Re: [galaxy-user] Cuffdiff output

[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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Re: [galaxy-user] Cuffdiff question

[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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> 

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Re: [galaxy-user] Cuffdiff question

[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  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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>> 
>> 
> 
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Re: [galaxy-user] Cuffdiff question

[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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Re: [galaxy-user] Cuffdiff version not apparent

[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"  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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Re: [galaxy-user] Cuffdiff changes

[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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Re: [galaxy-user] Cuffdiff changes

[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

[Jeremy Goecks]

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

[Jeremy Goecks]

Thanks for the information. 

I've added an option to the Cuffdiff tool so that the read group files can be 
output by Galaxy; this should make it possible to run Cuffdiff in Galaxy and 
then Cummerbund for replicate analysis. This change will make it out to our 
public server in a couple weeks.

Thanks,
J.

On Jul 31, 2013, at 5:39 PM, Mike Shamblott wrote:

> Thanks 
> 
> My tests confirm that cummerbund does require the run group files to do 
> replicate analyses. Therefore Galaxy cannot be used to do cuffdiff on 
> replicates when cummerbund is the next step in the workflow. 
> 
> Mike
> 
> 
> 
> Sent from my iPhone
> 
> On Jul 31, 2013, at 9:45 AM, Jeremy Goecks  wrote:
> 
>> 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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>> 

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

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

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

[Mohammad Heydarian]

Hi Jeremy,
The header of the Cuffdiff tool page says it is version 0.0.5

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



Cheers,
Mo Heydarian

PhD candidate
The Johns Hopkins School of Medicine
Department of Biological Chemistry
725 Wolfe Street
402 Biophysics
Baltimore, MD 21205


On Wed, Mar 13, 2013 at 12:41 PM, Jeremy Goecks wrote:

> 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"  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?

[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"  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?

[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"  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?

[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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Re: [galaxy-user] cuffdiff

[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

[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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Re: [galaxy-user] cuffdiff fpkm=0 "not significant"

[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

[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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Re: [galaxy-user] Cuffdiff no without replicates

[Sean Davis]

On Wed, Oct 3, 2012 at 7:35 AM, Ross  wrote:
> On Wed, Oct 3, 2012 at 9:11 PM, i b  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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Re: [galaxy-user] Cuffdiff no without replicates

[Ross]

On Wed, Oct 3, 2012 at 9:11 PM, i b  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 sample values assigned

[Jennifer Jackson]

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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Re: [galaxy-user] Cuffdiff errors

[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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Re: [galaxy-user] cuffdiff with three groups

[i b]

Sorry, I did not read carefully. All three samples are listed, just
down in the column I found the other sample.

ib

On Sat, Aug 11, 2012 at 7:41 PM, i b  wrote:
> 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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Re: [galaxy-user] cuffdiff: same gene listed with different FPKM values

[Jennifer Jackson]

Hello Irene,

These are RefSeq transcript identifiers. In some GTF file sources, 
transcript_id is also assigned to the gene_id attribute in the 9th 
field, which can cause confusion. Alternative GTF sources are Ensembl 
and iGenomes. However, examining differential expression at the 
transcript level may be best, as Leonor explained in response to your 
other recent post:

http://lists.bx.psu.edu/pipermail/galaxy-user/2012-July/004953.html

Thanks,

Jen
Galaxy team

On 7/26/12 1:59 PM, i b wrote:

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

[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  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

[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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Re: [galaxy-user] cuffdiff results missing

[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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Re: [galaxy-user] cuffdiff

[Jennifer Jackson]

Hello,

On 7/16/12 10:50 PM, i b wrote:

ok, so if I have 3 different samples I would use 3 different groups and
add my samples as replicate within each group.


Yes, where each sample represents an individual condition.


or did you mean to create just one group and add all my 3 samples as 3
replicates within that only group?


No, would be incorrect. Only replicates from the same sample/condition 
should be in the same group. This is not appear to be the case for your 
data, given the additional information.


