Re: [galaxy-user] Galaxy Pages - importing/downloading embedded data.

[Mohammad Heydarian]

Thanks so much, Jen!

Cheers,
Mo Heydarian

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


On Thu, Nov 7, 2013 at 5:09 PM, Jennifer Jackson  wrote:

>  Hi Mo,
>
> Datasets are a bit tricky for the download/import steps on Pages. Do this:
>
> 1 - click on the dataset name to expand it
> 2 - you will see a message "This dataset is large and only the first
> megabyte is shown below. | Show all".
> 3 - Click on the "Show all". On the resulting view, the import icon is in
> the upper right corner.
>
> I decided to open a ticket to make this more direct (have both download
> and import directly on a Page, to right of object, in the box, as it is for
> other embedded types). Here is the link so that you can track progress,
> should the team like the idea: https://trello.com/c/MOGOkexa
>
> Yes, publish all objects if this is going to be open for everyone (the
> History will include Datasets by default, then Workflows and
> Visualizations). You have to keep these around - don't delete!
>
> I now this is obvious, so is just general advice for whoever may read this
> post - but when Publishing - I also annotate very carefully and save a
> backup of anything important. Worth it :)
>
> Good question!
>
> Jen
> Galaxy team
>
>
> On 11/7/13 12:14 PM, Mohammad Heydarian wrote:
>
> Hi All,
> Hope things are groovy.
>
>  I am in the process of setting up a Galaxy page on the main server to
> host our NGS data. I am embedding the various datasets and they are showing
> up and viewable, but there is no way to import (or download) the data. Am I
> correct, or is the import button hidden on my screen? Would publishing the
> Page enable import of the data?
>
>  Many thanks in advance, we are looking forward to utilizing Galaxy Pages
> and making our data accessible.
>
>
> Cheers,
> Mo Heydarian
>
> PhD candidate
> The Johns Hopkins School of Medicine
> Department of Biological Chemistry
> 725 Wolfe Street
> 402 Biophysics
> Baltimore, MD 21205
>
>
> ___
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>
> --
> Jennifer Hillman-Jacksonhttp://galaxyproject.org
>
>
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[galaxy-user] Galaxy Pages - importing/downloading embedded data.

[Mohammad Heydarian]

Hi All,
Hope things are groovy.

I am in the process of setting up a Galaxy page on the main server to host
our NGS data. I am embedding the various datasets and they are showing up
and viewable, but there is no way to import (or download) the data. Am I
correct, or is the import button hidden on my screen? Would publishing the
Page enable import of the data?

Many thanks in advance, we are looking forward to utilizing Galaxy Pages
and making our data accessible.


Cheers,
Mo Heydarian

PhD candidate
The Johns Hopkins School of Medicine
Department of Biological Chemistry
725 Wolfe Street
402 Biophysics
Baltimore, MD 21205
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Re: [galaxy-user] Contents of a SAM file

[Mohammad Heydarian]

Hi Benjy,
To extract unmapped reads from a SAM file in Galaxy, you can use 'Filter
SAM' ('NGS: SAM Tools' -> 'Filter SAM'). Add a 'flag' and use the 'The read
is unmapped' type, then set the state for this flag to 'Yes'. This should
return a SAM file with your unmapped reads.



Cheers,
Mo Heydarian

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


On Thu, Nov 7, 2013 at 1:22 PM, Benjamin Osei-agyeman <
benjy_o...@yahoo.co.uk> wrote:

> Thanks a lot,
>
> If i want to extract the unmapped reads from the SAM file after bowtie has
> run do I have to use Samtools or can this be done straightaway using
> galaxy? What command should I use?
>
> Thanks
>
> Benjy
>
>  --
> * From: * Jeremy Goecks ;
> * To: * Benjamin Osei-agyeman ;
> * Cc: * ;
> * Subject: * Re: [galaxy-user] Contents of a SAM file
> * Sent: * Thu, Nov 7, 2013 1:12:44 PM
>
>   All reads are in the SAM file; you can filter to remove unmapped reads
> as needed.
>
> J.
>
> On Nov 7, 2013, at 5:36 AM, Benjamin Osei-agyeman 
> wrote:
>
>
> ___
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Re: [galaxy-user] Exceptionally high RPKM values of miRNA and other short genes in Cuffdiff's output

[Mohammad Heydarian]

Hi Thanh,
This is due to Cuffdiff correcting for the size of smaller transcripts, the
authors call it the "effective length correction". It is supposed to
correct the loss of shorter transcripts upon size selection in creating
your RNA-seq library. The default setting on Galaxy is to use the
"effective length correction".

