12878_RNAAligned.sortedByCoord.out_deduped.bam --CREATE_INDEX true --VALIDATION_STRINGENCY SILENT --METRICS_FILE NA12878_RNA_duplicates.metrics &

Output

NA12878_RNAAligned.sortedByCoord.out_deduped.bam, \ #deduplicated bam file NA12878_RNAAligned.sortedByCoord.out_deduped.bai, \ #deduplicated bam file index NA12878_RNA_duplicates.metrics #metrics

Input

reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta, #reference genome
NA12878_RNAAligned.sortedByCoord.out_deduped.bam #deduplicated bam file

Command

nohup gatk SplitNCigarReads -R reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta -I NA12878_RNAAligned.sortedByCoord.out_deduped.bam -O NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar.bam &

Output

NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar.bam,
NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar.bai

Input

reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta,
/workspace/RNAseq_data/NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp.bam,
/workspace/RNAseq_data/gatk_resource_bundle/hg38/Homo_sapiens_assembly38.dbsnp138.vcf.gz -known-sites,
/workspace/RNAseq_data/gatk_resource_bundle/hg38/Homo_sapiens_assembly38.known_indels.vcf.gz

Command

gatk BaseRecalibrator -R /workspace/RNAseq_data/reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta -I /workspace/RNAseq_data/NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp.bam --use-original-qualities -O NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal.bam -known-sites /workspace/RNAseq_data/gatk_resource_bundle/hg38/Homo_sapiens_assembly38.dbsnp138.vcf.gz -known-sites /workspace/RNAseq_data/gatk_resource_bundle/hg38/Homo_sapiens_assembly38.known_indels.vcf.gz &

Output

NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal.bam

Input

/workspace/RNAseq_data/reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta,
/workspace/RNAseq_data/NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp.bam,
NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal.bam

Command

nohup gatk ApplyBQSR --add-output-sam-program-record -R /workspace/RNAseq_data/reference_genome_hg38/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta -I /workspace/RNAseq_data/NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp.bam --use-original-qualities -O NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal_2.bam --bqsr-recal-file NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal.bam &

Output

NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal_2.bam,
NA12878_RNAAligned.sortedByCoord.out_deduped_postSplitNCigar_withReadGrp_recal_2.bai