Cuffdiff Results Missing

Question: Cuffdiff Results Missing

6.4 years ago by

i b • 330

i b • 330 wrote:

Hi, I ran cuffdiff using Refseq genes as GTF and two groups of BAM. Group one has two replicates (treated) and group two only one replicate (untreated). When looking at the outputs the following are empty (1 line): TSS group FPKM tracking TSS groups differential expression testing CDS FPKM tracking CDS FPKM differential expression testing CDS overloading diffential expression testing promoters differential expression testing splicing differential expression testing the other four outputs have data downloadable as excel. Is this normal? thanks, ib

rna-seq cuffdiff • 2.8k views

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modified 6.4 years ago by Jennifer Hillman Jackson ♦ 25k • written 6.4 years ago by i b • 330

6.4 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello Irene, Yes, this is can be the result if your source GTF data did not have the full compliment of attributes needed by Cuffdiff to perform these calculations. The primary tool documentation covers this information here: http://cufflinks.cbcb.umd.edu/manual.html#fpkm_track The iGenomes datasets are a popular choice for this reason. A version of UCSC RefGenes is available for certain genomes. Please see: http://cufflinks.cbcb.umd.edu/igenomes.html (scroll down on page in some browsers to find table) Galaxy has one of these already loaded, mm9 genes.gtf, in the "Shared Data -> Shared Libraries" section on the public Main server. More iGenomes .gtf files will likely be added here, sometime after the GCC2012 conference. For now, locally download to your own system/desktop, uncompress, and just load the GTF file to Galaxy. Consider FTP for larger datasets: http://wiki.g2.bx.psu.edu/FTPUpload) More resources include the author supported help email at tophat.cufflinks@gmail.com and seqanswers.com (where the authors often post). Hopefully this helps, Jen Galaxy team -- Jennifer Jackson http://galaxyproject.org

ADD COMMENT • link written 6.4 years ago by Jennifer Hillman Jackson ♦ 25k

6.4 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello Irene, This issue is similar to the original. The input GTF for this run (dataset #15) has tss_id populated, but not p_id. The p_id attribute is required for the CDS calculations. http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff_input (quote) Cuffdiff Input: Attribute Description p_id The ID of the coding sequence this transcript contains. This attribute is attached by Cuffcompare to the .combined.gtf records only when it is run with a reference annotation that include CDS records. Further, differential CDS analysis is only performed when all isoforms of a gene have p_id attributes, because neither Cufflinks nor Cuffcompare attempt to assign an open reading frame to transcripts. Dataset #15 was created from a CuffMerge run (which runs Cuffcompare as a component). Examining the selections used (clicking on the blue arrow "rerun" icon), shows that the option "Use Sequence Data:" was set to "No". Changing this to "Yes" and using "Choose the source for the reference list:" as "Locally cached" (and double checking that all inputs are assigned to hg19) will assign p_id. Note that this will be true only for those transcripts that are associated with reference annotation transcripts containing coding regions (in your data: 'nearest_ref "NM_XXXXX"', not "NR_XXXXX". NR_ human RefSeq transcripts are non-coding). Galaxy's CuffMerge tool form has this option labeled: (quote) Use Sequence Data: Use sequence data for some optional classification functions, including the addition of the p_id attribute required by Cuffdiff. Thanks! Jen Galaxy team -- Jennifer Jackson http://galaxyproject.org

ADD COMMENT • link written 6.4 years ago by Jennifer Hillman Jackson ♦ 25k

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