Re: [galaxy-user] Cuffdiff output

2014-03-12 Thread Ian Donaldson
I am quite new to RNA-seq analysis, but what I have learned so far is that 
replicates are important.  If you have this result with no replicates then 
P-values are more or less meaningless.  You can also gauge what is happening by 
looking at the modelled read count output.  If the counts are both less than 
50ish you are unlikely to have a robust result for that gene/transcript.

Ian 



From: galaxy-user-boun...@lists.bx.psu.edu 
[galaxy-user-boun...@lists.bx.psu.edu] on behalf of Malik, Shivani 
[shivani.ma...@ucsf.edu]
Sent: 11 March 2014 21:43
To: galaxy-user@lists.bx.psu.edu
Subject: [galaxy-user] Cuffdiff output

Hi,
I have a question about interpreting the cuffdiff data and how to pick up 
significant genes. I have genes which show ~8 fold change between 2 conditions: 
eg from FPKM of 0.08 to 28 and yet they are not significant. Is there is 
threshold of FPKM below which Cuffdiff does not consider it  an FPKM to be 
valid and hence significance in no? What downstream analysis should I use to 
extract a meaningful list of genes from the Cuffdiff data?

Also, I filtered out FPKMs which were below 5 in both conditions? Is that 
reasonable?

Thanks
Shivani



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[galaxy-user] Cuffdiff output

2014-03-11 Thread Malik, Shivani
Hi,
I have a question about interpreting the cuffdiff data and how to pick up 
significant genes. I have genes which show ~8 fold change between 2 conditions: 
eg from FPKM of 0.08 to 28 and yet they are not significant. Is there is 
threshold of FPKM below which Cuffdiff does not consider it  an FPKM to be 
valid and hence significance in no? What downstream analysis should I use to 
extract a meaningful list of genes from the Cuffdiff data? 

Also, I filtered out FPKMs which were below 5 in both conditions? Is that 
reasonable?

Thanks
Shivani



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Re: [galaxy-user] Cuffdiff question

2013-11-15 Thread Noa.sher
Dont use the - b parameter

Sent from my iPhone; please excuse any brevity or typos!

On Nov 15, 2013, at 2:51 PM, clare Hardman chard...@mrc-lmb.cam.ac.uk wrote:

 Hi Noa,
 
 Yes I did use Cufflinks so this sounds just like my problem. So how have you 
 dealt with the problem?
 
 Best wishes,
 
 Clare
 
 
 
 On 14 Nov 2013, at 18:17, Noa Sher wrote:
 
 Hi Clare
 We just ran into a similar issue about a week ago and were debugging with 
 the authors of cuffdiff
 Apparently there are issues with the -b parameter - were you using this in 
 cufflinks?
 If yes - this may be the cause - we switched the order of the replicates and 
 the values changed; as did PCA's of the samples, etc
 They are working on this issue for an upcoming version of cufflinks.
 I am interested in knowing whether this was indeed your problem or were you 
 using a pipeline that does not include cufflinks?
 Good luck,
 Noa
 
 
 On 14/11/2013 13:13, clare Hardman wrote:
 Hello,
 
 Could you please advise me on this probably naive question. When I compare 
 sample A and sample B by Ciffdiff and then separately compare Sample A to 
 Sample C by Cuffdiff too, should the FMPK value be the same for A in both 
 tests? At the moment mine does not seem to be!
 
 Best wishes
 
 Clare
 
 
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Re: [galaxy-user] Cuffdiff question

2013-11-15 Thread clare Hardman
Hi Noa,

Yes I did use Cufflinks so this sounds just like my problem. So how have you 
dealt with the problem?

Best wishes,

Clare



On 14 Nov 2013, at 18:17, Noa Sher wrote:

 Hi Clare
 We just ran into a similar issue about a week ago and were debugging with the 
 authors of cuffdiff
 Apparently there are issues with the -b parameter - were you using this in 
 cufflinks?
 If yes - this may be the cause - we switched the order of the replicates and 
 the values changed; as did PCA's of the samples, etc
 They are working on this issue for an upcoming version of cufflinks.
 I am interested in knowing whether this was indeed your problem or were you 
 using a pipeline that does not include cufflinks?
 Good luck,
 Noa
 
 
 On 14/11/2013 13:13, clare Hardman wrote:
 Hello,
 
 Could you please advise me on this probably naive question. When I compare 
 sample A and sample B by Ciffdiff and then separately compare Sample A to 
 Sample C by Cuffdiff too, should the FMPK value be the same for A in both 
 tests? At the moment mine does not seem to be!
 
 Best wishes
 
 Clare
 
 
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[galaxy-user] Cuffdiff question

2013-11-14 Thread clare Hardman
Hello,

Could you please advise me on this probably naive question. When I compare 
sample A and sample B by Ciffdiff and then separately compare Sample A to 
Sample C by Cuffdiff too, should the FMPK value be the same for A in both 
tests? At the moment mine does not seem to be!

Best wishes

Clare


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Re: [galaxy-user] Cuffdiff question

2013-11-14 Thread Noa Sher

  
  
Hi Clare
We just ran into a similar issue about a week ago and were debugging
with the authors of cuffdiff
Apparently there are issues with the -b parameter - were you using
this in cufflinks?
If yes - this may be the cause - we switched the order of the
replicates and the values changed; as did PCA's of the samples, etc
They are working on this issue for an upcoming version of cufflinks.
I am interested in knowing whether this was indeed your problem or
were you using a pipeline that does not include cufflinks?
Good luck,
Noa


On 14/11/2013 13:13, clare Hardman
  wrote:


  Hello,

Could you please advise me on this probably naive question. When I compare sample A and sample B by Ciffdiff and then separately compare Sample A to Sample C by Cuffdiff too, should the FMPK value be the same for A in both tests? At the moment mine does not seem to be!

Best wishes

Clare


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[galaxy-user] Cuffdiff question

2013-11-12 Thread Jennifer Jackson

Hello,

The transcript name when using RefSeq as a reference annotation is the 
NM_ type of identifier.


If you want to include a gene symbol, then the reference annotation 
should include the attribute gene_name. The iGenomes GTF files are an 
example of datasets that include this attribute.


http://wiki.galaxyproject.org/Support#Interpreting_scientific_results
See: /Example/?/*RNA-seq analysis*/*tools*
http://cufflinks.cbcb.umd.edu/faq.html#gtfs
http://cufflinks.cbcb.umd.edu/igenomes.html
http://cufflinks.cbcb.umd.edu/manual.html (search on page for 
gene_name to see where can be used/output)


Best,

Jen
Galaxy team

On 11/12/13 10:40 AM, Irene Bassano wrote:

Dear Jennifer,
I am about to start a new analysis and learning from my old mistakes would like 
to ask just one question:
I would like my Cufflinks and Cuffdiff results to give me the actual gene name or 
transcript name rather than NM_.. like I had last time i run the job.

At which stage and which option do I have ti use to get the proper names?

Thanks a lot!

Irene


--
Jennifer Hillman-Jackson
http://galaxyproject.org

--
Jennifer Hillman-Jackson
http://galaxyproject.org

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Re: [galaxy-user] Cuffdiff version not apparent

2013-11-05 Thread graham etherington (TSL)
Hi Cory,
A list of Galaxy dependancies can be found on the wiki at:
http://wiki.galaxyproject.org/Admin/Tools/Tool%20Dependencies
...although many tools allow a range of tool versions.

You can also identify the information about the specific tool versions by
clicking on the View Details Œi¹ icon of a history item created by that
tool and looking at the Tool Version field.
If you¹re using the Galaxy public server (https://usegalaxy.org/) then
clicking on the Œi¹ icon of a cuffdiff output file will show:

Tool Version:cuffdiff v2.1.1 (4046M)
Hope this helps.

Cheers,
Graham


Dr. Graham Etherington
Bioinformatics Support Officer,
The Sainsbury Laboratory,
Norwich Research Park,
Norwich NR4 7UH.
UK
Tel: +44 (0)1603 450601





On 04/11/2013 20:57, Cory Dunn cd...@ku.edu.tr wrote:

Dear Galaxy Staff:


I was wondering which version of Cuffdiff is currently running on Galaxy.
 The wrapper version is 0.0.6, but I did not see the actual version of
the underlying software under the Tool Version field (please see
attached screen grab).


Thanks for your help,
Cory Dunn












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[galaxy-user] Cuffdiff version not apparent

2013-11-04 Thread Cory Dunn
Dear Galaxy Staff:

I was wondering which version of Cuffdiff is currently running on Galaxy.
 The wrapper version is 0.0.6, but I did not see the actual version of the
underlying software under the Tool Version field (please see attached
screen grab).

Thanks for your help,
Cory Dunn


[image: Inline image 1]
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Re: [galaxy-user] Cuffdiff changes

2013-08-23 Thread Jeremy Goecks
 Where can I see which version are being used?

You can see both the Galaxy tool version and the Cuffdiff tool version (when 
available) by clicking on the 'view details' icon (the 'i' at the bottom of an 
expanded dataset). Right now the Cuffdiff version is not displayed, but that 
will change when our server is updated.

