Question: How To Get Ref.Gtf File For Cufflink
0
gravatar for raj.gzra
4.1 years ago by
raj.gzra30
raj.gzra30 wrote:

hi everyone i have assembled genome sequence, assembled transcriptome sequence and two sample of transcripts reads and i mapped these reads to transcriptome with BWA. i want to know how to calculate RPKM value after mapping.is there any software or tool which calculate RPKM value? please tell me. and second thing is that when i used cuffdiff it wants ref.gtf file and i don't have ref.gtf file. from where i can get it. please reply as soon as possible thank you.

gtf rpkm cufflinks mapping • 1.3k views
ADD COMMENTlink modified 3.3 years ago by Biostar ♦♦ 20 • written 4.1 years ago by raj.gzra30
0
gravatar for Nick
4.1 years ago by
Nick240
Spain
Nick240 wrote:

Since August I stopped using cuffdiff because some of the results produced by the latest version look quite improbable. I switched to using the following pipeline:

  1. tophat2
  2. htseq-count
  3. edgeR

There is a very nice tutorial ( http://www-huber.embl.de/pub/pdf/nprot.2013.099.pdf‎) - you just need to adapt the scripts from it and you will have a working pipeline. The pipeline won't directly compute the RPKM values but I am told that edgeR has a function (rpkm()) which would do this for you. Instead of using that function I used R script to compute the RPKM values which I picked from this post (http://seqanswers.com/forums/showpost.php?p=120485&postcount=5). By the way, RPKM is still a very popular metric but it has some downsides (http://www.ncbi.nlm.nih.gov/pubmed/22872506). You would be better off using the FDR and logFC computed by edgeR (or DESeq).

You would need a gtf file that corresponds to the genome version that you have used to align the reads. The best solution is to download them both from Ensemble (http://www.ensembl.org/info/data/ftp/index.html).

ADD COMMENTlink modified 4.1 years ago • written 4.1 years ago by Nick240

thank you nikolay. but i have genome of fungus which is unannotated yet and newly sequenced and assembled. then how to download files from Ensemble>>

ADD REPLYlink written 4.1 years ago by raj.gzra30

can i make gtf file by blast against genome with transcriptome???

ADD REPLYlink written 4.1 years ago by raj.gzra30
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