Cole Trapnell, the creator of the "Cuff-suite tools", discusses this length
correction here:
http://seqanswers.com/forums/showpost.php?p=76430&postcount=32

Some library preparation protocols don't include a size selection. The one
we favor, and Illumina recommends, ScriptSeq v2 from Epicentre (owned by
Illumina), does not include a size selection step. It would be great if
there was an option in the Cuffdiff wrapper in Galaxy to turn off the
effective length correction.



Cheers,
Mo Heydarian

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


On Thu, Jul 18, 2013 at 12:55 PM, Hoang, Thanh  wrote:

> Hi all,
> I have been analyzing my RNA-seq data on mouse tissues. My RNA-data is
> single-ended and 51 bp in length. I ran TopHat/Cufflink/Cuffdiff to test to
> differential gene expression
> In the Cuffdiff's output, I got very high RPKM value for some of miRNA and
> some other short genes ( less than 100bp). These genes are in the top genes
> with the highest RPKM. I think the RPKM values of these genes are probably
>  too high to be true.
>   *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*  *ENSMUSG0093077* *ENSMUSG0093077* *Mir5105* *
> 5:146231229-146302874* *Epithelium* *Fiber* *OK* *1.53E+06* *  445558* *
> -1.78097* *-355.367* *0.00715* *0.016986* *yes*  *ENSMUSG0093098* *
> ENSMUSG0093098* *Gm22641* *7:130162450-133124354* *Epithelium* *Fiber*
> *OK* *87894.1* * 36474.7* *-1.26887* *-0.59863* *0.4913* *0.587174* *no*
> *ENSMUSG0089855* *ENSMUSG0089855* *Gm15662* *
> 10:105187662-105583874* *Epithelium* *Fiber* *OK* *42868.9* * 21566.5* *
> -0.99114* *-20.7066* *0.0186* *0.039568* *yes*  *ENSMUSG0092984* *
> ENSMUSG0092984* *Mir5115* *2:73012853-73012927* *Epithelium* *Fiber* *
> OK* *21104.8* * 8317.49* *-1.34335* *-447.314* *0.0001* *0.000354* *yes*
> *ENSMUSG0086324* *ENSMUSG0086324* *Gm15564* *16:35926510-36037131*
> *Epithelium* *Fiber* *OK* *6443.35* * 3664.15* *-0.81433* *-1.52095* *
> 0.2129* *0.301429* *no*  *ENSMUSG0092981* *ENSMUSG0092981* *
> Mir5125* *17:23803186-23824739* *Epithelium* *Fiber* *OK* *5974.14* *
> 2390.75* *-1.32127* *-0.34111* *0.5746* *0.661937* *no*
>
>  I checked some forums and they said that this is the drawback of
> TopHat/Cufflink/Cuffdiff when dealing with short genes. But I am still not
> so clear about this. Anyone got the same problem? What can I do with this
> situation?
> Anyone suggests any other good tools to test for (1) differential gene
> expression OR (2) both differential gene expression and gene discovery?
>
> Thank you
> Thanh
>
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Re: [galaxy-user] [galaxy-dev] Connecting to new Galaxy Cloudman by FTP

[Mohammad Heydarian]

Hey Nate,
Thanks for the response and instructions.

I understand the last three steps of your protocol, but the first step is
difficult for me to understand. I'm guessing that, "1. Set 'use_pbkdf2 =
False' in universe_wsgi.ini anywhere in the [app:main] section", is telling
me to change a setting of Cloudman by command line. This is generally where
we get stuck in using Cloudman, because we aren't familiar with command
line (we get cold sweats and light palpitations) and are weary of making
changes to the Cloudman code.

I would ask our IT guys for help, but their expertise ends at updating
Office tools. I would bother a programmer or bioinformatician, but most of
them are so busy you need a formal collaboration to get on their radar. I
would ask people who vaguely "know" command line for help, but most of the
time their knowledge of command line is just higher than mine and we end up
troubleshooting an issue neither of us can really grasp.

So, is there a webcast, or video, or slideshow, that can show a newbie how
to command line into Cloudman and make the changes you outline in step 1?



