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.s...@gmail.com>
To: Bomba Dam <bomba....@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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