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Hi,
I am a medical student new to NGS. I'm currently trying to count the tumor mutational load from tumor samples (FFPE) to which I have no matched normal tissue. We are using a gene panel of less than 70 genes.
I have got the fastq files (2 files per sample, paired-end) and I tried to write a script in bash to generate recalibrated bam files ready for use with mutect2.
However, when I received the output vcf file from mutect2, there are some mutations but there is no information in CHROM, ID, FILTER and QUAL column.
I am attaching the commands I used. I suppose there might be a problem with alignment, but I failed to recognize where the problem is. As a complete beginner, I suppose maybe I do some kind of systemic error.
I would be grateful for any kind of hint. THanks.
I am a medical student new to NGS. I'm currently trying to count the tumor mutational load from tumor samples (FFPE) to which I have no matched normal tissue. We are using a gene panel of less than 70 genes.
I have got the fastq files (2 files per sample, paired-end) and I tried to write a script in bash to generate recalibrated bam files ready for use with mutect2.
However, when I received the output vcf file from mutect2, there are some mutations but there is no information in CHROM, ID, FILTER and QUAL column.
I am attaching the commands I used. I suppose there might be a problem with alignment, but I failed to recognize where the problem is. As a complete beginner, I suppose maybe I do some kind of systemic error.
I would be grateful for any kind of hint. THanks.
Code:
bwa index -a bwtsw hs37d5.fa | samtools faidx hs37d5.fa | picard CreateSequenceDictionary R=hs37d5.fa O=hs37d5.dict bwa mem -M ~/projects/patologie/hs37d5/hs37d5.fa ~/projects/patologie/01_raw_data/BRCA1_S1_L001_R1_001.fastq ~/projects/patologie/01_raw_data/BRCA1_S1_L001_R2_001.fastq > sample.sam samtools view -bS sample.sam > sample.bam picard SortSam I=sample.bam O=sorted_sample.bam SORT_ORDER=coordinate picard MarkDuplicates I=sorted_sample.bam O=sample_marked.bam M=marked_metrics.txt ASSUME_SORT_ORDER=coordinate picard AddOrReplaceReadGroups I=sample_marked.bam O=sample_rg.bam RGID=1 RGLB=lib1 RGPL=illumina RGPU=unit1 RGSM=1 gatk IndexFeatureFile -F= ~/projects/patologie/reference/1000G_phase1.indels.b37.vcf gatk IndexFeatureFile -F= ~/projects/patologie/reference/Mills_and_1000G_gold_standard.indels.b37.vcf gatk IndexFeatureFile -F= ~/projects/patologie/reference/dbsnp_138.b37.vcf gatk BaseRecalibrator -R ~/projects/patologie/hs37d5/hs37d5.fa -I sample_rg.bam --known-sites ~/projects/patologie/reference/1000G_phase1.indels.b37.vcf --known-sites ~/projects/patologie/reference/dbsnp_138.b37.vcf --known-sites ~/projects/patologie/reference/Mills_and_1000G_gold_standard.indels.b37.vcf -O recal_table gatk ApplyBQSR -bqsr recal_table -I sample_rg.bam -R ~/projects/patologie/hs37d5/hs37d5.fa -O sample_recal.bam gatk IndexFeatureFile -F= ~/projects/patologie/reference/af-only-gnomad.raw.sites.b37.vcf gatk Mutect2 -R ~/projects/patologie/hs37d5/hs37d5.fa -I ~/projects/patologie/analysis2/sample_recal.bam -tumor 1 -O sample_single.vcf --germline-resource ~/projects/patologie/reference/af-only-gnomad.raw.sites.b37.vcf