Hello Tao,

For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com.

Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq

For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build".

Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization

As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation.

Best,

Jen Galaxy team

On 8/18/11 3:15 PM, Peng, Tao wrote:

Hi jen, I have used BOWTIE to align my RNA-seq reads to HSV2 genome; out of 35,000,000 lines, only 621 lines left when I chose to have mapped reads only. How can visualize these aligned reads to HSV-2 genome?

In the panel of converted SAM to BAM, I tried to use the data in trickster, but I am not sure to how to build a HSV genome as a reference?

I appreciate your help,

tao

===> Please use "Reply All" when responding to this email! <===

Hi jen, I followed the GALAXY web cast to check the quality of RNA-seq data: one sample seem to have score above 20 in most bases (R2); but the other one is around 6-8 in most bases (R4) (see the attached PDF files).

Does this mean R4 RNA-seq data are BAD? What exactly does it mean anyway?

Thanks for your help,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thu 8/18/2011 3:46 PM To: galaxy-user@bx.psu.edu Cc: Peng, Tao Subject: visualization of alignment

Hello Tao,

For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com.

Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq

For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build".

Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization

As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation.

Best,

Jen Galaxy team

On 8/18/11 3:15 PM, Peng, Tao wrote:

Hi jen, I have used BOWTIE to align my RNA-seq reads to HSV2 genome; out of 35,000,000 lines, only 621 lines left when I chose to have mapped reads only. How can visualize these aligned reads to HSV-2 genome?

In the panel of converted SAM to BAM, I tried to use the data in trickster, but I am not sure to how to build a HSV genome as a reference?

I appreciate your help,

tao

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

Thanks, jen. I have asked informatic scientists at Hutch to do the QC for me and both R2 and R4 are ok from FASTQC analysis.

My question is: Do I still need to use the groomer in GALAXY and use the groomed data for further analysis such as TOPHAT? Should I skip the steps to compute quality statistics and draw boxplots using the groomed data?

Thanks,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Fri 8/26/2011 7:19 PM To: galaxy-user Cc: Peng, Tao Subject: Re: [galaxy-user] quality score

===> Please use "Reply All" when responding to this email!<===

Hello Tao,

The tool "NGS: QC and manipulation -> FastQC" (last tool in group) may be helpful for your project.

In general, sequence with quality scores this low would be considered unusable. Perhaps double check the options used with the Fastq Groomer tool? Or check/filter the data before grooming?

This may not be the case for your data, but just in case, please note that CASAVA 1.8+ now produces both filtered and unfiltered results and would need to be used with the "Sanger" option with the "Fastq Groomer" tool.

This prior Q&A explains the filtering: http://gmod.827538.n3.nabble.com/Filtering-Illumina-CASAVA-1-8-FASTQ-files-t...

Hopefully this helps. Please send future questions directly to the mailing list as the "to" recipient. There is no need to send directly "to" or as "cc" any of the Galaxy team directly. This helps us to track and address questions quickly and as a team.

Best,

Jen Galaxy team

Hi jen, I followed the GALAXY web cast to check the quality of RNA-seq data: one sample seem to have score above 20 in most bases (R2); but the other one is around 6-8 in most bases (R4) (see the attached PDF files).

Does this mean R4 RNA-seq data are BAD? What exactly does it mean anyway?

Thanks for your help,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thu 8/18/2011 3:46 PM To: galaxy-user@bx.psu.edu Cc: Peng, Tao Subject: visualization of alignment

Hello Tao,

For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com.

Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq

For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build".

Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization

As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation.

Best,

Jen Galaxy team

On 8/18/11 3:15 PM, Peng, Tao wrote:

...

Hi jen, I have used BOWTIE to align my RNA-seq reads to HSV2 genome; out of 35,000,000 lines, only 621 lines left when I chose to have mapped reads only. How can visualize these aligned reads to HSV-2 genome?

In the panel of converted SAM to BAM, I tried to use the data in trickster, but I am not sure to how to build a HSV genome as a reference?

I appreciate your help,

tao

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

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

===> Please use "Reply All" when responding to this email! <===

Hello Tao,

Sorry for the delayed reply, your question did not post to the mailing list since the "to" was not _only_ to galaxy-user.

Going forward, please leave off any "to" or "cc" to team members when asking a question. Send all questions directly "to" "galaxy-user@bx.psu.edu" and do not include any "Re" or "Fwd" text in the subject line.

