Inconsistency Between Cufflinks And Cuffdiff. This Is Killing Me.

Question: Inconsistency Between Cufflinks And Cuffdiff. This Is Killing Me.

6.5 years ago by

Jia Meng • 10 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?

rna-seq cufflinks fpkm expression cuffdiff • 2.1k views

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modified 18 months ago by Jennifer Hillman Jackson ♦ 25k • written 6.5 years ago by Jia Meng • 10

6.5 years ago by

Jeremy Goecks • 2.2k

Jeremy Goecks • 2.2k wrote:

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.

ADD COMMENT • link written 6.5 years ago by Jeremy Goecks • 2.2k

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

ADD REPLY • link written 6.5 years ago by Ateequr Rehman • 150

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 -- Jennifer Jackson http://galaxyproject.org

ADD REPLY • link written 6.5 years ago by Jennifer Hillman Jackson ♦ 25k

18 months ago by

vm.higareda • 0

vm.higareda • 0 wrote:

Hello I have the same trouble, Did you find a good answer?

ADD COMMENT • link written 18 months ago by vm.higareda • 0

The two tools calculate abundance differently, so the results from one are not expected to be the same as the other. The help link from several years ago (included in Jeremy's original reply) is now outdated and is not redirected for some reason at the author website. This is the current location of the tool's manual, usage guide, plus related publications: http://cole-trapnell-lab.github.io/cufflinks/

There is a google group for this tool if you want to discuss the scientific content with other tools users (details under "Help" at the same website link). There is also discussion at general bioinformatics Q&A website such as https://www.biostars.org/. These tools in Galaxy are really just wrappers around the original underlying tools, which means that the parameter usage and result interpretation is the same as when the tools are used line-command.

Next time, it is best to post a single comment or question, no need to post to multiple threads. Thanks! Jen, Galaxy team

ADD REPLY • link written 18 months ago by Jennifer Hillman Jackson ♦ 25k

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