 What does Cuffdiff(version 0.0.5) mean then?

That is the version of the Galaxy wrapper; the wrapper provides the interface 
between Cuffdiff and Galaxy.

 What version was it before?

I think Cuffdiff version was 1.3.1 previously.
 
 I look forward to the update, will that mean another version of Cuffdiff 
 again?

The wrapper will be updated but not Cuffdiff itself.

J.

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Re: [galaxy-user] Cuffdiff changes

2013-08-23 Thread Johanna Sandgren
Thanks for quick reply.

Where can I see which version are being used? It does not say in the attached 
(in my first e-mail) info-view  after the run.. What does Cuffdiff (version 
0.0.5) mean then? What version was it before? It is a great difference in 
number of DE genes..

I mostly meant it was the same now in August with more DE genes for all 
different type of analysis that I have previously done (consistent new results 
with no settings changed), I really do not see more DE genes when 5 vs 6 
samples compared to 1 vs 2 samples. But that might be due to more biological 
reasons.

I look forward to the update, will that mean another version of Cuffdiff again?

Kind regards,
Johanna

From: Jeremy Goecks [mailto:jeremy.goe...@emory.edu]
Sent: Thursday, August 22, 2013 8:04 PM
To: Johanna Sandgren
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Cuffdiff changes

I am wondering why Cuffdiff suddenly gives many more significant DE genes?

Cuffdiff was recently updated to version 2.1.0; this update likely explains the 
different results that you see.


I have rerun several analysis, also with more samples in each group, all give 
much more significant genes. Why?

More samples = more power to accurately estimate expression levels = more DE 
genes.


Also, will replicate information soon be included in output files?

Replicate data will be available when we next update our server. This should 
occur in about 3 weeks.

Best,
J.

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[galaxy-user] Cuffdiff changes

2013-08-22 Thread Johanna Sandgren
Hi,

I am wondering why Cuffdiff suddenly gives many more significant DE genes?
I have used same input data and now get approx 5x more significant genes, 
settings is same with the exception that you now included library normalization 
and dispersion estimation. See below for parameters.
I have rerun several analysis, also with more samples in each group, all give 
much more significant genes. Why?

Also, will replicate information soon be included in output files?

Kind regards,
Johanna


Tool: Cuffdiff

Tool: Cuffdiff

Name:

Cuffdiff on data 225, data 236, and others: splicing differential expression 
testing

Name:

Cuffdiff on data 225, data 236, and others: splicing differential expression 
testing

Created:

4-Apr-13

Created:

21-Aug-13

Filesize:

10.3 MB

Filesize:

10.2 MB

Dbkey:

hg19

Dbkey:

hg19

Format:

tabular

Format:

tabular

Galaxy Tool Version:

0.0.5

Galaxy Tool Version:

0.0.5

Tool Version:

Tool Version:

Tool Standard Output:

stdouthttps://main.g2.bx.psu.edu/datasets/bbd44e69cb8906b5355e4a035e92cd84/stdout

Tool Standard Output:

stdouthttps://main.g2.bx.psu.edu/datasets/bbd44e69cb8906b524bcf7e585f495b1/stdout

Tool Standard Error:

stderrhttps://main.g2.bx.psu.edu/datasets/bbd44e69cb8906b5355e4a035e92cd84/stderr

Tool Standard Error:

stderrhttps://main.g2.bx.psu.edu/datasets/bbd44e69cb8906b524bcf7e585f495b1/stderr

Tool Exit Code:

0

Tool Exit Code:

0

API ID:

bbd44e69cb8906b5355e4a035e92cd84

API ID:

bbd44e69cb8906b524bcf7e585f495b1


Input Parameter

Value

Note for rerun

Input Parameter

Value

Transcripts

261: Cuffmerge on data 258, data 135, and others: merged transcripts

Transcripts

261: Cuffmerge on data 258, data 135, and others: merged transcripts

Perform replicate analysis

Yes

Perform replicate analysis

Yes

Group name

s202

Group name

s202

Add file

194: Galaxy883-[MarkDups_Dupes_Marked_882_202.bam].bam

Add file

194: Galaxy883-[MarkDups_Dupes_Marked_882_202.bam].bam

Group name

Ctrls

Group name

Ctrls

Add file

236: MarkDups_Dupes Marked216.bam

Add file

236: MarkDups_Dupes Marked216.bam

Add file

225: MarkDups_Dupes Marked206.bam

Add file

225: MarkDups_Dupes Marked206.bam

Library normalization method

not used (parameter was added after this job was run)

Library normalization method

geometric

Dispersion estimation method

not used (parameter was added after this job was run)

Dispersion estimation method

pooled

False Discovery Rate

0.05

False Discovery Rate

0.05

Min Alignment Count

2

Min Alignment Count

2

Perform quartile normalization

No

Perform quartile normalization

No

Use multi-read correct

Yes

Use multi-read correct

Yes

Perform Bias Correction

Yes

Perform Bias Correction

Yes

Reference sequence data

cached

Reference sequence data

cached

Set Additional Parameters? (not recommended)

Yes

Set Additional Parameters? (not recommended)

Yes

Average Fragment Length

200

Average Fragment Length

200

Fragment Length Standard Deviation

80

Fragment Length Standard Deviation

80



..
Johanna Sandgren, PhD
Department of Oncology-Pathology
CCK, Karolinska Institutet
SE-171 76 Stockholm, Sweden
+46-8-517 721 35 (office),
+46-8- 321047(fax), +46-708 388476 (mobile)

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Re: [galaxy-user] Cuffdiff-cummerbund with biological replicates problem

2013-07-31 Thread Jeremy Goecks
In the past, others have had success using Cummerbund with Galaxy, and there's 
even a Cummerbund wrapper in the tool shed: 

http://toolshed.g2.bx.psu.edu/view/jjohnson/cummerbund

That said, it appears that replicate information is largely contained in the 
read group tracking files, which are not currently included in Galaxy's 
Cuffdiff outputs. I don't know if these files are required by Cummerbund to do 
replicate analysis. This would be a good question for the Cummerbund 
developers, as well as what the p and q values mean when doing replicate 
analysis.

If you find that Galaxy's lacking something for Cummerbund to function 
correctly, that would be very useful information to share with the list.

Best,
J.


On Jul 26, 2013, at 8:50 PM, Mike Shamblott wrote:

 I'm trying to run Cuffdiff on a set of 10 human samples with biological 
 replication then download the results for further analyses in 
 Cummerbund(v2.1.1).  It seems like a standard workflow but I cannot get 
 cummerbund to acknowledge replicates.  I download and rename the 11 cuffdiff 
 output files to the names expected by cummerbund.  Cummerbund builds a 
 CuffSet with no warnings and most analyses work as expected.  The problem 
 comes any time I try to see the results of replication.  For example, in 
 cummerbund, replicates() returns an empty set and any type of plot returns 
 an error when replicates=T is included as an argument.
 
 There is no evidence of replication data in any of the 11 cuffdiff output 
 files.  The data is presented with the group name only.  From this, I 
 conclude that the problem is with cuffdiff, since there is no replicate data 
 for cummerbund to build into its db.  I see that there are several read group 
 files that are produced by cuffdiff but cannot be downloaded in Galaxy.  Is 
 this the problem, and if so, how can Galaxy be used to generate data with 
 (essential) replication?  Are the p  and  q significance values reported in 
 the output files a result of replicate analysis?  
 
 I have tried to ask this question in several different forums without 
 success.  The responses I've gotten suggest its a Galaxy issue rather than 
 either cuffdiff or cummerbund.   I'm hoping someone here can help answer my 
 questions.
 
 Hopeful,
 
 Mike
 
 
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[galaxy-user] Cuffdiff-cummerbund with biological replicates problem

2013-07-29 Thread Mike Shamblott
I'm trying to run Cuffdiff on a set of 10 human samples with biological 
replication then download the results for further analyses in 
Cummerbund(v2.1.1).  It seems like a standard workflow but I cannot get 
cummerbund to acknowledge replicates.  I download and rename the 11 cuffdiff 
output files to the names expected by cummerbund.  Cummerbund builds a CuffSet 
with no warnings and most analyses work as expected.  The problem comes any 
time I try to see the results of replication.  For example, in cummerbund, 
replicates() returns an empty set and any type of plot returns an error when 
replicates=T is included as an argument.

There is no evidence of replication data in any of the 11 cuffdiff output 
files.  The data is presented with the group name only.  From this, I conclude 
that the problem is with cuffdiff, since there is no replicate data for 
cummerbund to build into its db.  I see that there are several read group files 
that are produced by cuffdiff but cannot be downloaded in Galaxy.  Is this the 
problem, and if so, how can Galaxy be used to generate data with (essential) 
replication?  Are the p  and  q significance values reported in the output 
files a result of replicate analysis?  


I have tried to ask this question in several different forums without success.  
The responses I've gotten suggest its a Galaxy issue rather than either 
cuffdiff or cummerbund.   I'm hoping someone here can help answer my questions.

Hopeful,

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Re: [galaxy-user] Cuffdiff statistical calculations are inconsistent?

