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Old 09-10-2013, 09:41 AM   #1
Location: Milwaukee

Join Date: Dec 2011
Posts: 72
Default CuffDiff cannot opn BAM file made in TopHat

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 \
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 \
gwilymh is offline   Reply With Quote
Old 09-11-2013, 04:12 PM   #2
Junior Member
Location: Kansas

Join Date: Jun 2012
Posts: 1
Default Help!! Cuffdiff cannot open alignment file for reading

Hi gwilymh!

I am also getting the exact same error message that you've gotten when trying to run cuffdiff

open: No such file or directory
File /Volumes/Data/2013-08-20_Gp_Cell_Cycle_Transcriptome/Olson_Samples_Run2/SampleXp1/accepted_hits.bam doesn't appear to be a valid BAM file, trying SAM...
Error: cannot open alignment file /Volumes/Data/2013-08-20_Gp_Cell_Cycle_Transcriptome/Olson_Samples_Run2/SampleXp1/accepted_hits.bam for reading

My accepted_hits.bam file was generated in Tophat v2.0.8. I used Cufflinks v2.1.1 but got an error when I tried to use cuffmerge, so I used a slightly older version of Cuffmerge (v2.0.2) to merge my transcript.gtf files generated by Cufflinks (I can provide this error message later if anyone is interested).

I have successfully used previous versions of the Tuxedo software to analyze my RNA-seq data (Tophat v2.0.8, Bowtie, and Cufflinks v2.0.2), so maybe it is a bug with the new version of Cufflinks?

I did notice that the version of samtools has changed to, where the analyses that I've done with the previous Tuxedo software used samtools Could it also be a samtools issue?

If anyone else is getting this error message, please help us!!!
tmarria is offline   Reply With Quote
Old 09-12-2013, 03:04 AM   #3
Devon Ryan
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480

The first thing to check is if you can "samtools view" the accepted_hits.bam file. If not, it was likely corrupted at some point. gwilymh's problem might be a bug in cuffdiff, it'd be good to make stripped down BAM and annotation files and see if the error persists.

I should note that the initial "open: No such file or directory" error would suggest that the path for the accepted_hits.bam file is simply being misspecified.

Last edited by dpryan; 09-12-2013 at 03:05 AM. Reason: I should really proof read before posting!
dpryan is offline   Reply With Quote

bam file, cuffdiff, cufflinks, cuffmerge, tophat

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