Cheers,
Mo Heydarian




On Mon, Jul 15, 2013 at 4:22 PM, Nate Coraor  wrote:

> On Jul 8, 2013, at 3:50 PM, Mohammad Heydarian wrote:
>
> > Hi,
> > I am having trouble setting up a FTP connection with the recently
> released version of Galaxy Cloudman (ami-118bfc78).
> >
> > I have instantiated the new version of Galaxy Cloudman with CloudLaunch
> and also through the AWS EC2 wizard (using the same security group settings
> as the previous versions) and neither instance will connect to my FTP
> connection.
> >
> > Has anyone else had this problem? Does anyone know what is preventing
> the FTP connection?
> >
> > Any help would be greatly appreciated.
>
> Hey Mo,
>
> This may be a case of the new password hashing algorithm's incompatibility
> with the provided ProFTPD config.  Could you try the following:
>
> 1. Set 'use_pbkdf2 = False' in universe_wsgi.ini anywhere in the
> [app:main] section
> 2. Restart Galaxy
> 3. Reset your password in the Galaxy UI
> 4. Test FTP again
>
> --nate
>
> >
> >
> > Cheers,
> > Mo Heydarian
> >
> > ___
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[galaxy-user] Connecting to new Galaxy Cloudman by FTP

[Mohammad Heydarian]

Hi,
I am having trouble setting up a FTP connection with the recently released
version of Galaxy Cloudman (ami-118bfc78).

I have instantiated the new version of Galaxy Cloudman with
CloudLaunch and
also through the AWS EC2 wizard (using the same security group settings as
the previous versions) and neither instance will connect to my FTP
connection.

Has anyone else had this problem? Does anyone know what is preventing the
FTP connection?

Any help would be greatly appreciated.


Cheers,
Mo Heydarian
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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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>> Galaxy analysis and other features on the public server
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>> use the Galaxy Development list:
>>
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>>
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>> please use the interface at:
>>
>>   http://lists.bx.psu.edu/
>>
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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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[galaxy-user] Counting RNA-seq reads per class.

[Mohammad Heydarian]

Hi All,
I have been trying to count the number of RNA-seq reads that fall into the
various Cufflinks class codes ('=', 'j', 'u', 'x', etc...) and I am curious
how others are determining how to count reads per class..

I tried first using the BedTools tool where you "count" the number of reads
overlapping another set of intervals and later realized that each interval
is extended1 kb up and downstream prior to the analysis (by default and not
adjustable on Galaxy), so the number of reads that were "counted" for all
of the classes was always much more than the amount of reads that I had for
my Bam file. I then tried to isolate reads from each class into separate
BAM files, using the BedTools "intersect" tool and there I consistently end
up with significantly less reads than I have in my sample.

I am very curious to find out how others are tackling this problem on
Galaxy.

Thanks for any input!

Cheers,
Mo Heydarian
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[galaxy-user] Counting intervals in one file overlapping intervals in another file - what are the hidden settings?

[Mohammad Heydarian]

Hi,
I am using the "Count intervals in one file overlapping intervals in
another file" tool (part of the bedTools package) to assess the number of
RNA-seq reads that map back to a specific region. (I am using this on the
Galaxy test server). I am finding that this tool returns many more reads
than it should.

In reading the bedTools manual, it seems like this tool is the "windowBed"
tool and it actually has many more parameters that are not shown on the
Galaxy interface. What are these (hidden) settings for these parameters
that are not shown?

Hopefully this can explain my incorrectly recorded reads.

Thanks in advance.


Cheers,
Mo Heydarian
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[galaxy-user] Generating pile-up files without spliced (intronic) regions.

[Mohammad Heydarian]

Hello,
Is there a way to generate a pile-up from a BAM file in SAMtools without
including spliced (intronic) regions? Alternatively, is there a way to
split spliced reads in a BAM file?

Thanks in advance!



Cheers,
Mo Heydarian
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[galaxy-user] Cuffmerge - "Use Sequence" option.

[Mohammad Heydarian]

Hi,
Can anyone shed light on what the "use sequence" parameter (Y/N) in
Cuffmerge is doing? The Cuffflinks manual says:

-s/--ref-sequence / This argument should point to the
genomic DNA sequences for the reference.

When "no" is chosen for the "use sequence" option I still get genomic
coordinates for each entry. Also, when "no" is chosen, I get less entries
than when "yes" is chosen.

Any insight would be greatly appreciated.

Have a great day.


Cheers,
Mo Heydarian
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