2013-03-15 Thread Jeremy Goecks
 The header of the Cuffdiff tool page says it is version 0.0.5

This version is the Galaxy tool wrapper version, not the tool version. (Yes, 
this is a usability issue.) You can find the tool version in the dataset's 
information panel by clicking on the 'i' icon.

 Is there a way, or setting, on Cuffdiff 2.0 to revert the parameters to be 
 more similar to Cuffdiff 1.3?

This isn't a parameter issue. The Cuffdiff algorithm has changed substantially, 
and it's not clear to me if/how (or whether it's a good idea at all) to modify 
parameters to obtain 1.3-esque results. 

Best,
J.
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[galaxy-user] Cuffdiff statistical calculations are inconsistent?

2013-03-13 Thread Jenna Smith
Hi,

I'll preface my concern by saying that I'm a novice to Cufflinks.  Back in
September, I performed a Cuffdiff analysis comparing a wild-type and mutant
condition.  The analysis returned ~800 transcripts differentially regulated
between the two with statistical significance.  Recently, I've rerun the
Cuffdiff analysis - using exactly the same files stored in Galaxy for all
inputs, and with all the same parameters - and only get a few dozen
statistically significant hits.  However, all of the data besides the p and
q values are essentially identical between these two runs, so I am really
unclear as to what is causing the difference.  Here is just one clear
example:

From run 1:
YFR026C
FPKM 1 = 17.2434
FPKM 2 = 196.735
log2(fold change) = 3.51214
p = 1.64E-8
q = 7.33E-6
significant = yes

From run 2:
YFR026C
FPKM 1 = 14.4489
FPKM 2 = 144.939
log2(fold change) = 3.32641
p = 0.000170034
q = 0.0719964
significant = no

The second Cuffdiff analysis shows there is still a ~10-fold difference
between conditions, but this is not statistically significant.  Has the
version of Cuffdiff on Galaxy been updated such that some parameters have
changed, that could explain this difference?  Or, is there some setting I
am missing that would cause very large changes to fail statistical
significance testing?  Any help or input would be appreciated, I am really
at a loss for why executing what should be exactly the same task is giving
vastly different results.  I could just be overlooking something very
fundamental that is obvious to someone with more experience with this
program.  Thanks.

-Jenna Smith
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Re: [galaxy-user] Cuffdiff statistical calculations are inconsistent?

2013-03-13 Thread Mohammad Heydarian
We are having the exact same issue, on the main server and our (recent)
cloud instances.

Were some of the hidden Cuffdiff parameters modified since fall 2012?

Cheers,
Mo Heydarian
On Mar 13, 2013 11:02 AM, Jenna Smith jes...@case.edu wrote:

 Hi,

 I'll preface my concern by saying that I'm a novice to Cufflinks.  Back in
 September, I performed a Cuffdiff analysis comparing a wild-type and mutant
 condition.  The analysis returned ~800 transcripts differentially regulated
 between the two with statistical significance.  Recently, I've rerun the
 Cuffdiff analysis - using exactly the same files stored in Galaxy for all
 inputs, and with all the same parameters - and only get a few dozen
 statistically significant hits.  However, all of the data besides the p and
 q values are essentially identical between these two runs, so I am really
 unclear as to what is causing the difference.  Here is just one clear
 example:

 From run 1:
 YFR026C
 FPKM 1 = 17.2434
 FPKM 2 = 196.735
 log2(fold change) = 3.51214
 p = 1.64E-8
 q = 7.33E-6
 significant = yes

 From run 2:
 YFR026C
 FPKM 1 = 14.4489
 FPKM 2 = 144.939
 log2(fold change) = 3.32641
 p = 0.000170034
 q = 0.0719964
 significant = no

 The second Cuffdiff analysis shows there is still a ~10-fold difference
 between conditions, but this is not statistically significant.  Has the
 version of Cuffdiff on Galaxy been updated such that some parameters have
 changed, that could explain this difference?  Or, is there some setting I
 am missing that would cause very large changes to fail statistical
 significance testing?  Any help or input would be appreciated, I am really
 at a loss for why executing what should be exactly the same task is giving
 vastly different results.  I could just be overlooking something very
 fundamental that is obvious to someone with more experience with this
 program.  Thanks.

 -Jenna Smith


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Re: [galaxy-user] Cuffdiff statistical calculations are inconsistent?

2013-03-13 Thread Jeremy Goecks
This is likely due to the upgrade from Cufflinks 1.3.x to Cufflinks 2.0.x; 
Cufflinks 2.0 introduced a new algorithm for Cuffdiff in particular. You can 
read about these changes on the website:
http://cufflinks.cbcb.umd.edu/ (and there's a manuscript describing the changes 
as well).

You might consider writer to to the tool authors directly for more details: 
tophat.cuffli...@gmail.com Of course, please consider sharing anything you 
learn with members of this list as well.

Best,
J.



On Mar 13, 2013, at 12:06 PM, Mohammad Heydarian wrote:

 We are having the exact same issue, on the main server and our (recent) cloud 
 instances.
 
 Were some of the hidden Cuffdiff parameters modified since fall 2012? 
 
 Cheers,
 Mo Heydarian
 
 On Mar 13, 2013 11:02 AM, Jenna Smith jes...@case.edu wrote:
 Hi,
 
 I'll preface my concern by saying that I'm a novice to Cufflinks.  Back in 
 September, I performed a Cuffdiff analysis comparing a wild-type and mutant 
 condition.  The analysis returned ~800 transcripts differentially regulated 
 between the two with statistical significance.  Recently, I've rerun the 
 Cuffdiff analysis - using exactly the same files stored in Galaxy for all 
 inputs, and with all the same parameters - and only get a few dozen 
 statistically significant hits.  However, all of the data besides the p and q 
 values are essentially identical between these two runs, so I am really 
 unclear as to what is causing the difference.  Here is just one clear example:
 
 From run 1:
 YFR026C
 FPKM 1 = 17.2434
 FPKM 2 = 196.735
 log2(fold change) = 3.51214
 p = 1.64E-8
 q = 7.33E-6
 significant = yes
 
 From run 2:
 YFR026C
 FPKM 1 = 14.4489
 FPKM 2 = 144.939
 log2(fold change) = 3.32641
 p = 0.000170034
 q = 0.0719964
 significant = no
 
 The second Cuffdiff analysis shows there is still a ~10-fold difference 
 between conditions, but this is not statistically significant.  Has the 
 version of Cuffdiff on Galaxy been updated such that some parameters have 
 changed, that could explain this difference?  Or, is there some setting I am 
 missing that would cause very large changes to fail statistical significance 
 testing?  Any help or input would be appreciated, I am really at a loss for 
 why executing what should be exactly the same task is giving vastly different 
 results.  I could just be overlooking something very fundamental that is 
 obvious to someone with more experience with this program.  Thanks.
 
 -Jenna Smith
 
 
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Re: [galaxy-user] Cuffdiff tracking file does not report all genes and trancrips from reference annotation?

2012-11-30 Thread Jennifer Jackson

Hello Wei,

The results do sound strange. The best advice to start with is to make 
sure that you are up-to-date with both Galaxy and the RNA-seq tools and 
using the best possible inputs.


1 . Make sure that you are running the latest distribution
 http://wiki.galaxyproject.org/DevNewsBriefs

2.  Update to use the current version of CuffDiff
 http://wiki.galaxyproject.org/Admin/Tools/Tool%20Dependencies

3.  Use the iGenomes GTF annotation to make full use of the 
functionality in Cuffdiff

 http://cufflinks.cbcb.umd.edu/igenomes.html

A good test to see if your set-up is correct would be to run the RNA-seq 
tutorial locally as a test case.

http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise

The reverse can also be done, if you have a problem locally, try running 
it as a small test (that still demonstrates the issue) on the Public 
Main server and see if the results can be duplicated. This can help 
determine if problem is with data inputs/settings or a problem with tool 
set-up/installation. It can also be a way to share your data with us if 
you need feedback.


But, hopefully after updating the issue clears up!

Jen
Galaxy team

On 11/29/12 11:51 AM, Wei Liao wrote:

Hi, Galaxy user.
I ran into a problem when using Cuffdiff 1.2.1 in Galaxy local 
instance to check differential expressed genes in my samples.

I have 3 normals and 6 cancers samples, I did the following:
- After tophat for each samples, run cufflink with refseq annotation 
which has 25266 genes and 43091 transcripts

- cuffmerge all cufflink outputs contains 58112 lines
- run cuffdiff with 3 normals as triplicate and compare to each cancer 
sample.
Suprisingly, I fould out that the tanscripts tracking file, gene 
tracking, CDS tracking only has 2000 genes and 4000 transcripts. So 
the cufflink only compare 2000 genes and 4000 transcripts between 
samples.
The question I want to ask here is that *why are the rest of the genes 
and transcripts not being tested and included in the tracking files?* 
Do you know what cause this kind of problem?

Thanks,
Wei

--
Wei Liao
Research Scientist,
Brentwood Biomedical Research Institute
16111 Plummer St.
Bldg 7, Rm D-122
North Hills, CA 91343
818-891-7711 ext 7645



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[galaxy-user] Cuffdiff tracking file does not report all genes and trancrips from reference annotation?

