Question: Cuffdiff crashing suddenly
0
gravatar for VY
17 months ago by
VY120
United States
VY120 wrote:

Hello,

I'm running cuffdiff and this error comes up when it runs.

Fatal error: Matched on error
[01:53:06] Loading reference annotation.
Warning: No conditions are replicated, switching to 'blind' dispersion method
[01:53:14] Inspecting maps and determining fragment length distributions.
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute

( ### omitted a large number of identical lines ###)


Processed 34319 loci.                       
Performed 7764 isoform-level transcription difference tests
Performed 7543 tss-level transcription difference tests
Performed 6910 gene-level transcription difference tests
Performed 0 CDS-level transcription difference tests
Performed 0 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests
Writing isoform-level FPKM tracking
Writing TSS group-level FPKM tracking
Writing gene-level FPKM tracking
Writing CDS-level FPKM tracking
Writing isoform-level count tracking
Writing TSS group-level count tracking
Writing gene-level count tracking
Writing CDS-level count tracking
Writing isoform-level read group tracking
Writing TSS group-level read group tracking
Writing gene-level read group tracking
Writing CDS-level read group tracking
Writing read group info
Writing run info
Loading required package: BiocGenerics
Loading required package: parallel

Attaching package: 'BiocGenerics'

The following objects are masked from 'package:parallel':

    clusterApply, clusterApplyLB, clusterCall, clusterEvalQ,
    clusterExport, clusterMap, parApply, parCapply, parLapply,
    parLapplyLB, parRapply, parSapply, parSapplyLB

The following object is masked from 'package:stats':

    xtabs

The following objects are masked from 'package:base':

    Filter, Find, Map, Position, Reduce, anyDuplicated, append,
    as.data.frame, as.vector, cbind, colnames, do.call, duplicated,
    eval, evalq, get, intersect, is.unsorted, lapply, mapply, match,
    mget, order, paste, pmax, pmax.int, pmin, pmin.int, rank, rbind,
    rep.int, rownames, sapply, setdiff, sort, table, tapply, union,
    unique, unlist, unsplit

Loading required package: RSQLite
Loading required package: DBI
Loading required package: ggplot2
Loading required package: reshape2
Loading required package: fastcluster

Attaching package: 'fastcluster'

The following object is masked from 'package:stats':

    hclust

Loading required package: rtracklayer
Loading required package: GenomicRanges
Loading required package: S4Vectors
Loading required package: stats4
Loading required package: IRanges
Loading required package: GenomeInfoDb
Loading required package: Gviz
Loading required package: grid

Attaching package: 'cummeRbund'

The following object is masked from 'package:GenomicRanges':

    promoters

The following object is masked from 'package:IRanges':

    promoters

The following object is masked from 'package:BiocGenerics':

    conditions

Creating database ./cummeRbund.sqlite
Reading Run Info File ./run.info
Writing runInfo Table
Reading Read Group Info  ./read_groups.info
Writing replicates Table
Reading Var Model Info  ./var_model.info
Writing varModel Table
Reading ./genes.fpkm_tracking
Checking samples table...
Populating samples table...
Writing genes table
Reshaping geneData table
Recasting
Writing geneData table
Reading ./gene_exp.diff
Writing geneExpDiffData table
Reading ./promoters.diff
Writing promoterDiffData table
Reading ./genes.count_tracking
Reshaping geneCount table
Recasting
Writing geneCount table
Reading read group info in ./genes.read_group_tracking
Writing geneReplicateData table
Reading ./isoforms.fpkm_tracking
Checking samples table...
OK!
Writing isoforms table
Reshaping isoformData table
Recasting
Writing isoformData table
Reading ./isoform_exp.diff
Writing isoformExpDiffData table
Reading ./isoforms.count_tracking
Reshaping isoformCount table
Recasting
Writing isoformCount table
Reading read group info in ./isoforms.read_group_tracking
Writing isoformReplicateData table
Reading ./tss_groups.fpkm_tracking
Checking samples table...
OK!
Writing TSS table
Reshaping TSSData table
Recasting
Writing TSSData table
Reading ./tss_group_exp.diff
Writing TSSExpDiffData table
Reading ./splicing.diff
Writing splicingDiffData table
Reading ./tss_groups.count_tracking
Reshaping TSSCount table
Recasting
Writing TSSCount table
Reading read group info in ./tss_groups.read_group_tracking
Writing TSSReplicateData table
Reading ./cds.fpkm_tracking
Checking samples table...
OK!
Writing CDS table
No records found in ./cds.fpkm_tracking
CDS FPKM tracking file was empty.
Reading ./cds_exp.diff
No records found in ./cds_exp.diff
Reading ./cds.diff
No records found in ./cds.diff
Reading ./cds.count_tracking
No records found in ./cds.count_tracking
Reading read group info in ./cds.read_group_tracking
No records found in ./cds.read_group_tracking
Indexing Tables...
Warning messages:
1: attributes are not identical across measure variables; they will be dropped 
2: attributes are not identical across measure variables; they will be dropped 
3: attributes are not identical across measure variables; they will be dropped 
4: attributes are not identical across measure variables; they will be dropped 
5: attributes are not identical across measure variables; they will be dropped 
6: attributes are not identical across measure variables; they will be dropped

