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.
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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 2.1.0.0, 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 0.1.19.0, where the analyses that I've done with the previous Tuxedo software used samtools 0.1.18.0. Could it also be a samtools issue?
If anyone else is getting this error message, please help us!!!Leave a comment:
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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 \
/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
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Differential Expression and Data Visualization: Recommended Tools for Next-Level Sequencing Analysis
After covering QC and alignment tools in the first segment and variant analysis and genome assembly in the second segment, we’re wrapping up with a discussion about tools for differential gene expression analysis and data visualization. In this article, we include recommendations from the following experts: Dr. Mark Ziemann, Senior Lecturer in Biotechnology and Bioinformatics, Deakin University; Dr. Medhat Mahmoud Postdoctoral Research Fellow at Baylor College of Medicine;...05-23-2023, 12:26 PM -
Continuing from our previous article, we share variant analysis and genome assembly tools recommended by our experts Dr. Medhat Mahmoud, Postdoctoral Research Fellow at Baylor College of Medicine, and Dr. Ming "Tommy" Tang, Director of Computational Biology at Immunitas and author of From Cell Line to Command Line.
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With new tools and computational resources being released regularly, it can be hard to determine which are best suited for the analysis process and which older tools continue to be maintained. In an effort to assist the sequencing community, we interviewed three highly skilled bioinformaticians about their recommended tools for several important analysis applications.
Quality control and preprocessing tools
“Garbage in, garbage out” is a popular...05-16-2023, 10:11 AM
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