Re: [galaxy-user] GFF not recognized in CUFFLINK

2012-09-27 Thread Qian Dong 
Dear Jennifer,

My .fasta reference genome is like this:

'>gi|289546492|ref|NC_011420.2| Rhodospirillum centenum SW chromosome,
complete genome'

and in the SAM file generated by BOWTIE it says:

@SQ SN:gi|289546492|ref|NC_011420.2|LN:4355543

So I think they are all the same as "NC_011420.2". Is there anything
else I can try?

Thank you,

Qian


On Wed, Sep 26, 2012 at 3:35 PM, Jennifer Jackson  wrote:

> Hello,
>
> The first thing to double check is that the chromosome identifier is an
> exact match between the reference genome and the reference annotation.
>
> The GFF3 file is naming the chromosome "NC_011420.2".
>
> The reference annotation chromosome should be named exactly the same way.
> Check this in the input BAM/SAM datasets or the original .fasta reference
> genome.
>
> Hopefully this finds the problem. Correcting mismatched names (due to
> various reasons) is the most common solution to this sort of issue:
> 'Tools on the Main server: Example', bullet item #2:
> http://wiki.g2.bx.psu.edu/**Support#Interpreting_**scientific_results
>
> Best,
>
> Jen
> Galaxy team
>
>
>
> On 9/26/12 8:46 AM, Qian Dong wrote:
>
>> Dear Team,
>>
>> I've been having a problem with cufflink regarding GFF files. I tried
>> searching the mailing list first and failed to find an answer. Could you
>> help me look at this?
>>
>> I downloaded my genome annotation GFF file from NCBI (soon I realized
>> NCBI format may be a problem) for my bacterial RNA-seq data analysis. My
>> GFF file looks like the following:
>>
>> '
>> ##gff-version 3
>> #!gff-spec-version 1.20
>> #!processor NCBI annotwriter
>> ##sequence-region NC_011420.2 1 4355543
>> ##species http://www.ncbi.nlm.nih.gov/**Taxonomy/Browser/wwwtax.cgi?**
>> id=414684
>> NC_011420.2 RefSeq  region  1   4355543 .   +   .
>> ID=id0;Dbxref=taxon:414684;Is_**circular=true;culture-**
>> collection=ATCC:51521;gb-**synonym=Rhodocista
>> centenaria SW;gbkey=Src;genome=**chromosome;mol_type=genomic
>> DNA;strain=SW%3B ATCC 51521
>> NC_011420.2 RefSeq  gene11  3343.   +   .
>> ID=gene0;Name=RC1_0011;Dbxref=**GeneID:7008893;gbkey=Gene;**
>> locus_tag=RC1_0011
>> NC_011420.2 RefSeq  CDS 11  3343.   +   0
>> ID=cds0;Name=YP_002296275.1;**Parent=gene0;Note=Contains a type I
>> secretion target ggxgxdxxx repeat %282 copies%29 domain%3B Contains a
>> