I’m having a similar issue with DexSeq to count differential exon usage. Source files from DexSeq-Count and GFT annotate look fine. I get this error:
Fatal error: Error in the R script.
Tool generated the following standard error:
Warning message:
In Sys.setlocale(“LC_MESSAGES”, “en_US.UTF-8”) :
OS reports request to set locale to “en_US.UTF-8” cannot be honored
Hi @ken67
You can share more details of your analysis and we can try to help.
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Consider Sharing your History or posting content from the Job Information 
view as described in Troubleshooting errors.
Downloaded SRA files and mapped with Galaxy RNA-STAR using the D. melanogaster genome 6.46 FASTA and 6.46.110 gtf file.
Generated a flattened annotation file of dm 6.46.110 using ‘annotation’ mode of DEXSeq-COUNT, which was then paired with STAR .bam output files with ‘count’ mode of DEXSeq-COUNT. Output files were two columns, exon:counts, as expected.
Problem arises when I use the prepared annotation of the GTF to analyze differential exon usage between the two DEXSeq count files.
Error messages:
[1] “” genotype dataset_b4e0c04f-dbbc-4deb-af5d-2507eadd5078.dat 2_tai dataset_fcebf0ac-4b39-4701-87fb-e94b03ecd359.dat 1_gfp ~sample + exon + genotype:exon ~sample + exon [1] “genotype” [1] "/jetstr
and
Fatal error: An undefined error occured, please check your input carefully and contact your administrator.
Fatal error: Error in the R script.
Tool generated the following standard error:
Warning message:
In Sys.setlocale("LC_MESSAGES", "en_US.UTF-8") :
OS reports request to set locale to "en_US.UTF-8" cannot be honored
Warning message:
package 'DEXSeq' was built under R version 4.2.3
OK, thanks for explaining.
Would you please send in a bug report from the error? Use the bug icon in any of the red output datasets to send that in.
We made a few changes at the UseGalaxy.org server earlier this week, and this looks like a server problem we should address. If you could put the link to this topic post in the comments field, that would be helpful.
Sure.
An error occurred while running the tool toolshed.g2.bx.psu.edu/repos/iuc/dexseq/dexseq/1.44+galaxy0.
Execution resulted in the following messages:
Fatal error: An undefined error occured, please check your input carefully and contact your administrator.
Fatal error: Error in the R script.
Tool generated the following standard error:Warning message:
In Sys.setlocale(“LC_MESSAGES”, “en_US.UTF-8”) :
OS reports request to set locale to “en_US.UTF-8” cannot be honored
Warning message:
package ‘DEXSeq’ was built under R version 4.2.3
converting counts to integer mode
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Warning message:
In DESeqDataSet(rse, design, ignoreRank = TRUE) :
some variables in design formula are characters, converting to factors
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, ‘’ and ‘.’. It is recommended (but not required) to use
only letters, numbers, and delimiters '’ or ‘.’, as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Error: BiocParallel errors
8 remote errors, element index: 1, 2, 3, 4, 5, 6, …
0 unevaluated and other errors
first remote error:
Error in estimateDispersionsGeneEst(x, maxit = maxit, quiet = quiet, modelMatrix = modelMatrix, : the number of samples and the number of model coefficients are equal,
i.e., there are no replicates to estimate the dispersion.
use an alternate design formula
The wrapper is trying to “guess” how to reformat the labels, but you could clean those up yourself. There is much discussion about those at this forum in context of other Bioconductor tools, and it usually helps.
Most Bioconductor tools also require replicates. That comes directly from the tool authors and how they wrote the tools.
FAQ: Extended Help for Differential Expression Analysis Tools
It is easier to review everything in context, especially when looking at someone else’s experiment! But address those two items first anyway. If you run into more problems, share back the history or actually send in the bug report please.
Thanks. Very helpful. I’ll try to clean up the inputs.
One issue that I may not be able to fix: I only have single replicates of the exon count datasets to input into DEXSeq. Can I configure preferences to accommodate this?
Thanks. Helpful
OK, I obtained a second replicate of DEXSeq-count data and ran DEXSeq analysis. All the errors disappeared but this one:
Error in rownames<-(*tmp*, value = lf[[1]][, 1]) :
attempt to set ‘rownames’ on an object with no dimensions
Calls: DEXSeqDataSetFromHTSeq → rownames<- → rownames<-
Do I just need to add named headers to the “gene_ID:exon” and “count” columns?
Hi @ken67
Yes, probably. Be sure to format those in the same simple way as the other labels. Each should be unique.