Regarding RNA-seq analysis and reference GTF files, the place to incorporate the GTF file is in the Cufflinks step, the option to select the GTF file from your history is on the tool's form. If you have questions about the tools that are not addressed by these help links:

http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise

then contacting the tool authors would be the next step: email tophat.cufflinks@gmail.com

To visualize the data, the available options will be links associated with each dataset (expand the dataset box to locate these). The Galaxy Track Browser (GTB) aka "Trackster", UCSC Genome Browser, Ensembl, and GeneTrack are potential options; the datatype will determine which links are provided.

Hopefully this helps,

Best,

Jen Galaxy team

-------- Original Message -------- Subject: run tophat in galaxy Date: Sun, 28 Aug 2011 08:50:04 -0700 From: Peng, Tao tpeng@fhcrc.org To: Jennifer Jackson jen@bx.psu.edu, galaxy-user galaxy-user@lists.bx.psu.edu

Hi how can I specify a GTF gene annotation file when running tophat to guide the alignment to human genome? What is the best way to visualize the tophat results in the context of annotated human genome, i.e. RefSeq?

Thanks,

tao

Hi when I am done with cuffdiff analysis in GALAXY, I got many tabular data output for gene, transcript, promoter and CDS differential expression testing, I wonder if there are some systematic ways to look at the data output from cuffdiff analysis?

Thanks,

tao

Jen, thank you for following up on my question. Is any tool in GALAXY to visualize the coverage of aligned reads from TopHat on human chromosomes (histogram or density plot)?

Tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thursday, September 15, 2011 2:37 PM To: galaxy-user Cc: Peng, Tao Subject: Re: [galaxy-user] run tophat in galaxy

===> Please use "Reply All" when responding to this email! <===

Hi Tao,

I made an error in my prior reply, it is possible to guide assembly in TopHat. To do this, on the TopHat form, change "TopHat settings to use:"

from "Use Defaults" to "Full parameter list". In the expanded form:

1 - change "Use Own Junctions:" to be "yes". 2 - change "Use Gene Annotation Model:" to be "yes" 3 - in the new pull-down menu, select the GTF file from your history

Great question! Glad that we were able to provide you with the correct instruction,

Best,

Jen Galaxy team

On 9/15/11 1:38 PM, Jennifer Jackson wrote:

===> Please use "Reply All" when responding to this email! <===

Hello Tao,

Sorry for the delayed reply, your question did not post to the mailing list since the "to" was not _only_ to galaxy-user.

Going forward, please leave off any "to" or "cc" to team members when asking a question. Send all questions directly "to" "galaxy-user@bx.psu.edu" and do not include any "Re" or "Fwd" text in the subject line.

Regarding RNA-seq analysis and reference GTF files, the place to incorporate the GTF file is in the Cufflinks step, the option to

select

the GTF file from your history is on the tool's form. If you have questions about the tools that are not addressed by these help links:

http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise

then contacting the tool authors would be the next step: email tophat.cufflinks@gmail.com

To visualize the data, the available options will be links associated with each dataset (expand the dataset box to locate these). The Galaxy Track Browser (GTB) aka "Trackster", UCSC Genome Browser, Ensembl, and GeneTrack are potential options; the datatype will determine which

links

are provided.

Hopefully this helps,

Best,

Jen Galaxy team

-------- Original Message -------- Subject: run tophat in galaxy Date: Sun, 28 Aug 2011 08:50:04 -0700 From: Peng, Tao tpeng@fhcrc.org To: Jennifer Jackson jen@bx.psu.edu, galaxy-user galaxy-user@lists.bx.psu.edu

Hi how can I specify a GTF gene annotation file when running tophat to guide the alignment to human genome? What is the best way to visualize the tophat results in the context of annotated human genome, i.e.

RefSeq?

Thanks,

tao

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

Thanks a lot. Please see the attached screen shot from using Browser in GALAXY for viewing results of Tophat. What do those numbers mean? Is there any way to adjust Y-axis to have the bar taller so it will be easier to see?