Thanks!

Jen
Galaxy team

thanks a lot
ngs-ib

From: Jennifer Jackson [j...@bx.psu.edu <mailto:j...@bx.psu.edu>]
Sent: Tuesday, July 17, 2012 1:58 AM
To: Irene Bassano
Cc: galaxy-user@lists.bx.psu.edu <mailto:galaxy-user@lists.bx.psu.edu>
Subject: Re: [galaxy-user] cuffdiff

Hello Irene,

On 7/16/12 4:11 PM, Irene Bassano wrote:
 > 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

Change the tool form for the option "Perform replicate analysis:" to be
"Yes" (the default is "No"). Use two conditions (Groups).

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

GTF output from any of these may be appropriate.

Please see the tutorial on Galaxy main at:
http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise
FAQ:
http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq

And the "Common uses" protocols on the Cufflinks web site plus their new
paper (also linked at this site, from side bar, as "Protocol"):
http://cufflinks.cbcb.umd.edu/tutorial.html

Best,

Jen
Galaxy team

 >
 >
 > Thanks a lot!
 >
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 > Galaxy analysis and other features on the public server
 > at usegalaxy.org <http://usegalaxy.org>. Please keep all replies on
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 >
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 >

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Re: [galaxy-user] cuffdiff

[Jennifer Jackson]

Hello Irene,

On 7/16/12 4:11 PM, Irene Bassano wrote:

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


Change the tool form for the option "Perform replicate analysis:" to be 
"Yes" (the default is "No"). Use two conditions (Groups).




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


GTF output from any of these may be appropriate.

Please see the tutorial on Galaxy main at:
http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise
FAQ:
http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq

And the "Common uses" protocols on the Cufflinks web site plus their new 
paper (also linked at this site, from side bar, as "Protocol"):

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

Best,

Jen
Galaxy team




Thanks a lot!

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Re: [galaxy-user] Cuffdiff

[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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Re: [galaxy-user] Cuffdiff

[Carlos Borroto]

Hi Ateequr,

I don't think there is an easy answer to your question. I would highly
recommend you take a look to this recently released paper from Tophat
and Cufflinks authors:
Differential gene and transcript expression analysis of RNA-seq
experiments with TopHat and Cufflinks.
http://www.ncbi.nlm.nih.gov/pubmed/22383036

The somehow short answer to your question is, you should run cuffdiff
with the transcript file from cuffmerge, although I don't quite
understand what you mean by "and concatenate data sets". You should
not concatenate the BAM files, or you won't be able to run cuffdiff
correctly.

Depending on how you ran cufflinks, by using the transcript file from
cuffmerge you will get expression values for possible new transcripts
not present in the databases. This is commonly desired. If you aren't
interested in possible new transcripts, then you could simply use a
GFF file with transcripts definitions for the genome you are using. In
this case you will have the advantage of getting the results with IDs
compatibles with the database source the GFF came from. If you use the
cuffmerge file you might have the results using cufflinks generated
IDs.

Hope it helps,
Carlos


On Tue, Apr 17, 2012 at 11:00 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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Re: [galaxy-user] Cuffdiff result P and q values

[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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Re: [galaxy-user] Cuffdiff errors

[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 


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

[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 
> client.  For discussion of local Galaxy instances and the Galaxy 
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> use the interface at:
>
>http://lists.bx.psu.edu/

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

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Re: [galaxy-user] Cuffdiff question about using an unspecified (?) database/build

[Jennifer Jackson]

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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--
Jennifer Jackson
http://usegalaxy.org
http://galaxyproject.org/Support
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Re: [galaxy-user] Cuffdiff Question

[Kurinji Pandiyan]

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,

Thanks!
Kurinji

On Tue, Jun 28, 2011 at 6:13 AM, Jeremy Goecks wrote:

> 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

[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 Question

[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 gene fpkm tracking file.

[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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