2012-11-29 Thread Wei Liao
Hi, Galaxy user.
I ran into a problem when using Cuffdiff 1.2.1 in Galaxy local instance to
check differential expressed genes in my samples.
I have 3 normals and 6 cancers samples, I did the following:
- After tophat for each samples, run cufflink with refseq annotation which
has 25266 genes and 43091 transcripts
- cuffmerge all cufflink outputs contains 58112 lines
- run cuffdiff with 3 normals as triplicate and compare to each cancer
sample.
Suprisingly, I fould out that the tanscripts tracking file, gene tracking,
CDS tracking only has 2000 genes and 4000 transcripts. So the cufflink only
compare 2000 genes and 4000 transcripts between samples.
The question I want to ask here is that *why are the rest of the genes and
transcripts not being tested and included in the tracking files?* Do you
know what cause this kind of problem?
Thanks,
Wei

-- 
Wei Liao
Research Scientist,
Brentwood Biomedical Research Institute
16111 Plummer St.
Bldg 7, Rm D-122
North Hills, CA 91343
818-891-7711 ext 7645
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[galaxy-user] Cuffdiff without gene annotation

2012-11-29 Thread Ianiri, Giuseppe
Hello guys,
I went through the RNAseq workflow (I didn't do Cuffmerge) and from the 
Cuffdiff output gene and transcript differential expression testing I filtered 
some data. For example, for two samples I got about 400 gene and 900 transcript 
differential expressed with fold change 2. Since I am working with a fungus 
whose genome annotation is in a format (gff) not accepted by Cuffmerge or 
Cuffcompare in Galaxy (the accepted one is GTF2), the Cuffdiff output tells me 
only the position of relevant genes on the scaffolds.
Going to genome browser and see which gene is in that position is fine for few 
genes, but doing that for all 400 or 900 is something probably impossible.
Does anybody have a helpful suggestion on what I can do? It would be great if 
there was a program where based on the position of the genes on the scaffold 
(Cuffdiff output) I can get their information using the annotation file. I have 
also the gene annotation file in gene bank format (gbk) but I don't see a way 
to use it for what I need.
Thanks


Giuseppe Ianiri, Ph.D.
Division of Cell Biology and Biophysics
School of Biological Sciences
5100 Rockhill Road
University of Missouri-Kansas City
Kansas City, MO 64110
Email: iani...@umkc.edu

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[galaxy-user] cuffdiff

2012-11-12 Thread Vevis, Christis
Hi,

I got confused while trying to perform Cuffdiff for my RNA sequencing analysis. 
So I have five different samples which were sequenced. I used tophat to create 
the bam files and cufflink to create the assembled trancripts. Then I uded 
Cuffmerge to merge them in one file and then I wanted to do Cuffdiff with that 
merged file. What shall I choose for the ''SAM or BAM file of aligned RNA-Seq'' 
option? I have the 5 options from the 5 tophat actions on my 5 samples. All I 
want in the end is an excel table showing the number of hits from each sample 
(and not necessary a comparison of them).

Regards

Kristis Vevis, PhD Student
Cell Biology
UCL Institute of Ophthalmology
11-43 Bath Street
London
EC1V 9EL, UK
020 7608 4067

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Re: [galaxy-user] cuffdiff

2012-11-12 Thread Jeremy Goecks
Use the replicates option (yes, a bit of a misnomer) and put each Tophat run in 
its own group. This will produce a tabular file with FPKM for each group/run.

Best,
J.  

On Nov 12, 2012, at 10:05 AM, Vevis, Christis wrote:

 Hi,
  
 I got confused while trying to perform Cuffdiff for my RNA sequencing 
 analysis. So I have five different samples which were sequenced. I used 
 tophat to create the bam files and cufflink to create the assembled 
 trancripts. Then I uded Cuffmerge to merge them in one file and then I wanted 
 to do Cuffdiff with that merged file. What shall I choose for the ‘’SAM or 
 BAM file of aligned RNA-Seq’’ option? I have the 5 options from the 5 tophat 
 actions on my 5 samples. All I want in the end is an excel table showing the 
 number of hits from each sample (and not necessary a comparison of them).
  
 Regards  
  
 Kristis Vevis, PhD Student
 Cell Biology
 UCL Institute of Ophthalmology
 11-43 Bath Street
 London
 EC1V 9EL, UK
 020 7608 4067
  
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Re: [galaxy-user] cuffdiff values different for same sample

2012-10-17 Thread Jennifer Jackson

Hello,

Thank you for sharing your history. The difference in FPKM values can be 
explained by the use of the -N option (Perform quartile normalization: 
Yes). Set this to No to avoid the variable per-run normalization.


This has also been discussed at seqanswers.com:
http://seqanswers.com/forums/showthread.php?t=4606

Hopefully this helps!

Jen
Galaxy team

On 10/12/12 9:43 AM, i b wrote:

Hello forum,
I have ran cuffdiff with two samples, treated vs untreated, and had
certain values as expressed in the output (isoform differential
expression).

When I ran it again, using a different treated sample, but SAME
UNTREATED SAMPLE, the values assigned to the untreated are different.

Why is this if values are calculated from a unique FPKM? This happend
for other jobs where I have ran the same untreated vs other treated
samples...

Thanks a lot,
ib
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--
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Re: [galaxy-user] cuffdiff fpkm=0 not significant

2012-10-16 Thread Haiping Hao

ib,

Look at the status column.  I suspect that for this example you given 
the status is HiData.  Cuffdiff considers the expression are very high 
and no statistic testing would have been done for the gene.  fpkm 2=0 
could be misleading, as it may not be actually 0. I have encountered in 
a data set where several genes are expected to be highly expressed in 
all samples, but one of the fpkm values were all given as 0.  Hope this 
helps.


Haiping

--

JHMI Deep Sequencing and Microarray Core Facility at BRB
- Your source for quality service on Microarray studies, NextGen Sequencing, 
and Data Analysis.
http://www.microarray.jhmi.edu/

--


Haiping Hao Ph.D.
Associate Director
Johns Hopkins University Deep Sequencing and Microarray Core
Edward D. Miller Research Building
733 N. Broadway, Rm 359
Baltimore, MD 21205
h...@jhmi.edu
Phone: 443-287-9056
Fax: 410-502-

On 10/15/2012 5:36 PM, i b wrote:

Dear forum,
whoever knows this please tell me!

Given two fpkm  . e.g. fpkm 1=2922828 and fpkm 2=0, why cuffdiff does
not calculate differential expression. those genes are listed in
cuffdiff as not significant and the log2 fold change is infinite ,
either negative or positve...how do i consider them when i want to
pull out the genes that are differentially expressed between treated
and untreated samples?

how do i interpret these data?

thanks,
ib
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Re: [galaxy-user] cuffdiff values different for same sample

2012-10-15 Thread Jennifer Jackson

Hello,

Would you be able to share a history containing these data? Use 
Options (gear icon) - Share or Publish, generate the share link, then 
copy and paste that into a reply email sent to me directly. Please note 
the dataset #'s for the Cuffdiff runs that you are comparing and make 
sure that all inputs are undeleted.


Best,

Jen
Galaxy team

On 10/12/12 9:43 AM, i b wrote:

Hello forum,
I have ran cuffdiff with two samples, treated vs untreated, and had
certain values as expressed in the output (isoform differential
expression).

When I ran it again, using a different treated sample, but SAME
UNTREATED SAMPLE, the values assigned to the untreated are different.

Why is this if values are calculated from a unique FPKM? This happend
for other jobs where I have ran the same untreated vs other treated
samples...

Thanks a lot,
ib
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--
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[galaxy-user] cuffdiff values different for same sample

2012-10-12 Thread i b
Hello forum,
I have ran cuffdiff with two samples, treated vs untreated, and had
certain values as expressed in the output (isoform differential
expression).

When I ran it again, using a different treated sample, but SAME
UNTREATED SAMPLE, the values assigned to the untreated are different.

Why is this if values are calculated from a unique FPKM? This happend
for other jobs where I have ran the same untreated vs other treated
samples...

Thanks a lot,
ib
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Re: [galaxy-user] Cuffdiff no without replicates

2012-10-03 Thread Ross
On Wed, Oct 3, 2012 at 9:11 PM, i b ibse...@gmail.com wrote:
 Dear all,
 how reliable is running Cuffdiff without replicates? e.g.one samples
 agains another one?

 Is it statistically makign any difference when using replicates?

Seqanswers might be a better place to ask this very interesting
technical question that goes way beyond Galaxy...

My 2c: Statistically speaking, sequencing and biology are both noisy.
Replicates provide information about non-experimental (technical and
biological) variation. That variation is usually not the variation you
are looking for, but if you want to remove it, you have to model it
and that requires information from replicates (or really good
guesswork). In some situations (eg extreme experimental conditions),
I'm sure you'll find biologically meaningful signal without them but
in my experience, they can really help to decrease non-experimental
noise, particularly where the experimental condition induces only
subtle changes in transcript abundance.

