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Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
08-May-2018, 11:35 AM
Post: #1
Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
Hi,
I did a "Differential Translation Analysis" under RiboseqR (samples are aligned to yeast transcriptome). I notice for the same gene ID, some of the genes are shown more than once in the list, e.g., there are more than one “YEL071W” in "Riboseq file 1" with read counts shown as “6”, “33”, "79", .....and “23”. (Please kindly see the snapshot of the data in the attachment). Are they coming from multiple segments of "start codons (ATG) - stop codons (TAG, TAA, TGA)"? I read the publication of RiboseqR, but I cannot figure out how should I process for TE calculation for each Gene since I have multiple outputs for the same gene, shall I take the sum (sum =”6” + “33” + “79”+...+"23") for “YEL071W”? Or these counts are corresponding to different length ORFs of “YEL071W” (If in this case, where can I find the corresponding length of these ORFs?)

Thanks a lot for your advice!


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08-May-2018, 12:43 PM
Post: #2
RE: Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
Hi,

It is not clear why your output counts contains several row entries for the same gene ID. Are there several isoform entries for e.g. “YEL071W” in your transcriptome FASTA file?

Would you mind sending an email to ribogalaxy@gmail.com so that we can check on your RiboGalaxy history and try to determine why there are several entries for some genes?

Thanks,
Audrey
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08-May-2018, 02:21 PM
Post: #3
RE: Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
(08-May-2018 12:43 PM)audrey Wrote:  Hi,

It is not clear why your output counts contains several row entries for the same gene ID. Are there several isoform entries for e.g. “YEL071W” in your transcriptome FASTA file?

Would you mind sending an email to ribogalaxy@gmail.com so that we can check on your RiboGalaxy history and try to determine why there are several entries for some genes?

Thanks,
Audrey
Thanks Audrey! I just send the transcriptome FASTA file and send the history link to ribogalaxy@gmail.com. Thanks for your support!
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13-Jun-2018, 06:10 PM
Post: #4
RE: Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
Hi,
I'm using ribogalaxy docker and I tried to run a differential translation analysis for my control and treatment samples, that I have put together in the same Prepare RiboSeqR input. I have put both Control and treatment (RiboSeq and RNASeq alignments) to the same Prepare RiboSeqR input. Triplet periodicity gave me a graph for only the control, but Metagene analysis gave me plots for both control and treatment. When I run differential translation I get the following error:
Fatal error: Exit code 1 (Error)
Traceback (most recent call last):
File "/shed_tools/toolshed.ribogalaxy.ucc.ie/repos/ribogalaxy/riboseqr_wrapper/7b9cb1091196/riboseqr_wrapper/riboseqr/difftrans.py", line 150, in <module>
html_file=args.html_file, output_path=args.output_path)
File "/shed_tools/toolshed.ribogalaxy.ucc.ie/repos/ribogalaxy/riboseqr_wrapper/7b9cb1091196/riboseqr_wrapper/riboseqr/difftrans.py", line 70, in get_counts
run_rscript(cmd)
File "/shed_tools/toolshed.ribogalaxy.ucc.ie/repos/ribogalaxy/riboseqr_wrapper/7b9cb1091196/riboseqr_wrapper/riboseqr/difftrans.py", line 20, in run_rscript
output = R(command)
File "/export/tool_deps/rpy2/2.3.10/ribogalaxy/riboseqr_wrapper/7b9cb1091196/lib/python/rpy2/robjects/__init__.py", line 240, in __call__
res = self.eval(p)
File "/export/tool_deps/rpy2/2.3.10/ribogalaxy/riboseqr_wrapper/7b9cb1091196/lib/python/rpy2/robjects/functions.py", line 86, in __call__
return super(SignatureTranslatedFunction, self).__call__(*args, **kwargs)
File "/export/tool_deps/rpy2/2.3.10/ribogalaxy/riboseqr_wrapper/7b9cb1091196/lib/python/rpy2/robjects/functions.py", line 35, in __call__
res = super(Function, self).__call__(*new_args, **new_kwargs)
rpy2.rinterface.RRuntimeError: Error in .local(.Object, ...) :
All vectors in '@groups' slot must equal number of columns of '@data' slot.

Any ideas what I'm doing wrong? I'd really appreciate any feedback

Thanks a lot,

Katerina
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14-Sep-2018, 05:01 PM
Post: #5
RE: Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
Hi everyone,
so I managed to run the Differential Translation analysis -and I tested it only with one biological replicate of Control and Mutant for a start- but I get weird results in the TopCounts output. I'm attaching a screenshot of what I get but basically the numbers in the Control column don't make sense as they seem to be really low and do not correlate with the number of RiboCounts and RNACounts that look normal.

Has that happened to anybody before? Any ideas???

Thanks for any feedback!!


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21-Sep-2018, 02:56 PM
Post: #6
RE: Differential analysis(RiboseqR): Multiple read counts for the same gene identifier
Hi Katerina,

Did you check the 'Normalisation' option in the BaySeq tool in RiboSeqR for the differential expression analysis? If so, this would account for differences bewteen RiboCounts counts and the output of BaySeq.
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