Thanks,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Tuesday, September 27, 2011 1:13 PM To: Peng, Tao Cc: galaxy-user Subject: Re: [galaxy-user] run tophat in galaxy

Hi Tao,

Yes, the resulting SAM dataset can be converted to BAM and viewed in the

GTB (Galaxy Track Browser).

http://galaxyproject.org/wiki/Learn -> scroll to "Visualization" to find: http://galaxyproject.org/wiki/Learn/Visualization

The GTB can be reached through a different links, but one quick way to do this is:

1 - start with TopHat's output SAM dataset 2 - use the tool "NGS: SAM Tools -> SAM-to-BAM" 3 - hover over "Visualization" in the top menu bar then click on "New Track Browser" 4 - at the prompt, name the visualization and specify the reference genome and click on "Continue" 5 - once the browser opens, click on the "Add Datasets to Visualization"

prompt to add datasets. Histories and Libraries can be navigated, selected, then individual datasets selected and loaded. 6 - default view for BAM datasets a coverage histogram. 7 - adding more tracks and other functions can be performed by using the

left menu "Actions" on the GTB interface. Be sure to use "Save" before navigating away if you want to use the same browser again.

If datasets are not available to add to a browser, then likely there is a mismatch between the browser's reference database and the database assigned to the dataset. In some cases this can and should be adjusted (perhaps database was unassigned after an analysis).

The SAM file can also be converted to interval, then BED format for visualization, but BAM is the most direct route and preserves the sequence content when zoomed in at the base level.

Hopefully this helps you and others to learn more about the GTB. This tool is under active development. Screencasts and more example documentation is on the way soon to offer more help.

Best,

Jen Galaxy team

On 9/27/11 12:45 PM, Peng, Tao wrote:

Jen, thank you for following up on my question. Is any tool in GALAXY to visualize the coverage of aligned reads from TopHat on human chromosomes (histogram or density plot)?

Tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thursday, September 15, 2011 2:37 PM To: galaxy-user Cc: Peng, Tao Subject: Re: [galaxy-user] run tophat in galaxy

===> Please use "Reply All" when responding to this email!<===

Hi Tao,

I made an error in my prior reply, it is possible to guide assembly in TopHat. To do this, on the TopHat form, change "TopHat settings to

use:"

from "Use Defaults" to "Full parameter list". In the expanded form:

1 - change "Use Own Junctions:" to be "yes". 2 - change "Use Gene Annotation Model:" to be "yes" 3 - in the new pull-down menu, select the GTF file from your history

Great question! Glad that we were able to provide you with the correct instruction,

Best,

Jen Galaxy team

On 9/15/11 1:38 PM, Jennifer Jackson wrote:

...

===> Please use "Reply All" when responding to this email!<===

Hello Tao,

Sorry for the delayed reply, your question did not post to the

mailing

...

list since the "to" was not _only_ to galaxy-user.

Going forward, please leave off any "to" or "cc" to team members when asking a question. Send all questions directly "to" "galaxy-user@bx.psu.edu" and do not include any "Re" or "Fwd" text in the subject line.

Regarding RNA-seq analysis and reference GTF files, the place to incorporate the GTF file is in the Cufflinks step, the option to

select

...

the GTF file from your history is on the tool's form. If you have questions about the tools that are not addressed by these help links:

http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise

then contacting the tool authors would be the next step: email tophat.cufflinks@gmail.com

To visualize the data, the available options will be links associated with each dataset (expand the dataset box to locate these). The

Galaxy

...

Track Browser (GTB) aka "Trackster", UCSC Genome Browser, Ensembl,

and

...

GeneTrack are potential options; the datatype will determine which

links

...

are provided.

Hopefully this helps,

Best,

Jen Galaxy team

-------- Original Message -------- Subject: run tophat in galaxy Date: Sun, 28 Aug 2011 08:50:04 -0700 From: Peng, Taotpeng@fhcrc.org To: Jennifer Jacksonjen@bx.psu.edu, galaxy-user galaxy-user@lists.bx.psu.edu

Hi how can I specify a GTF gene annotation file when running tophat

to

...

guide the alignment to human genome? What is the best way to

visualize

...

the tophat results in the context of annotated human genome, i.e.

RefSeq?

...

Thanks,

tao

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

Tao,

Cuffdiff output is tabular data, and hence any statistical tool that supports tabular data can be used to analyze/visualize Cuffdiff outputs. In Galaxy, using tools in the categories Statistics and Graph/Display Data should enable you to visualize basic aspects of your data.

Good luck, J.

On Oct 4, 2011, at 4:05 PM, Peng, Tao wrote:

Hi I wonder how I can quantitatively visualize expression of a gene/transcript which has a significant p-value/q value from splicing differential testing from Cuffdiff analysis? I tried track browser in GALAXY and UCSC genome browser and none of them really provide a quantitative analysis of gene/transcript expression from Cuffdiff results.