You could always analyse a data set with replicates and compare the
results with and without those replicates yourself to see what happens
- it would be a nice paper I'm sure.


 Thanks,
 ib
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Re: [galaxy-user] Cuffdiff no without replicates

2012-10-03 Thread Sean Davis
On Wed, Oct 3, 2012 at 7:35 AM, Ross ross.laza...@gmail.com wrote:
 On Wed, Oct 3, 2012 at 9:11 PM, i b ibse...@gmail.com wrote:
 Dear all,
 how reliable is running Cuffdiff without replicates? e.g.one samples
 agains another one?

 Is it statistically makign any difference when using replicates?

 Seqanswers might be a better place to ask this very interesting
 technical question that goes way beyond Galaxy...

 My 2c: Statistically speaking, sequencing and biology are both noisy.
 Replicates provide information about non-experimental (technical and
 biological) variation. That variation is usually not the variation you
 are looking for, but if you want to remove it, you have to model it
 and that requires information from replicates (or really good
 guesswork). In some situations (eg extreme experimental conditions),
 I'm sure you'll find biologically meaningful signal without them but
 in my experience, they can really help to decrease non-experimental
 noise, particularly where the experimental condition induces only
 subtle changes in transcript abundance.

 You could always analyse a data set with replicates and compare the
 results with and without those replicates yourself to see what happens
 - it would be a nice paper I'm sure.

A bit off-topic, but you might take a look here:

http://www.ncbi.nlm.nih.gov/pubmed/21747377

In short, one needs replication in biology, regardless of the
technology used.  In particular, one would never suggest running a
microarray experiment without replicates; one should follow
approximately the same rules for sequencing (and sequence data
analysis).

Sean


 Thanks,
 ib
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[galaxy-user] cuffdiff sample values assigned

2012-08-21 Thread Jennifer Jackson

On 8/21/12 4:33 AM, i b wrote:

Thanks Jen,
useful link. But I did not understand one thing.
I have the following FPKM in cufflinks for two samples:
s1 (untreated): 1234106
s2 (treated): 159713

cuffdiff of the two samples gives me the following values:
value_1:5.4
value_2:20.9

and it is not significant (!). My two question:

1. how is this not significant?
2. what is the realtion between the high fpkm in cufflinks and the low
values in cuffdiff?I  read the manual: is this part of the statistical
method adopted?e.g are these  numbers (cuffdiff values) derived from
the formula adopted?

thanks a lot,
ib


On Thu, Aug 16, 2012 at 11:26 PM, Jennifer Jackson j...@bx.psu.edu wrote:

Hello,

A very similar question came up a few days ago and Jeremy had some good
advice for how to approach learning to interpret this data:

http://lists.bx.psu.edu/pipermail/galaxy-user/2012-August/004985.html

Best,

Jen
Galaxy team


On 8/15/12 8:49 AM, i b wrote:


Dear all,
in cuffdiff outputs e.g. transcript differential expression, I find for
example:
value_1 value_2 log2(fold_change)
7.77183 0   -1.79769e+308

or

value_1 value_2 log2(fold_change)
0   14.5972 1.79769e+308

for many many rows.


if I sort in excel my data by fold change column (big to small ), all
the rows with -1.79769e+308 or +1.79769e+308 are on the top.
How can be sure that these on the top are really the most up-regulated
or down regulated transcripts if I don't know the real value of one of
the two samples (is 0 really zero?)?
I was told that the zero in one if the two samples is very small
number and Cuffdiff simply writes 0, but it is not absolutely zero,
otherwise it would not be possible ot have -1.79769e+308 or
1.79769e+308

Could you please tell me then how can I extrapolate the highest fold
change? (up and down regualted)?or of what is done by sorting by log
fold chnage is correct?

Thanks,
ib
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[galaxy-user] Cuffdiff errors

2012-08-16 Thread Yan He
Hello,

 

I am having a problem running Cuffdiff on some RNA-seq data.  I want to
compare 2 samples (A and B). I did Cufflinks and Cuffmerge before running
Cuffdiff. I ran Cuffdiff with the following options: Cuffmerge + Bowtie A, B
(sorted required by Cufflinks after mapped with Bowtie). But I got the
following error message:

 

An error occurred running this job: cuffdiff v1.3.0 (3022)
cuffdiff --no-update-check -q -p 8 -c 10 --FDR 0.05
/galaxy/main_pool/pool4/files/004/800/dataset_4800173.dat
/galaxy/main_pool/pool3/files/004/799/dataset_4799827.dat
/galaxy/main_pool/pool4/files/004/799/dataset_4799831.dat

 

Where did I do wrong? Thanks very much for your help!

 

Yan

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Re: [galaxy-user] Cuffdiff errors

2012-08-16 Thread Jennifer Jackson

Hi Yan,

Would you please submit this as a bug report? It helps if you leave all 
inputs undeleted in your history. Instructions:


http://wiki.g2.bx.psu.edu/Support#Reporting_tool_errors

Thanks!

Jen
Galaxy team

On 8/16/12 6:18 AM, Yan He wrote:

Hello,

I am having a problem running Cuffdiff on some RNA-seq data.  I want to
compare 2 samples (A and B). I did Cufflinks and Cuffmerge before
running Cuffdiff. I ran Cuffdiff with the following options: Cuffmerge +
Bowtie A, B (sorted required by Cufflinks after mapped with Bowtie). But
I got the following error message:

*An error occurred running this job: /cuffdiff v1.3.0 (3022)
cuffdiff --no-update-check -q -p 8 -c 10 --FDR 0.05
/galaxy/main_pool/pool4/files/004/800/dataset_4800173.dat
/galaxy/main_pool/pool3/files/004/799/dataset_4799827.dat
/galaxy/main_pool/pool4/files/004/799/dataset_4799831.dat/*

*//*

Where did I do wrong? Thanks very much for your help!

Yan



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[galaxy-user] cuffdiff with three groups

2012-08-11 Thread i b
Dear all,
I ran Cuffdiff with 3 groups: A, B, C each with 2, 5 and 1 replicates
respectively.
When looking  at the transcripts dif.exp.testing, I have only sample A
and B and redpective values.
What happened to sample C?

Thanks for any help.
ib
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[galaxy-user] cuffdiff: same gene listed with different FPKM values

2012-07-26 Thread i b
Dear all,
has anything like this happened to you?

I compared two samples with cuffdiff and when I look at the
differentially expressed genes values I have the same gene listed for
5 times with different values.

E.g.
sample1sample2gene
71.6837 9.76435 NM_005514
87.6456 27.3965 NM_005514
115.333 4.81687 NM_005514
38.1879 5.2753  NM_005514
69.4197 5.84387 NM_005514
112.964 3.89226 NM_005514


What does this mean? And how do I know which one represents the real
expression level for that gene for the two samples?

Thanks,
ib
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Re: [galaxy-user] cuffdiff results missing

2012-07-22 Thread Jennifer Jackson

Hello Irene,

This issue is similar to the original. The input GTF for this run 
(dataset #15) has tss_id populated, but not p_id. The p_id attribute is 
required for the CDS calculations.


http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff_input

   (quote) Cuffdiff Input:

   AttributeDescription
   p_id The ID of the coding sequence this
   transcript contains. This attribute is attached by Cuffcompare
   to the .combined.gtf records only when it is run with a reference
   annotation that include CDS records. Further, differential CDS
   analysis is only performed when all isoforms of a gene have p_id
   attributes, because neither Cufflinks nor Cuffcompare attempt to
   assign an open reading frame to transcripts.

Dataset #15 was created from a CuffMerge run (which runs Cuffcompare as 
a component). Examining the selections used (clicking on the blue arrow 
rerun icon), shows that the option Use Sequence Data: was set to 
No. Changing this to Yes and using Choose the source for the 
reference list: as Locally cached (and double checking that all 
inputs are assigned to hg19) will assign p_id. Note that this will be 
true only for those transcripts that are associated with reference 
annotation transcripts containing coding regions (in your data: 
'nearest_ref NM_X', not NR_X. NR_ human RefSeq transcripts 
are non-coding).


   Galaxy's CuffMerge tool form has this option labeled:

   (quote) Use Sequence Data:
   Use sequence data for some optional classification
   functions, including the addition of the p_id attribute
   required by Cuffdiff.


Thanks!

Jen
Galaxy team

On 7/21/12 11:04 PM, i b wrote:

Hi,
I ran again cuffdiff using the cuffmerge as gtf.
The following dataset were empty:

128: Cuffdiff on data 12, data 14, and others: CDS FPKM tracking

  127: Cuffdiff on data 12, data 14, and others: CDS FPKM differential
expression testing

126: Cuffdiff on data 12, data 14, and others: CDS overloading
diffential expression testing

The others have data downloadable as excel.

any explanation???

Thanks,
ib

On Fri, Jul 20, 2012 at 12:10 AM, Jennifer Jackson j...@bx.psu.edu wrote:

Hello Irene,

Yes, this is can be the result if your source GTF data did not have the full
compliment of attributes needed by Cuffdiff to perform these calculations.

