Hi Ateeq

I am running either bowtie and then cufflinks or tophat and then cufflinks. With bacteria there is not much of a reason to use tophat since you arent expecting alternative splicing. If you DO decide to do tophat and then cufflinks, play with the intron size parameters.

For bowtie I have been using a genome in FASTA format. When you then go to cufflinks you can give it your reference gene annotation in gtf format so it recognizes the genes that were aligned to the genome.

Hope that helps

Noa

Dear Bomba

I am bit confused , Are you running bowtie ---> tophat ----> Cufflink

For bowtie run you are using reference genome annotation  in gff3 format

 I am curremntly totally unable to figure it out correctly, how i should analyse my RNA seq data

Best

Ateeq

 

---

From: Noa Sher <noa.sher@gmail.com>
To: Bomba Dam <bomba.dam@visva-bharati.ac.in>
Cc: galaxy-user@lists.bx.psu.edu
Sent: Wednesday, February 22, 2012 7:28 AM
Subject: Re: [galaxy-user] Cufflinks related problem

Hi Bomba

I cant know for sure without seeing your files but I originally had the same problem and it ended up being because the way the genome was named in the fasta genome file was not the same as the way it was named in column 1 of my gtf file. I would check that first.  Also- with bacteria you dont want to run cufflinks with default parameters- use the galaxy browser to check how your data looks after tophat and you will most likely see very strange gene-spanning introns. Change the max intron size to 1000-1500 and the min distnace to 101-5nt and you should get results that make much more sense.

Good luck

Noa

On 22/02/2012 00:12, Bomba Dam wrote:

Dear Noa,

Can you please  tell me the parameters that I should keep while mapping bacterial transcripts using cuffkins. I have kept the default parameters as in Cufflinks and used my custom genome annotation  in gff3 format. The cufflinks seems to work Ok but all the FPKM values in these files are zero. As suggested by other users I have checked the correctness of my GFF3 files. The corresponding fasta file was used for mapping the transcripts using Bowtie. Are there any special trick for mapping bacterial transcriptome.

regards,

Bomba

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