Can you share some of the image as text for easier understanding?
It seems like there might be an issue with your BAM file or the region you are trying to call variants on. To help diagnose the issue, please follow these steps:
1. Check if your BAM file is sorted and indexed:
samtools sort -o test_sorted.bam test.bam
samtools index test_sorted.bam
2. Check if your reference genome (hg19_GRCh37_p13_full.fasta) is indexed for freebayes:
samtools faidx hg19_GRCh37_p13_full.fasta
3. Run freebayes again with the sorted and indexed BAM file:
software/freebayes_v0.9.21-19-gc003c1e/freebayes/bin/freebayes --use-duplicate-reads --min-alternate-count 2 --min-alternate-fraction 0.01 --min-coverage 5 --use-mapping-quality -b test_sorted.bam -f hg19_GRCh37_p13_full.fasta -v test.vcf
4. If you still encounter the error, try to inspect the BAM file to see if there are any alignments:
samtools view test_sorted.bam | head
If you see alignments in the BAM file but still encounter the error with freebayes, it might be helpful to narrow down the region you are trying to call variants on. You can use the -r option in freebayes to specify a specific region, for example:
software/freebayes_v0.9.21-19-gc003c1e/freebayes/bin/freebayes --use-duplicate-reads --min-alternate-count 2 --min-alternate-fraction 0.01 --min-coverage 5 --use-mapping-quality -b test_sorted.bam -f hg19_GRCh37_p13_full.fasta -r chr1:100000-200000 -v test.vcf
Replace chr1:100000-200000 with the region you are interested in. If this works, it might indicate that the issue is specific to certain regions in your BAM file.
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Thank you Sasha for the steps to diagnose. You were right, there was an issue with the bam file alignment.