Thanks,

tao

-----Original Message----- From: Peng, Tao Sent: Friday, September 30, 2011 3:13 PM To: 'Jennifer Jackson' Cc: galaxy-user Subject: RE: [galaxy-user] run tophat in galaxy

Thanks a lot. Please see the attached screen shot from using Browser in GALAXY for viewing results of Tophat. What do those numbers mean? Is there any way to adjust Y-axis to have the bar taller so it will be easier to see?

Thanks,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Tuesday, September 27, 2011 1:13 PM To: Peng, Tao Cc: galaxy-user Subject: Re: [galaxy-user] run tophat in galaxy

Hi Tao,

Yes, the resulting SAM dataset can be converted to BAM and viewed in the

GTB (Galaxy Track Browser).

http://galaxyproject.org/wiki/Learn -> scroll to "Visualization" to find: http://galaxyproject.org/wiki/Learn/Visualization

The GTB can be reached through a different links, but one quick way to do this is:

1 - start with TopHat's output SAM dataset 2 - use the tool "NGS: SAM Tools -> SAM-to-BAM" 3 - hover over "Visualization" in the top menu bar then click on "New Track Browser" 4 - at the prompt, name the visualization and specify the reference genome and click on "Continue" 5 - once the browser opens, click on the "Add Datasets to Visualization"

prompt to add datasets. Histories and Libraries can be navigated, selected, then individual datasets selected and loaded. 6 - default view for BAM datasets a coverage histogram. 7 - adding more tracks and other functions can be performed by using the

left menu "Actions" on the GTB interface. Be sure to use "Save" before navigating away if you want to use the same browser again.

If datasets are not available to add to a browser, then likely there is a mismatch between the browser's reference database and the database assigned to the dataset. In some cases this can and should be adjusted (perhaps database was unassigned after an analysis).

The SAM file can also be converted to interval, then BED format for visualization, but BAM is the most direct route and preserves the sequence content when zoomed in at the base level.

Hopefully this helps you and others to learn more about the GTB. This tool is under active development. Screencasts and more example documentation is on the way soon to offer more help.

Best,

Jen Galaxy team

On 9/27/11 12:45 PM, Peng, Tao wrote:

...

Jen, thank you for following up on my question. Is any tool in GALAXY to visualize the coverage of aligned reads from TopHat on human chromosomes (histogram or density plot)?

Tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thursday, September 15, 2011 2:37 PM To: galaxy-user Cc: Peng, Tao Subject: Re: [galaxy-user] run tophat in galaxy

===> Please use "Reply All" when responding to this email!<===

Hi Tao,

I made an error in my prior reply, it is possible to guide assembly in TopHat. To do this, on the TopHat form, change "TopHat settings to

use:"

...

from "Use Defaults" to "Full parameter list". In the expanded form:

1 - change "Use Own Junctions:" to be "yes". 2 - change "Use Gene Annotation Model:" to be "yes" 3 - in the new pull-down menu, select the GTF file from your history

Great question! Glad that we were able to provide you with the correct instruction,

Best,

Jen Galaxy team

On 9/15/11 1:38 PM, Jennifer Jackson wrote:

...

===> Please use "Reply All" when responding to this email!<===

Hello Tao,

Sorry for the delayed reply, your question did not post to the

mailing

...

...

list since the "to" was not _only_ to galaxy-user.

Going forward, please leave off any "to" or "cc" to team members when asking a question. Send all questions directly "to" "galaxy-user@bx.psu.edu" and do not include any "Re" or "Fwd" text in the subject line.

Regarding RNA-seq analysis and reference GTF files, the place to incorporate the GTF file is in the Cufflinks step, the option to

select

...

the GTF file from your history is on the tool's form. If you have questions about the tools that are not addressed by these help links:

http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise

then contacting the tool authors would be the next step: email tophat.cufflinks@gmail.com

To visualize the data, the available options will be links associated with each dataset (expand the dataset box to locate these). The

Galaxy

...

...

Track Browser (GTB) aka "Trackster", UCSC Genome Browser, Ensembl,

and

...

...

GeneTrack are potential options; the datatype will determine which

links

...

are provided.

