Hello,
I am working with a paired-end data set of rhesus macaque reads aligned with STAR against a current rhesus .fasta reference and .gtf annotation. I am interested in doing some quality control measures on my .bam file with RNAseqC to determine numbers of reads that map to non-genic, exonic, or intronic regions.
For now, I'm trying to bypass the command-line execution and opting to perform the run on the web-based GenePattern interface offered by Broad institute. For those familiar, there are a number of inputs required for RNAseqC, all of which I believe that I have provided:
-indexed .bam with read groups created with Picard tools -.gtf file -indexed .fasta -dictionary of .fasta created with Picard tools
Unfortunately there is no accompanying 18S rRNA information for rhesus I am able to provide.
Anyways, the program seems to complete successfully, however for all of my transcripts I get this message in the output:
WARNING: Transcript has no coverage: ACTB_transcript_01
This is peculiar to me, because if I load the .bam, .gtf, and .fasta in IGV, there are clearly an abundant number of reads mapping to the ACTB gene.
I am wondering why RNAseqC is not picking up any reads to exons, or anything really. If anyone has encountered similar issues it would be greatly appreciated. From what I have seen, the chromosomes are annotated the same in both the .gtf and .bam file.
thank you!
For what it's worth, I'm getting the following ERRORs in the output log when running the GATK depth of coverage analysis:
Running GATK Depth of Coverage Analysis ....
Arguments: -T DepthOfCoverage -R ref_input/MacaM_Rhesus_Genome_v7.fasta -I bam_input/JB37_sorted_ReadGroup.bam -o .//JB37_sorted_ReadGroup.bam/lowexpr//perBaseDoC.out -L .//JB37_sorted_ReadGroup.bam/lowexpr/intervals.list -l ERROR
Arguments Array: [-T, DepthOfCoverage, -R, ref_input/MacaM_Rhesus_Genome_v7.fasta, -I, bam_input/JB37_sorted_ReadGroup.bam, -o, .//JB37_sorted_ReadGroup.bam/lowexpr//perBaseDoC.out, -L, .//JB37_sorted_ReadGroup.bam/lowexpr/intervals.list, -l, ERROR]
GATK command result code: 0
Depth of Coverage run time: 1 min
... GATK Depth of Coverage Analysis DONE
Running GATK Depth of Coverage Analysis ....
Arguments: -T DepthOfCoverage -R ref_input/MacaM_Rhesus_Genome_v7.fasta -I bam_input/JB37_sorted_ReadGroup.bam -o .//JB37_sorted_ReadGroup.bam/medexpr//perBaseDoC.out -L .//JB37_sorted_ReadGroup.bam/medexpr/intervals.list -l ERROR
Arguments Array: [-T, DepthOfCoverage, -R, ref_input/MacaM_Rhesus_Genome_v7.fasta, -I, bam_input/JB37_sorted_ReadGroup.bam, -o, .//JB37_sorted_ReadGroup.bam/medexpr//perBaseDoC.out, -L, .//JB37_sorted_ReadGroup.bam/medexpr/intervals.list, -l, ERROR]
GATK command result code: 0
Depth of Coverage run time: 2 min
... GATK Depth of Coverage Analysis DONE
Running GATK Depth of Coverage Analysis ....
Arguments: -T DepthOfCoverage -R ref_input/MacaM_Rhesus_Genome_v7.fasta -I bam_input/JB37_sorted_ReadGroup.bam -o .//JB37_sorted_ReadGroup.bam/highexpr//perBaseDoC.out -L .//JB37_sorted_ReadGroup.bam/highexpr/intervals.list -l ERROR
Arguments Array: [-T, DepthOfCoverage, -R, ref_input/MacaM_Rhesus_Genome_v7.fasta, -I, bam_input/JB37_sorted_ReadGroup.bam, -o, .//JB37_sorted_ReadGroup.bam/highexpr//perBaseDoC.out, -L, .//JB37_sorted_ReadGroup.bam/highexpr/intervals.list, -l, ERROR]
GATK command result code: 0
Depth of Coverage run time: 7 min
... GATK Depth of Coverage Analysis DONE