Replicate analysis via Cuffdiff can yield different results as compared to single sample analysis via Cufflinks. See this FAQ for details: http://cufflinks.cbcb.umd.edu/howitworks.html#reps Best, J. On May 31, 2012, at 10:55 AM, Jia Meng wrote:

<<Problem>> The cufflinks and cuffdiff results are not consistent with each other. This is killing me. Does it make sense?

<< Obseration >> We have 3 control samples and 3 treated sample. For many genes, their FPKM in cufflinks and cuffdiff are far from consistent. In cufflinks result, for a gene’s FPKM are control group (sample: 1,2,3)：0， 0， 4.8 treated group(sample: 1,2,3)：0， 0， 6.0

In cuffdiff, the estimated FPKM are control group： 12.6 treated group：2.0

<<Method>> Use ucsc gene annotation gtf file, mm9, downloaded from UCSC table database

Use cufflinks on each individual sample. Cufflinks: galaxy mirror at cistrome, minimal count:10, no quantile normalization, use gtf as reference, no background correction

Use cufflinks on treated groups (3 biological replicates) and control groups (3 biological replicates) Cuffdiff: galaxy mirror at cistrome, minimal count:10, no normalization, use gtf as reference, no background correction

<<Additional Comments>> Cufflinks returns 55350 transcripts, while cuffdiff return 55418 transcripts, even though they use the same gene annotation gtf file. For the 6 cufflinks results (corresponding to 6 samples), the transcript ids are all the same, but the order are not,

<<Question>> Does it make sense? Or did I do anything wrong? ___________________________________________________________ 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:

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Hello Ateeq, Custom reference genomes are loaded in FASTA format. Help for preparing the data is in our wiki: http://wiki.g2.bx.psu.edu/Support#Custom_reference_genome http://wiki.g2.bx.psu.edu/Learn/CustomGenomes Best, Jen Galaxy team On 6/14/12 9:15 AM, Ateequr Rehman wrote:

Dear All

I am analysing bacterial RNA seq (illumina). as my reference genome is not in galaxy. so i need to downlaoded the reference genome from NCBI and upload to my history

Should i use reference genome with gene features or single fasta file with out any annotation information. even after download should i need to modify or it should be used as it is.

Thanks in advance Ateeq

___________________________________________________________ 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:

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-- Jennifer Jackson http://galaxyproject.org