The primary tool documentation covers this information here:
http://cufflinks.cbcb.umd.edu/manual.html#fpkm_track

The iGenomes datasets are a popular choice for this reason. A version of
UCSC RefGenes is available for certain genomes. Please see:
http://cufflinks.cbcb.umd.edu/igenomes.html (scroll down on page in some
browsers to find table)

Galaxy has one of these already loaded, mm9 genes.gtf, in the Shared Data
- Shared Libraries section on the public Main server. More iGenomes .gtf
files will likely be added here, sometime after the GCC2012 conference. For
now, locally download to your own system/desktop, uncompress, and just load
the GTF file to Galaxy.
Consider FTP for larger datasets: http://wiki.g2.bx.psu.edu/FTPUpload)

More resources include the author supported help email at
tophat.cuffli...@gmail.com and seqanswers.com (where the authors often
post).

Hopefully this helps,

Jen
Galaxy team


On 7/19/12 1:38 PM, i b wrote:


Hi,
I ran cuffdiff using Refseq genes as GTF and two groups of BAM. Group
one has two replicates (treated) and group two only one replicate
(untreated).

When looking at the outputs the following are empty (1 line):

TSS group FPKM tracking
TSS groups differential expression testing
CDS FPKM tracking
CDS FPKM differential expression testing
CDS overloading diffential expression testing
promoters differential expression testing
splicing differential expression testing

the other four outputs have data downloadable as excel.

Is this normal?

thanks,
ib
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Re: [galaxy-user] cuffdiff failed

2012-07-20 Thread Jennifer Jackson

Hello Irene,

There appears to be a problem with the information entered into the tool 
form for the labels (e.g. Group name). The command string only shows 
one group label value when there are two group data sets.


You submitted a bug report for this same issue, so I will take a look 
there at the exact error (usually is very specific about problem) and at 
the exact settings entered into tool form and provide feedback there.


Thanks,

Jen
Galaxy team

On 7/20/12 9:19 AM, i b wrote:

Hi,
I had  3 samples (2replicates treated (A-B) and one untreated (C) ).

I did cufflnks and cuffmerge (all 3 cufflinks)

I run cuffdiff with the following options:
cuffmerge
+ tophats from A, B, C (2 groups: gr.one with A, B), gropu2: C.

I had the following message:

0 bytes
An error occurred running this job: cuffdiff v1.3.0 (3022)
cuffdiff --no-update-check -q -p 8 -c 10 --FDR 0.05 -N -b
/galaxy/data/hg19/sam_index/hg19.fa --labels +
/galaxy/main_pool/pool4/files/004/645/dataset_4645857.dat
/galaxy/main_pool/pool3/files/004/623/dataset_4623286.dat,/galaxy

Where did I do wrong? What does it mean?

Cheers,
ib
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[galaxy-user] cuffdiff results missing

2012-07-19 Thread i b
Hi,
I ran cuffdiff using Refseq genes as GTF and two groups of BAM. Group
one has two replicates (treated) and group two only one replicate
(untreated).

When looking at the outputs the following are empty (1 line):

TSS group FPKM tracking
TSS groups differential expression testing
CDS FPKM tracking
CDS FPKM differential expression testing
CDS overloading diffential expression testing
promoters differential expression testing
splicing differential expression testing

the other four outputs have data downloadable as excel.

Is this normal?

thanks,
ib
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Re: [galaxy-user] cuffdiff results missing

2012-07-19 Thread Jennifer Jackson

Hello Irene,

Yes, this is can be the result if your source GTF data did not have the 
full compliment of attributes needed by Cuffdiff to perform these 
calculations.


The primary tool documentation covers this information here: 
http://cufflinks.cbcb.umd.edu/manual.html#fpkm_track


The iGenomes datasets are a popular choice for this reason. A version of 
UCSC RefGenes is available for certain genomes. Please see: 
http://cufflinks.cbcb.umd.edu/igenomes.html (scroll down on page in some 
browsers to find table)


Galaxy has one of these already loaded, mm9 genes.gtf, in the Shared 
Data - Shared Libraries section on the public Main server. More 
iGenomes .gtf files will likely be added here, sometime after the 
GCC2012 conference. For now, locally download to your own 
system/desktop, uncompress, and just load the GTF file to Galaxy.

Consider FTP for larger datasets: http://wiki.g2.bx.psu.edu/FTPUpload)

More resources include the author supported help email at 
tophat.cuffli...@gmail.com and seqanswers.com (where the authors often 
post).


Hopefully this helps,

Jen
Galaxy team

On 7/19/12 1:38 PM, i b wrote:

Hi,
I ran cuffdiff using Refseq genes as GTF and two groups of BAM. Group
one has two replicates (treated) and group two only one replicate
(untreated).

When looking at the outputs the following are empty (1 line):

TSS group FPKM tracking
TSS groups differential expression testing
CDS FPKM tracking
CDS FPKM differential expression testing
CDS overloading diffential expression testing
promoters differential expression testing
splicing differential expression testing

the other four outputs have data downloadable as excel.

Is this normal?

thanks,
ib
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[galaxy-user] cuffdiff

2012-07-16 Thread Irene Bassano
Hi, 
just few questions about cuffdiff if anyone can answer:

1.how can I load more than two sam/bam files?Galaxy gives spaceonly for two 
files

2.what to use as input: cufflinks, cuffcompare or cuffmerge?


Thanks a lot!

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Re: [galaxy-user] Cuffdiff

2012-04-24 Thread Jennifer Jackson

Hi Ateequr,

This post from today has information another member found at 
seqanswers.com, directly from the CuffLinks/Merge/Diff tool author:

http://user.list.galaxyproject.org/Re-1-cuffcompare-or-cuffmerge-td4581029.html

Best,

Jen
Galaxy team

On 4/17/12 8:00 AM, Ateequr Rehman wrote:

Dear All
I have simple and question for cuffdiff
should we run cuffdif on merge transcript file (produced by cuffmerge)
and concatenate data sets
or directly on cufflink produced files, in the later case, i have two
transcript files resulting from cufflink on sample 1 and 2 respectively,
result using sample 1 as transcripts are not the same when i am suing
sample 2 as transcript

i am bit confused what should be the correct way

any help is very much welcomed

Best
ateeq
Ateequr Rehman
House No. 2 ground floor
Blauenstr. 10
79115 Freiburg im Breisgau


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[galaxy-user] Cuffdiff

2012-04-17 Thread Ateequr Rehman
Dear All
I have simple and question for cuffdiff
should we run cuffdif on merge transcript file (produced by cuffmerge) and 
concatenate data sets
or directly on cufflink produced files, in the later case, i have two 
transcript files resulting from cufflink on sample 1 and 2 respectively,
result using sample 1 as transcripts are not the same when i am suing sample 2 
as transcript 


i am bit confused what should be the correct way

any help is very much welcomed

Best
ateeq

 
Ateequr Rehman
House No. 2 ground floor
Blauenstr. 10
79115 Freiburg im Breisgau___
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Re: [galaxy-user] Cuffdiff result P and q values

2012-03-20 Thread Jennifer Jackson

Hello Ateeq,

It looks like you are working with a bacterial genome. There has been 
some limited discussion on the Galaxy mailing list about using RNA-seq 
tools with circular genomes, but the best resources are probably the 
tool documentation itself (e.g. 
http://cufflinks.cbcb.umd.edu/manual.html#gene_exp_diff  
http://cufflinks.cbcb.umd.edu/howitworks.html#hdif), the tool author's 
Q/A email tophat.cuffli...@gmail.com, and seqanswers.com.


From a quick check, it seems that the 'not significant' result is due 
to the value of Test status being NOTEST.


Definition in documentation link above:
NOTEST (not enough alignments for testing)

Hopefully this helps,

Best,

Jen
Galaxy team

On 3/19/12 11:03 AM, Ateequr Rehman wrote:

Dear galaxy user
After running cuffdiff on my two samples (SAM files from bowtie)
i got a list with p and q values, and löast colum is saying abou
significance
with P value, it seems like the comparison should be significant, but in
Q value is 1, and last coumn is saying not significant

any one have an idea, how to interpret it , should we take any
comparsion with less than 0.05 p value as significant or not

