Paired-End Reads and Variant Calling
I am trying to call variants from paired-read whole genome reseq data. I have two fastq files (forward and reverse) for each sample. I got to the MergeBamAlignment command and no matter what I try, I cannot get the file to generate. The code itself runs in Linux, it is just my mergealignment.bam file never appears so I cannot proceed to the next step. Does anyone have any advice?
Things I've tried: I've used ValidateSamFile to make sure all of my files have no errors and particularly making sure that the Read Groups were included at each stage. I tried using RevertSam to revert my .sam files to unaligned files, however, I cannot get a RevertSam file to generate either.
a) GATK version used =188.8.131.52
b) Exact GATK commands used
#Generate the .sam files (one for each read direction)
../scripts/Read_Mapping_softwares/bwa/bwa mem -M -t 40 ../scripts/Read_Mapping_softwares/ref_bwa/Sbicolor_454_v3.0.1.fa read1.fq read2.fq > paired_aligned_reads_bwa_mem.sam
#The code below is where I can run the code but it does not generate the output file.
java -Xmx16G -jar picard.jar MergeBamAlignment ALIGNED=paired_aligned_reads_bwa_mem.sam UNMAPPED=unaligned_reads.bam O=paired_aligned_reads_mergebamalignment.bam R=../scripts/Read_Mapping_softwares/ref_bwa/Sbicolor_454_v3.0.1.fa CREATE_INDEX=true CLIP_ADAPTERS=false INCLUDE_SECONDARY_ALIGNMENTS=true MAX_INSERTIONS_OR_DELETIONS=-1 PRIMARY_ALIGNMENT_STRATEGY=BestMapq ATTRIBUTES_TO_RETAIN=XS CLIP_OVERLAPPING_READS=true
Take a look at this workspace in Terra that provides readymade workflows to convert fastq and bam to ubam: https://app.terra.bio/#workspaces/help-gatk/Sequence-Format-Conversion
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