Hi, Another approach you can try is to use DESeq or EdgeR from Bioconductor to assess differential expression.
I personally like these two methods LOTS better than Cuff* mainly because they are a lot closer to tried and true statistical methods developed for microarrays. I esp. like how both methods let you test different factors. For example, if you are testing a treatment (drug or no drug) and a genotype (mutant vs. wildtype) you can find out which genes' expression depends on having a wild-type copy of the gene by testing an "interaction term." Both methods start with simple counts - numbers of reads overlapping annotated genes. Probably there is a Galaxy workflow that can calculate counts of reads per gene, but I don't know if Galaxy currently incorporates R/Bioconductor tools. If you can get Galaxy to calculate reads per gene, then you can then download the file and run it through edgeR or DESeq. R is free but it does take some time to master it. But it is incredibly powerful and well worth the effort! To get started with R, I recommend doing the free-of-charge O'Reilly Press "try R" tutorial which is on-line here: http://tryr.codeschool.com/ I hope this will be helpful! Best wishes, Ann Loraine ------------------------------- Ann Loraine, Ph.D. Associate Professor Department of Bioinformatics and Genomics University of North Carolina at Charlotte North Carolina Research Campus 600 Laureate Way Kannapolis, NC 28081 704-250-5750 alora...@uncc.edu http://www.transvar.org http://www.bioviz.org http://www.uncc.edu On 1/7/13 8:27 PM, "Jennifer Hillman-Jackson" <j...@bx.psu.edu> wrote: >Hello Wei, > >The contents of the reference GTF files (original, before analysis) will >probably provide some explanation. My guess is that GTF files have >different contents and are not directly comparable - RefSeq with full >transcripts and Ensembl with full transcripts + potentially partial >predictions and/or predicted splice sites. Alternative versions of each >may be available. When possible, you most likely will want to be using a >reference GTF file that represents complete transcripts. > >I don't know what genome you are using, but you can check the source >notes at Ensembl (& NCBI) to find out what each annotation build >contains. A raw count on the number of entries in the GTF files can also >be a clue - if greatly different, then you very likely have different >populations in the two files. > >Good luck with your project! > >Jen >Galaxy team > >On 1/7/13 1:47 PM, Wei Liao wrote: >> Hi all, >> >> I am analyzing significant differential expressed genes for a pair of >> normal V.S tumor, using Cuffdiff 2.0.2. >> I noticed that by using ensemble GTF and refseq GTF, the results showed >> a big difference on the number of genes being significant expressed. >> >> For ensemble GTF, there are only 250 genes differential expressed. >> But for refseq GTF, there are about 1000 genes. >> >> I am running these data on Galaxy server and with the same workflow. >> >> Can anyone explain what is going on here? so which result should I >>trust? >> >> Thanks. >> >> >> -- >> Wei Liao >> Research Scientist, >> Brentwood Biomedical Research Institute >> 16111 Plummer St. >> Bldg 7, Rm D-122 >> North Hills, CA 91343 >> 818-891-7711 ext 7645 >> >> >> ___________________________________________________________ >> The Galaxy User list should be used for the discussion of >> Galaxy analysis and other features on the public server >> at usegalaxy.org. Please keep all replies on the list by >> using "reply all" in your mail client. For discussion of >> local Galaxy instances and the Galaxy source code, please >> use the Galaxy Development list: >> >> http://lists.bx.psu.edu/listinfo/galaxy-dev >> >> To manage your subscriptions to this and other Galaxy lists, >> please use the interface at: >> >> http://lists.bx.psu.edu/ >> > >-- >Jennifer Hillman-Jackson >Galaxy Support and Training >http://galaxyproject.org >___________________________________________________________ >The Galaxy User list should be used for the discussion of >Galaxy analysis and other features on the public server >at usegalaxy.org. Please keep all replies on the list by >using "reply all" in your mail client. For discussion of >local Galaxy instances and the Galaxy source code, please >use the Galaxy Development list: > > http://lists.bx.psu.edu/listinfo/galaxy-dev > >To manage your subscriptions to this and other Galaxy lists, >please use the interface at: > > http://lists.bx.psu.edu/ ___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/