Hopefully this helps,

Best,

Jen Galaxy team

-------- Original Message -------- Subject: run tophat in galaxy Date: Sun, 28 Aug 2011 08:50:04 -0700 From: Peng, Taotpeng@fhcrc.org To: Jennifer Jacksonjen@bx.psu.edu, galaxy-user galaxy-user@lists.bx.psu.edu

Hi how can I specify a GTF gene annotation file when running tophat

to

...

...

guide the alignment to human genome? What is the best way to

visualize

...

...

the tophat results in the context of annotated human genome, i.e.

RefSeq?

...

Thanks,

tao

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

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

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

===> Please use "Reply All" when responding to this email! <===

Hello,

Chromosome names will need to be modified once the file is imported, as explained in #5 of the FAQ: http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq

Hopefully this helps,

Best, Jen Galaxy team

On 9/15/11 8:22 PM, shamsher jagat wrote:

I have related question If I have to use Ensembl mouse GTF file (Mus_musculus.NCBIM37.64) Do I have to download and reformat it or Galaxy can take it from the source directly?

Thanks

On Sun, Aug 28, 2011 at 8:50 AM, Peng, Tao <tpeng@fhcrc.org mailto:tpeng@fhcrc.org> wrote:

  ===> Please use "Reply All" when responding to this email! <===


__

Hi how can I specify a GTF gene annotation file when running tophat
to guide the alignment to human genome? What is the best way to
visualize the tophat results in the context of annotated human
genome, i.e. RefSeq?

Thanks,

tao


___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org <http://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 source code, please
use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists,
please use the interface at:

http://lists.bx.psu.edu/

If using Ensembl make sure it is properly formatted. Vasu

--- On Tue, 8/30/11, Peng, Tao tpeng@fhcrc.org wrote:

From: Peng, Tao tpeng@fhcrc.org Subject: [galaxy-user] annotation file for cufflinks To: "Jennifer Jackson" jen@bx.psu.edu, "galaxy-user" galaxy-user@lists.bx.psu.edu Date: Tuesday, August 30, 2011, 1:52 PM

Hi jen and galaxy users, when I try to use cufflinks in GALAXY and select "use reference annotation", I am NOT sure how to get the annotation file in GTF format for cufflinks.

I was trying to down-load the annotation file from Illumina donated package in Cufflink website, I am NOT sure this is the right way to go for?

Thanks,

tao

-----Inline Attachment Follows-----

___________________________________________________________ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org.  Please keep all replies on the list by using "reply all" in your mail client.  For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

Hi I have down-loaded UCSC HG19 annotation files from Cufflinks; Should I just load genes.GTF file (about 100 MB) to galaxy for cufflink analysis?

Thanks,

tao

-----Original Message----- From: vasu punj [mailto:punjv@yahoo.com] Sent: Tue 8/30/2011 5:52 PM To: Jennifer Jackson; galaxy-user; Peng, Tao Subject: Re: [galaxy-user] annotation file for cufflinks

If using Ensembl make sure it is properly formatted. Vasu

--- On Tue, 8/30/11, Peng, Tao tpeng@fhcrc.org wrote:

From: Peng, Tao tpeng@fhcrc.org Subject: [galaxy-user] annotation file for cufflinks To: "Jennifer Jackson" jen@bx.psu.edu, "galaxy-user" galaxy-user@lists.bx.psu.edu Date: Tuesday, August 30, 2011, 1:52 PM

Hi jen and galaxy users, when I try to use cufflinks in GALAXY and select "use reference annotation", I am NOT sure how to get the annotation file in GTF format for cufflinks.

I was trying to down-load the annotation file from Illumina donated package in Cufflink website, I am NOT sure this is the right way to go for?

Thanks,

tao

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I had a similar question, after i downloaded a bacterial ref genome GFF file and loaded it into Galaxy, the "use reference annotation" feature of Cufflinks kept telling it need to be the GTF file instead, When I check the Cufflinks manual at http://cufflinks.cbcb.umd.edu/manual.html It looks like both GFF and GTF should be ok. Any insight? Gary

On Aug 30, 2011, at 12:52 PM, Peng, Tao wrote:

Hi jen and galaxy users, when I try to use cufflinks in GALAXY and select "use reference annotation", I am NOT sure how to get the annotation file in GTF format for cufflinks.

I was trying to down-load the annotation file from Illumina donated package in Cufflink website, I am NOT sure this is the right way to go for?