tables in excel looks like it

Nay help is welcome

best
Ateeq


test_id gene_id genelocus   sample_1sample_2
status  value_1
value_2 log2(fold_change)   test_stat   p_value q_value 
significant
CUFF.428.1  CUFF.428-
gi|190572091|ref|NC_010943.1|:1575813-1577629   q1  q2  NOTEST  171.773
605.136 -150.518588.996 3,86E-051   no
CUFF.462.1  CUFF.462-
gi|190572091|ref|NC_010943.1|:1680283-1681214   q1  q2  NOTEST  696.628
322.149 -111.266538.062 7,42E-031   no
CUFF.635.1  CUFF.635-
gi|190572091|ref|NC_010943.1|:2343969-2346219   q1  q2  NOTEST  396.469
223.951 -0.824027   476.902 1,85E-011   no
CUFF.512.1  CUFF.512-
gi|190572091|ref|NC_010943.1|:1840464-1843486   q1  q2  NOTEST  136.314
70.604  -0.949109   422.322 2,41E-011   no
CUFF.632.1  CUFF.632-
gi|190572091|ref|NC_010943.1|:2346561-2347408   q1  q2  NOTEST  351.508
167.567 -106.882415.844 3,20E-011   no
CUFF.941.1  CUFF.941-
gi|190572091|ref|NC_010943.1|:3664426-3665364   q1  q2  NOTEST  282.247
133.798 -10.769 412.254 3,75E-011   no
CUFF.616.1  CUFF.616-
gi|190572091|ref|NC_010943.1|:2301552-2303180   q1  q2  NOTEST  169.682
744.885 -118.774462.107 3,82E-011   no
CUFF.617.1  CUFF.617-
gi|190572091|ref|NC_010943.1|:2295763-2297758   q1  q2  NOTEST  225.933
112.178 -101.011454.517 5,49E-011   no
CUFF.9.1CUFF.9  -   gi|190572091|ref|NC_010943.1|:41597-42402   
q1
q2  OK  1729.08 2797.07 0.693913-4.461  
8,16E-010.000179474 yes
CUFF.956.1  CUFF.956-
gi|190572091|ref|NC_010943.1|:3665445-3669232   q1  q2  NOTEST  518.525
323.653 -0.679966   444.565 8,76E-011   no
CUFF.549.1  CUFF.549-
gi|190572091|ref|NC_010943.1|:2043111-2043664   q1  q2  OK  7148.23
11816.4 0.725138-421.4432,50E+000.000275446 
yes
CUFF.872.1  CUFF.872-
gi|190572091|ref|NC_010943.1|:3489557-3490326   q1  q2  NOTEST  220.274
840.662 -13.897 416.179 3,16E+001   no
CUFF.636.1  CUFF.636-
gi|190572091|ref|NC_010943.1|:2348784-2352394   q1  q2  NOTEST  114.384
601.415 -0.927447   414.807 3,35E+001   no
CUFF.605.1  CUFF.605-
gi|190572091|ref|NC_010943.1|:2271979-2275960   q1  q2  NOTEST  217.007
133.837 -0.697264   409.373 4,24E+001   no
CUFF.568.1  CUFF.568-
gi|190572091|ref|NC_010943.1|:2160538-2164415   q1  q2  NOTEST  74.365
377.013 -0.980011   395.097 7,78E+001   no
CUFF.597.1  CUFF.597-
gi|190572091|ref|NC_010943.1|:2250029-2250918   q1  q2  NOTEST  229.389
105.246 -112.403386.937 0.000109116 1   no

Ateequr Rehman
House No. 2 ground floor
Blauenstr. 10
79115 Freiburg im Breisgau


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[galaxy-user] Cuffdiff result P and q values

2012-03-19 Thread Ateequr Rehman
Dear galaxy user
After running cuffdiff on my two samples (SAM files from bowtie)
i got a list with p and q values, and löast colum is saying abou significance

with P value, it seems like the comparison should be significant, but in Q 
value is 1, and last coumn is saying not significant

any one have an idea, how to interpret it , should we take any comparsion with 
less than 0.05 p value as significant or not 


tables in excel looks like it

Nay help is welcome

best
Ateeq



test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 
log2(fold_change) test_stat p_value q_value significant 
CUFF.428.1 CUFF.428 - gi|190572091|ref|NC_010943.1|:1575813-1577629 q1 q2 
NOTEST 171.773 605.136 -150.518 588.996 3,86E-05 1 no 
CUFF.462.1 CUFF.462 - gi|190572091|ref|NC_010943.1|:1680283-1681214 q1 q2 
NOTEST 696.628 322.149 -111.266 538.062 7,42E-03 1 no 
CUFF.635.1 CUFF.635 - gi|190572091|ref|NC_010943.1|:2343969-2346219 q1 q2 
NOTEST 396.469 223.951 -0.824027 476.902 1,85E-01 1 no 
CUFF.512.1 CUFF.512 - gi|190572091|ref|NC_010943.1|:1840464-1843486 q1 q2 
NOTEST 136.314 70.604 -0.949109 422.322 2,41E-01 1 no 
CUFF.632.1 CUFF.632 - gi|190572091|ref|NC_010943.1|:2346561-2347408 q1 q2 
NOTEST 351.508 167.567 -106.882 415.844 3,20E-01 1 no 
CUFF.941.1 CUFF.941 - gi|190572091|ref|NC_010943.1|:3664426-3665364 q1 q2 
NOTEST 282.247 133.798 -10.769 412.254 3,75E-01 1 no 
CUFF.616.1 CUFF.616 - gi|190572091|ref|NC_010943.1|:2301552-2303180 q1 q2 
NOTEST 169.682 744.885 -118.774 462.107 3,82E-01 1 no 
CUFF.617.1 CUFF.617 - gi|190572091|ref|NC_010943.1|:2295763-2297758 q1 q2 
NOTEST 225.933 112.178 -101.011 454.517 5,49E-01 1 no 
CUFF.9.1 CUFF.9 - gi|190572091|ref|NC_010943.1|:41597-42402 q1 q2 OK 1729.08 
2797.07 0.693913 -4.461 8,16E-01 0.000179474 yes 
CUFF.956.1 CUFF.956 - gi|190572091|ref|NC_010943.1|:3665445-3669232 q1 q2 
NOTEST 518.525 323.653 -0.679966 444.565 8,76E-01 1 no 
CUFF.549.1 CUFF.549 - gi|190572091|ref|NC_010943.1|:2043111-2043664 q1 q2 OK 
7148.23 11816.4 0.725138 -421.443 2,50E+00 0.000275446 yes 
CUFF.872.1 CUFF.872 - gi|190572091|ref|NC_010943.1|:3489557-3490326 q1 q2 
NOTEST 220.274 840.662 -13.897 416.179 3,16E+00 1 no 
CUFF.636.1 CUFF.636 - gi|190572091|ref|NC_010943.1|:2348784-2352394 q1 q2 
NOTEST 114.384 601.415 -0.927447 414.807 3,35E+00 1 no 
CUFF.605.1 CUFF.605 - gi|190572091|ref|NC_010943.1|:2271979-2275960 q1 q2 
NOTEST 217.007 133.837 -0.697264 409.373 4,24E+00 1 no 
CUFF.568.1 CUFF.568 - gi|190572091|ref|NC_010943.1|:2160538-2164415 q1 q2 
NOTEST 74.365 377.013 -0.980011 395.097 7,78E+00 1 no 
CUFF.597.1 CUFF.597 - gi|190572091|ref|NC_010943.1|:2250029-2250918 q1 q2 
NOTEST 229.389 105.246 -112.403 386.937 0.000109116 1 no 

 
Ateequr Rehman
House No. 2 ground floor
Blauenstr. 10
79115 Freiburg im Breisgau___
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[galaxy-user] Cuffdiff errors

2012-01-20 Thread Erin Shanle
Hello
I am having a problem running Cuffdiff on some RNA-seq data.  I want to
compare 2 of my conditions.  I successfully used Cuffdiff three days ago to
compare two sets of data that are processed the exact same way (align with
Tophat and use Picard to confirm adequate alignment). I am using Galaxy
through MAIN and I tried Cuffdiff with these samples on 1/18 and 1/19.
Here's the error:

An error occurred running this job: *cuffdiff v1.3.0 (3022)
cuffdiff --no-update-check -q -p 8 -c 1000 --FDR 0.05 -b
/galaxy/data/hg19/sam_index/hg19.fa --labels DMSO,E2
/galaxy/main_pool/pool2/files/003/607/dataset_3607369.dat
/galaxy/main_pool/pool2/files/003/590/dataset_3590726.dat,/g*

Thanks for the help.
-- 
Erin Shanle

Graduate Research Assistant
Molecular and Environmental Toxicology
425 McArdle Laboratory for Cancer Research
1400 University Ave
Madison, WI 53706
608 262 9834
sha...@wisc.edu
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Re: [galaxy-user] Cuffdiff errors

2012-01-20 Thread Jennifer Jackson

Hello Erin,

This was a temporary problem due to a new filesystem we installed during 
this time frame, that has since been resolved. Please try again and if 
the problem persists, please send a bug report from the error dataset 
using the green bug icon. This allows us to gain access to the inputs 
and job parameters to diagnose the root cause of the problem.


http://wiki.g2.bx.psu.edu/Support#Error_from_tools
http://wiki.g2.bx.psu.edu/Support#Unexpected_scientific_result

Thank you!

Jen
Galaxy team

On 1/20/12 6:51 AM, Erin Shanle wrote:

Hello
I am having a problem running Cuffdiff on some RNA-seq data.  I want to
compare 2 of my conditions.  I successfully used Cuffdiff three days ago
to compare two sets of data that are processed the exact same way (align
with Tophat and use Picard to confirm adequate alignment). I am using
Galaxy through MAIN and I tried Cuffdiff with these samples on 1/18 and
1/19.  Here's the error:

An error occurred running this job: /cuffdiff v1.3.0 (3022)
cuffdiff --no-update-check -q -p 8 -c 1000 --FDR 0.05 -b
/galaxy/data/hg19/sam_index/hg19.fa --labels DMSO,E2
/galaxy/main_pool/pool2/files/003/607/dataset_3607369.dat
/galaxy/main_pool/pool2/files/003/590/dataset_3590726.dat,/g/

Thanks for the help.
--
Erin Shanle

Graduate Research Assistant
Molecular and Environmental Toxicology
425 McArdle Laboratory for Cancer Research
1400 University Ave
Madison, WI 53706
608 262 9834
sha...@wisc.edu mailto:sha...@wisc.edu


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--
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http://usegalaxy.org
http://galaxyproject.org/wiki/Support
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[galaxy-user] Cuffdiff question about using an unspecified (?) database/build

2011-08-19 Thread David K Crossman
Hello!