My first question is if anyone knows why this is happening and secondly,

I can still look at the tabular file though for the differential gene expression testing and it still has all the genes and if they are significant or not and their calculated FPKM values? Can I still trust that?

Thank you :)

rna-seq chip-seq cuffdiff • 645 views
ADD COMMENTlink written 17 months ago by VY120
2

Hi Val, Are you setting a 'library type' in Cuffdiff? A similar error was reported on BioStars (https://www.biostars.org/p/118904/) and setting the 'library type' in Cuffdiff fixed the issue in that case.

Cheers, Mo Heydarian

ADD REPLYlink written 17 months ago by Mo Heydarian790

Val, I see the re-run going with the Advanced Settings: Library prep parameter now set (it was not originally - thanks Mo!). If you have time, please let us know if this works or not. Jen

ADD REPLYlink written 17 months ago by Jennifer Hillman Jackson23k

Dear Mo and Jen,

I've tried re-running it using the library prep parameter but the same error has come up unfortunately. Val

ADD REPLYlink written 17 months ago by VY120

Ok, thanks Val. Our admin is looking into this. I'll move all this back to a comment until there is a definitive explanation.

I am also doing some other quick checks on your data, but I doubt that is a factor from my review so far.

Jen

ADD REPLYlink written 17 months ago by Jennifer Hillman Jackson23k

Hi Val,

How did you align your data?

Cheers, Mo Heydarian

ADD REPLYlink written 17 months ago by Mo Heydarian790

Hi Mo,

It was alligned using HISAT.

Val

ADD REPLYlink written 17 months ago by VY120

Hi Val,

Did you set the 'library type' (unstranded, firststrand, secondstrand) when you ran HISAT?

'Library type' is the Tophat/Cuffdiff nomenclature, HISAT refers to this as 'specify strand specific information' (under the 'spliced alignment parameters' options).

If I run HISAT without assigning the 'specify strand specific information' and run Cuffdiff on the output I get the same error you are seeing. When I run HISAT and assign the correct 'specify strand specific information' parameter, I no longer see errors.

Cheers, Mo

ADD REPLYlink modified 17 months ago • written 17 months ago by Mo Heydarian790

Hi Mo,

Thanks for all the suggestions! I'm not sure why this keeps happening. I had originally uploaded the bam files directly after aligning the data locally. Having uploaded the fastq files and aligning within galaxy has made the error disappear even though all of the parameters are the same. No idea why this might be happening, hopefully Jen might be able to let us know.

Thanks for all the help though! Val

ADD REPLYlink written 17 months ago by VY120

Hello - I haven't seen this exact message before. Where are you using Galaxy?

If at http://usegalaxy.org, or the error can be reproduced there, please submit a bug report following these instructions. Include a link to this Biostars posts in the comments.

If elsewhere, please share more about that: another public site (URL?), local (what Galaxy version?), or Cloudman?

Thanks, Jen, Galaxy team

ADD REPLYlink written 17 months ago by Jennifer Hillman Jackson23k

Hi Jen,

Thanks for getting back to me. Yes it's on the public galaxy server. I've Submitted the bug report and added in the link to this thread.

thanks Val

ADD REPLYlink written 17 months ago by VY120
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