Question: how to merge multiple cufflink data in one file
0
gravatar for Amalendu.Ghosh
15 months ago by
Amalendu.Ghosh10 wrote:

how to merge multiple cufflink data in one file. what i am expecting is rows will represent gene name and column will represent the FPKM values. i tried cuffdiff too. and all the time i am getting error: Error: atal error: Matched on error [13:22:33] Loading reference annotation. [13:22:36] 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 i am using mm10 gtf file for annotation from i genome. please let me know what is the fix! Please!

rna-seq • 747 views
ADD COMMENTlink modified 14 months ago • written 15 months ago by Amalendu.Ghosh10
1
gravatar for Jennifer Hillman Jackson
15 months ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

The root problem might be a genome mismatch problem. Was the data mapped within Galaxy against the mm10 natively indexed genome with HISAT? (or optionally, Tophat - which has been deprecated and is not recommended, or RNA STAR - which does not have indexes yet but will soon, by end of the week is the plan).

If the genome mapped against is not exactly the same as that in the other input datasets, this kind of result can occur (with any tool - or it may just manifest as an "exceeding compute resources" error).

CuffMerge or Stringtie Merge are the tools to use with Cufflinks/Stringtie output (gtf) and an optional reference GTF (example: iGenomes) to produce a merged GTF result.

Cuffdiff will give these warnings if the XS attribute is not present in the input BAM datasets (example: if Bowtie was used). Using HISAT will avoid the problem.

Support: https://galaxyproject.org/support/ (see Troubleshooting)

Tutorials: https://galaxyproject.org/learn/ (see RNA-seq)

Thanks! Jen, Galaxy team

ADD COMMENTlink written 15 months ago by Jennifer Hillman Jackson25k
1
gravatar for Amalendu.Ghosh
14 months ago by
Amalendu.Ghosh10 wrote:

Hi Jen Thanks for following up! i used mm10 iGenomes GTF file with the tool along with the natively indexed mm10 genome/and mm10 genome uploaded by me which is actually came from igenome. the problem remains. can i send you the link so that you can see what is happening! thanksenter link description here

https://usegalaxy.org/?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fdevteam%2Fsamtools_mpileup%2Fsamtools_mpileup%2F2.1.3&version=2.1.3&__identifer=ihyzg3byzpm

ADD COMMENTlink written 14 months ago by Amalendu.Ghosh10
0
gravatar for Amalendu.Ghosh
15 months ago by
Amalendu.Ghosh10 wrote:

Firs of all Thank you Jennifer! yes i did alignment using in built mm10 genome and then did the cufflink using the igenome gtf file which i uploaded into the system. lets try to upload mm10 genome from i genome and use that as refference genome with HISAT. I am not using UCSC annotation file because that does not produce gene name! Thanks for your suggestion. very helpful! Amalendu

ADD COMMENTlink written 15 months ago by Amalendu.Ghosh10

If the mm10 iGenomes GTF file was used with the tool along with the natively indexed mm10 genome at http://usegalaxy.org, then the mismatch problem is unlikely to be the root problem. The iGenomes file to use is mm10 "genes.gtf" (from the UCSC archive). This is different than using a gtf from the UCSC Table Browser which does not have the extra attributes needed by the tool, as you noticed. This may be what you have done - I just wanted to clarify that we are discussing the same file :)

Re-sorting the HISAT BAM dataset should not be needed (if you originally mapped with HISAT - it is not clear), but you could try that if it was. I also suggest double checking that mm10 was actually used as the target genome when mapping. Sometimes the wrong database is accidentally selected on the tool form.

If you cannot determine the problem and are working at http://usegalaxy.org or can reproduce the problem there, a bug report can be sent in and we can help to troubleshoot. Be sure to leave the input and output datasets undeleted and include a link to this post so we can associate the two.

Help:

ADD REPLYlink written 15 months ago by Jennifer Hillman Jackson25k
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