Hello Jen,

Thanks for your reply and input.  The genes which have"0 FPKM" have different 
tags: some are LOWDATA, some are HIDATA and some are OK...  

We tried changing the number of reads used as input into Cufflinks - when we 
used many reads (~12,000,000 on a genome with ~2,400 genes) most of the genes 
have 0 FPKM and "HIDATA".  When we used fewer reads (1,000,000) on the same 
genome many of the genes still had "0 FPKM" but now with different flags (OK or 
LOWDATA).  

We tried using quartile normalization and this didn't seem to help much.  

As in the previous cases, looking at the reads using IGV showed that genes with 
0 FPKM, even if they have "OK" as their tag, do indeed have reads associated 
with them.

Any suggestions?  Could this be due to the fact that these are bacterial 
genomes without introns?  If so, any suggestions what parameters to change?

Thanks
Dikla and Daniel


-----Original Message-----
From: galaxy-user-boun...@lists.bx.psu.edu 
[mailto:galaxy-user-boun...@lists.bx.psu.edu] On Behalf Of Jennifer Jackson
Sent: שבת 23 פברואר 2013 11:03
To: דקלה אהרונוביץ
Cc: דניאל שר; galaxy-u...@bx.psu.edu
Subject: Re: [galaxy-user] Why don't we get FPKMs from this gene?

Hello Dikla,

Information in the sixth field of these same files will provide 
information about why the calculations for individual genes/transcripts 
were not performed. Possible values are explained in the tool documentation:
http://cufflinks.cbcb.umd.edu/manual.html#gene_exp_diff

This particular region appears to have low coverage compared to the 
surrounding regions (e.g. low abundance), but this is of course only a 
small sample, and it is difficult to know about other criteria 
considered by the tool from the graphic (proper pairing, multiple map 
locations, etc.).

But if you believe that higher abundance transcripts are preventing 
lower abundance transcripts from being evaluated, or even just suspect 
that and want to test, you could try running Cufflinks with the option 
"Perform quartile normalization: Yes". Using "Perform Bias Correction: 
Yes" is also another parameter to explore (requires a reference genome).

The Cufflinks web site is a great resource to learn more about these 
parameters and the Galaxy tool form has each included as an option.

Hopefully this helps,

Jen
Galaxy team


On 2/14/13 1:48 AM, Dikla Aharonovich wrote:
> Hello,
>
> We are using BAM to map Illumina reads to a bacterial genome, followed
> by Cufflinks o get the FPKMs.  We have come across many genes for which
> we get FPKM=0 (using both gene and transcript expression) even though
> there are reads mapping to these gene IDs (e.g. the region between the
> dashed lines in the attached screenshot).  Can anyone suggest a
> reason/fix for this?
>
> Thanks
>
> Dikla
>
>
>
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-- 
Jennifer Hillman-Jackson
Galaxy Support and Training
http://galaxyproject.org
___________________________________________________________
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___________________________________________________________
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
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