Thanks,

tao

---

The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list:

http://lists.bx.psu.edu/listinfo/galaxy-dev

To manage your subscriptions to this and other Galaxy lists, please use the interface at:

http://lists.bx.psu.edu/

Hi I am NOT sure why running cufflinks failed here. Thanks for your suggestion,

tao

----------------------- Tool: Cufflinks Name: Cufflinks on data 6 and data 26: assembled transcripts Created: Sep 01, 2011 Filesize: 81.3 Mb Dbkey: hg19 Format: gtf Tool Version:

Input Parameter Value SAM or BAM file of aligned RNA-Seq reads 6: Tophat for R4_CG_wh_accepted_hits Max Intron Length 300000 Min Isoform Fraction 0.05 Pre MRNA Fraction 0.05 Perform quartile normalization Yes Conditional (reference_annotation) 1 Reference Aonnotation 26: Homo_sapiens.GRCh37.63.gtf Conditional (bias_correction) 0 Conditional (seq_source) 0 Conditional (singlePaired) 0

----------------------------------------------------------

Message from History panel in GALAXY:

An error occurred running this job: cufflinks v1.0.3 cufflinks -q --no-update-check -I 300000 -F 0.050000 -j 0.050000 -p 8 -N -b /galaxy/data/hg19/sam_index/hg19.fa Error running cufflinks. [18:40:45] Inspecting reads and determining fragment length distribution. Processed 915556 loci.

Hi I had 3 GTF files from ensemble, UCSC and NCBI for annotation; ONLY ensemble were recognized by GALAXY cufflinks as a GTF file although they all have .GTF. I am NOT sure why UCSC and NCBI GTF files were seen as GFF files?

Thx,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thursday, September 01, 2011 5:08 PM To: galaxy-user Cc: Peng, Tao Subject: running cufflinks

===> Please use "Reply All" when responding to this email <===

Hello,

This is the same reply as for the bug report, but for others who may run

into the same problem job that fails with this error:

terminate called after throwing an instance of 'std::bad_alloc' what(): std::bad_alloc

the reason is explained in #3 in the RNA-seq FAQ: http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq#faq3

A local or cloud instance may be the solution. These options are explained here: http://galaxyproject.org/wiki/Big%20Picture/Choices

Our apologies for any inconvenience,

Best,

Jen Galaxy team

On 9/1/11 4:55 PM, Peng, Tao wrote:

Hi I am NOT sure why running cufflinks failed here. Thanks for your suggestion,

tao

---

Tool: Cufflinks Name: Cufflinks on data 6 and data 26: assembled transcripts Created: Sep 01, 2011 Filesize: 81.3 Mb Dbkey: hg19 Format: gtf Tool Version:

Input Parameter Value SAM or BAM file of aligned RNA-Seq reads 6: Tophat for R4_CG_wh_accepted_hits Max Intron Length 300000 Min Isoform Fraction 0.05 Pre MRNA Fraction 0.05 Perform quartile normalization Yes Conditional (reference_annotation) 1 Reference Aonnotation 26: Homo_sapiens.GRCh37.63.gtf Conditional (bias_correction) 0 Conditional (seq_source) 0 Conditional (singlePaired) 0

---

Message from History panel in GALAXY:

An error occurred running this job: cufflinks v1.0.3 cufflinks -q --no-update-check -I 300000 -F 0.050000 -j 0.050000 -p 8

-N

-b /galaxy/data/hg19/sam_index/hg19.fa Error running cufflinks. [18:40:45] Inspecting reads and determining fragment length distribution. Processed 915556 loci.

Hi Jen, I have been using the following link for learning to visualize TOPHAT results:

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

How can get the RefGENE for the whole human genome instead of Ch19 listed in tutorial?

Thanks,

tao

-----Original Message----- From: Jennifer Jackson [mailto:jen@bx.psu.edu] Sent: Thu 8/18/2011 3:46 PM To: galaxy-user@bx.psu.edu Cc: Peng, Tao Subject: visualization of alignment

Hello Tao,

For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com.

Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq

For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build".

Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization

As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation.

Best,

Jen Galaxy team

On 8/18/11 3:15 PM, Peng, Tao wrote:

Hi jen, I have used BOWTIE to align my RNA-seq reads to HSV2 genome; out of 35,000,000 lines, only 621 lines left when I chose to have mapped reads only. How can visualize these aligned reads to HSV-2 genome?

In the panel of converted SAM to BAM, I tried to use the data in trickster, but I am not sure to how to build a HSV genome as a reference?

I appreciate your help,

tao