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stuck: triplet periodicity
08-Feb-2016, 05:00 PM
Post: #1
stuck: triplet periodicity
Hello,

I am trying to work on some riboseq data and trying to use the ribosome analysis triplet periodicity function but, I always get a graph with null values for all variables. I have read previous posts on these problems and I think I have seen to those problems. Any suggestions would be greatly appreciated.

P.S my history is shared on data. My data is from S. cerevisae and I am using the fasta and gff3 files from ENSEMBL.
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08-Feb-2016, 05:29 PM
Post: #2
RE: stuck: triplet periodicity
Hi there,

The error is likely originating from the spaces in your fasta headers (>I space dna ... etc) because in your SAM file, the alignments are to the chromosomes (i.e. the entries in your fasta headers before the first space).

Could you try removing all spaces/text after the chromosome number in your fasta headers and re-running the Prepare riboSeqR input and the Triplet Periodicity tool with the modified fasta file (it should not be necessary to re-align your raw data).

If it is necessary to keep all the text in the fasta headers, you will need to join the text with e.g. the underscore character so that there are no spaces in the fasta headers. You will then need to re-align your raw data to the modified fasta file.

If you can let us know if this resolves your issue?

Kind Regards,
Audrey
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21-Apr-2017, 06:15 AM (This post was last modified: 21-Apr-2017 07:24 AM by jjb91.)
Post: #3
RE: stuck: triplet periodicity
(08-Feb-2016 05:29 PM)audrey Wrote:  Hi there,

The error is likely originating from the spaces in your fasta headers (>I space dna ... etc) because in your SAM file, the alignments are to the chromosomes (i.e. the entries in your fasta headers before the first space).

Could you try removing all spaces/text after the chromosome number in your fasta headers and re-running the Prepare riboSeqR input and the Triplet Periodicity tool with the modified fasta file (it should not be necessary to re-align your raw data).

If it is necessary to keep all the text in the fasta headers, you will need to join the text with e.g. the underscore character so that there are no spaces in the fasta headers. You will then need to re-align your raw data to the modified fasta file.

If you can let us know if this resolves your issue?

Kind Regards,
Audrey

Hello,

I am stuck with a similar issue. I have aligned my processed reads to the SacCer3 transcriptome fasta file I obtained from UCSC (a screen shot is attached). When I run the sam file through riboSeqR step 1, I receive a file that has strand sense, gene name, number, then the read. When I pass the R file through the triplet periodicity software, I receive a graph with 0 values. I was hoping for some guidance in fixing this issue.

EDIT: Please disregard. I figured out how to remove the spacer sequences as suggested, and it has solved my problem. Smile

EDIT 2: For future reference, if anyone lacking sufficient coding skills is having a similar issue they can fix the transcriptomic file with the following Python 2.7 code.

Code:
#### Opens a fasta file and removes any characters seperated by a space in the
#### identifier line

NewFasta = open('sacCer3-transcriptome_fixed.fasta', 'w')
#new file to write to

with open('sacCer3-transcriptome.fasta') as file:
    #original file that needs to be fixed
    for line in file:
        if line.startswith('>'):
            identifier = line.split(' ')[0]
            #split the line at spaces and store only the gene name
            NewFasta.write(identifier + '\n')
            #write the fixed line to the new file
        else:
            NewFasta.write(line)
            #write the sequence-containing lines to the new file

NewFasta.close()

EDIT 3: I am receiving an error for the Metagene analysis that states "Please check if the correct RDA file is selected". I am selecting the file generated from step 2.


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21-Apr-2017, 09:04 AM
Post: #4
RE: stuck: triplet periodicity
Hello,
I saw your post, but it was not quite clear to me if you still have a problem re your EDIT 3.
Anyhow Step 1 is a preparation step to obtain files with the correct input format.
Step 2 produces a Triplet Periodicity (R data file) which you should then use for Step 3.
Probably you have to select this file from the drop down as there is also another file Triplet Periodicity (HTML report) produced by Step 2.
If you are still having an issue with this, please contact us again.

Kind Regards
jamesp
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21-Apr-2017, 01:43 PM
Post: #5
RE: stuck: triplet periodicity
(21-Apr-2017 09:04 AM)jamesp Wrote:  Hello,
I saw your post, but it was not quite clear to me if you still have a problem re your EDIT 3.
Anyhow Step 1 is a preparation step to obtain files with the correct input format.
Step 2 produces a Triplet Periodicity (R data file) which you should then use for Step 3.
Probably you have to select this file from the drop down as there is also another file Triplet Periodicity (HTML report) produced by Step 2.
If you are still having an issue with this, please contact us again.

Kind Regards
jamesp

Hi James,

Thanks for the response. The error I am receiving is in a snapshot I provided below. It occurs below the drop-down arrow. I am selecting the file generated from step 2.


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21-Apr-2017, 04:21 PM
Post: #6
RE: stuck: triplet periodicity
Hello,
I can see the issue you are having but have not seen this case previously.
Please let us know if you repeated all the steps right from the beginning including mapping (alignment) after you uploaded the fixed fasta file (sacCer3-transcriptome_fixed.fasta).
If you can reassure us you did that, then we will need to make a more detailed analysis which will take more time, and if you could provide us the names of the input files to start the analysis, that would be much appreciated.

Kind Regards
jamesp
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22-Apr-2017, 02:01 AM
Post: #7
RE: stuck: triplet periodicity
(21-Apr-2017 04:21 PM)jamesp Wrote:  Hello,
I can see the issue you are having but have not seen this case previously.
Please let us know if you repeated all the steps right from the beginning including mapping (alignment) after you uploaded the fixed fasta file (sacCer3-transcriptome_fixed.fasta).
If you can reassure us you did that, then we will need to make a more detailed analysis which will take more time, and if you could provide us the names of the input files to start the analysis, that would be much appreciated.

Kind Regards
jamesp

Thank you. Redoing the alignment with the fixed transcriptome solved the problem.
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05-Sep-2017, 08:53 AM
Post: #8
RE: stuck: triplet periodicity
Hi there,
I'm trying to run the triplet periodicity function but when I put my "Prepare RiboSeqR input" (the R data file) and set the parameters it just does not seem to recognise the file and keeps saying "Please check if the correct RDA file is selected". This happens a lot, and the "Prepare RiboSeq input" files seem to be ok, no problem is reported. Has anyone had the same issue?

Thanks for any ideas..I'm really stuck..
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05-Sep-2017, 03:12 PM
Post: #9
RE: stuck: triplet periodicity
Hi Katerina-D,
I am only aware of this issue when files were downloaded and uploaded again later. The issue usually arises because the file has the wrong name or wrong extension. So perhaps if you changed the name of the file this error would occur. If that is your case, I suggest you try processing from one step to the next without changing any of the attributes to see if that works better for you. And then you could rename the files afterwards (for example if you want to download them or to be able to identify them at at later stage).
Let me know if this helps.

Kind Regards
jamesp
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