I have an RNA-Seq project which consists of 5 samples from the 
species tree shrew.  When uploading these fastq files into Galaxy, I chose 
unspecified (?) for the database/build since the latest tree shrew version is 
not in the drop down list.  When using TopHat, Cufflinks/Compare I have 
selected a reference genome from my history instead of using a built-in index, 
as well as a gtf annotation file for Cufflinks/Compare and everything has been 
working fine.  Now, I am at the Cuffdiff step and I am running into an error 
when setting it up to perform replicate analysis.  When I select my TopHat 
accepted hits bam file I see a red X and the error: Unspecified genome build, 
click the pencil icon in the history item to set the genome build.  Here's a 
screenshot of what I'm seeing:

[cid:image001.png@01CC5E4E.76F37AF0]

Since the latest reference genome for tree shrew wasn't listed, 
that's why I chose unspecified (?).  Should I go back and edit these accepted 
hits bam files to say the Database/Build from the drop down list is Tree shrew 
Dec. 2006 (Broad/tupBel1) (tupBel1)?  I know that this is simple to change, 
but will this affect my results in any way?  Any help/info would be greatly 
appreciated.

Thanks,
David
inline: image001.png___
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Re: [galaxy-user] Cuffdiff question about using an unspecified (?) database/build

2011-08-19 Thread David K Crossman
Jen,

Thank you very much for the reply.  I'm glad to know it is a known bug 
and not something on my side of things.  So, would my analysis be affected if I 
did change the bam file Database/Build to the older tree shrew version found 
in the drop down list?  What significance does this Database/Build box have 
in downstream analysis if you have your own fasta reference genome file and gtf 
annotation file that is being referenced instead of a locally cached one?  I'm 
just trying to obtain a better understanding of the Database/Build box for 
analyses where I provide the fasta and gtf file.

Thanks,
David


-Original Message-
From: Jennifer Jackson [mailto:j...@bx.psu.edu] 
Sent: Friday, August 19, 2011 9:20 AM
To: David K Crossman
Cc: galaxy-user (galaxy-user@lists.bx.psu.edu)
Subject: Re: [galaxy-user] Cuffdiff question about using an unspecified (?) 
database/build

Hello David,

This is a known bug. The correction is planned to be moved out onto the public 
Galaxy instance at the next update (within a week).

Sorry for the current inconvenience,

Best,

Jen
Galaxy team

On 8/19/11 7:00 AM, David K Crossman wrote:
 Hello!

 I have an RNA-Seq project which consists of 5 samples from the species 
 tree shrew. When uploading these fastq files into Galaxy, I chose 
 unspecified (?) for the database/build since the latest tree shrew 
 version is not in the drop down list. When using TopHat, 
 Cufflinks/Compare I have selected a reference genome from my history 
 instead of using a built-in index, as well as a gtf annotation file 
 for Cufflinks/Compare and everything has been working fine. Now, I am 
 at the Cuffdiff step and I am running into an error when setting it up 
 to perform replicate analysis. When I select my TopHat accepted hits 
 bam file I see a red X and the error: Unspecified genome build, click 
 the pencil icon in the history item to set the genome build. Here's a 
 screenshot of what I'm seeing:

 Since the latest reference genome for tree shrew wasn't listed, that's 
 why I chose unspecified (?). Should I go back and edit these 
 accepted hits bam files to say the Database/Build from the drop down 
 list is Tree shrew Dec. 2006 (Broad/tupBel1) (tupBel1)? I know that 
 this is simple to change, but will this affect my results in any way? 
 Any help/info would be greatly appreciated.

 Thanks,

 David



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 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 
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 source code, please use the Galaxy Development list:

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--
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http://galaxyproject.org/Support

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Re: [galaxy-user] Cuffdiff Question

2011-06-28 Thread Jeremy Goecks
Hello Kurinji,

 I was at your USC Galaxy seminar last week, which I found very helpful - 
 thank you!

Glad to hear that you found the workshop helpful. As a reminder, please email 
questions about using Galaxy and its tools to the galaxy-user mailing list 
(which I've cc'd). You may get quicker and different responses from community 
members, and everyone will benefit from the discussion.

 I used my recently generated RNAseq data in Galaxy (which was pre-aligned 
 using tophat and already had cufflinks run on it) - I ran cuffcompare with 
 all the gtf files and then cuffdiff for the three pairs (there is 1 control 
 and 3 different drug treatments - no replicates). I got several output files, 
 as expected, but decided just to look at the gene differential expression as 
 a start. Some questions I have are - 
 
 1. (very basic question!) which is sample 1 (and corresponding value 1) and 
 sample 2 (and corresponding value 2)in my output file. This is what my output 
 file is called - 
 
 90: Cuffdiff on data 37, data 38, and data 60: gene differential expression 
 testing 33,969 lines
 
 Is 37 sample one or sample two? Given the data - I would expect sample 37 to 
 correspond to value 2 - but I could be wrong. Please let me know!

The best way to figure out which dataset corresponds with Cuffdiff's labels is 
to click the rerun button in the dataset: sample names correspond directly to 
the reads datasets (i.e. BAM files) provided as input to Cuffdiff.

 2. How do I find the UCSC gene names corresponding with start/end sites - I 
 did input the hg18 UCSC gtf file as a reference


You'll need to use a reference annotation (GTF file) that has the gene_name 
attribute as input for Cufflinks/compare/difff. Typically Ensembl annotations 
have this attribute; however, you'll need to prepend 'chr' to each 
line--really, to each chromosome name--in order to bring Ensembl notation in 
line with UCSC/Galaxy notation.

 Actually, I noticed that value 1 in this particular output file is all 0 - no 
 idea why. It is not this way in the other files, making me wonder if there is 
 an error somewhere. I am sure the bam file is okay as I viewed it on IGV and 
 saw the patterns I would expect for some candidate genes I looked at.

It's difficult for me to comment without seeing your analysis. Some output 
files depend on particular attributes being set correctly in the annotation 
file. You may want to search through our mailing list archives and see if your 
question has already been answered: 
http://gmod.827538.n3.nabble.com/Galaxy-Users-f815892.html

Good luck,
J.___
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Re: [galaxy-user] Cuffdiff Question

2011-06-28 Thread Jeremy Goecks

 Thanks for the reply. I tried to use the script provided on a previous galaxy 
 thread for adding the chr on to the gtf file on the mac terminal but I keep 
 getting this error - 
 
 awk: can't open file ensembl.gtf
  source line number 1
 
 I am very new to using the terminal so please let me know if there is 
 something basic that I am not doing right,

Try this Galaxy workflow:

http://main.g2.bx.psu.edu/u/jeremy/w/make-ensembl-gtf-compatible-with-cufflinks

It simply prepends 'chr' to the chromosome name, which is needed if you're 
using an Ensemble reference annotation and want to use it with 
Cufflinks/compare/diff in Galaxy.

Best,
J.
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Re: [galaxy-user] CuffDiff gene fpkm tracking file.

2011-02-03 Thread Jeremy Goecks
 
 this is an example of my CuffDiff gene fpkm tracking file.
 
 tracking_id class_code  nearest_ref_id  gene_short_name tss_id  locus 
   q1_FPKM q1_conf_lo  q1_conf_hi  q2_FPKM q2_conf_lo  q2_conf_hi
 XLOC_01 -   -   MT-ND5  -   chrM:0-1657112484.2 
 12260.8 12707.7 11447   11233.1 11661
 XLOC_02 -   -   USP14   TSS1,TSS2,TSS3  chr18:148586-236453   
   16.7235 9.41244 24.0346 19.437  11.7368 27.1371
 XLOC_03 -   -   SMCHD1  
 TSS10,TSS11,TSS12,TSS4,TSS5,TSS6,TSS7,TSS8,TSS9 chr18:2719322-2728540   
 28.2493 17.5093 38.9892 27.2263 16.6263 37.8262
 XLOC_04 -   -   EMILIN2 TSS13,TSS14 chr18:2880607-2882469 
   3.98118 0   7.99721 4.62875 0.2785198.97899
 
 I this is normal, how can I find the class code of transcript listed in the 
 CuffDiff gene expression file?
 


Hi Samuele,

Without seeing your history, it's difficult to say for certain what your 
problem is. However, I'd guess that the GTF file that you're providing to 
Cuffdiff does not have the p_id attribute. You can produce a GTF file with both 
tss_id and p_id attributes by running Cuffcompare and using sequence data.

Thanks,
J.
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