I am running a number of Tuxedo pipelines, starting with TopHat and ending in CuffDiff. Most of these pipelines work fine. However, I encountered the following error in CuffDiff for one of my pipelines:
open: No such file or directory
File /gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam doesn't appear to be a valid BAM file, trying SAM...
Error: cannot open alignment file /gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam for reading
I have checked over the input file carefully, and cannot find any errors in the file paths.
The accepted_hits.bam file was made in TopHat. I have tried remaking it in TopHat and get the same result. I have looked over it in SamTools and can see no obvious errors.
The only thing that separates this pipelines from my others is that I am using a gff file (made in TopHat from a Flybase GFF3 file) that contains exon and CDS data. Other GFF files with different data combinations seem to work (e.g. exons only, CDS + UTR). Has anyone encountered a similar problem? Is this a bug in CuffDiff?
My full CuffDiff and CuffMerge pipeline is below.
module load tophat/2.0.9
module load bowtie/2.1.0
module load cufflinks/2.1.1
cuffmerge \
-o /gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/Test_ARDM1005 \
-g /gpfs/group1/f/flyinv/working_index/ExonCDS_.gff \
-s /gpfs/group1/f/flyinv/RNASeq/Dpse3_0.fasta \
/gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/CuffLinks_Output_GTF_List_ARDM1005.txt
cuffdiff \
-o "/gpfs/group1/f/flyinv/Outputs_CuffDiff/exonCDS/Test_ARDM1005" \
-L AR_DM1005_Male,AR_DM1005_Female \
--total-hits-norm \
--frag-bias-correct /gpfs/group1/f/flyinv/working_index/Dpse3_0_1.fa \
--multi-read-correct \
--library-norm-method classic-fpkm \
/gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/Test_ARDM1005/merged.gtf \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_ExonCDS/AR_DM1005_Male/accepted_hits.bam \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam
open: No such file or directory
File /gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam doesn't appear to be a valid BAM file, trying SAM...
Error: cannot open alignment file /gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam for reading
I have checked over the input file carefully, and cannot find any errors in the file paths.
The accepted_hits.bam file was made in TopHat. I have tried remaking it in TopHat and get the same result. I have looked over it in SamTools and can see no obvious errors.
The only thing that separates this pipelines from my others is that I am using a gff file (made in TopHat from a Flybase GFF3 file) that contains exon and CDS data. Other GFF files with different data combinations seem to work (e.g. exons only, CDS + UTR). Has anyone encountered a similar problem? Is this a bug in CuffDiff?
My full CuffDiff and CuffMerge pipeline is below.
module load tophat/2.0.9
module load bowtie/2.1.0
module load cufflinks/2.1.1
cuffmerge \
-o /gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/Test_ARDM1005 \
-g /gpfs/group1/f/flyinv/working_index/ExonCDS_.gff \
-s /gpfs/group1/f/flyinv/RNASeq/Dpse3_0.fasta \
/gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/CuffLinks_Output_GTF_List_ARDM1005.txt
cuffdiff \
-o "/gpfs/group1/f/flyinv/Outputs_CuffDiff/exonCDS/Test_ARDM1005" \
-L AR_DM1005_Male,AR_DM1005_Female \
--total-hits-norm \
--frag-bias-correct /gpfs/group1/f/flyinv/working_index/Dpse3_0_1.fa \
--multi-read-correct \
--library-norm-method classic-fpkm \
/gpfs/group1/f/flyinv/Outputs_CuffMerge/exonCDS/Test_ARDM1005/merged.gtf \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_ExonCDS/AR_DM1005_Male/accepted_hits.bam \
/gpfs/group1/f/flyinv/Outputs_TopHat/transcriptomeSequence_exonCDS/AR_DM1005_Female/accepted_hits.bam
Comment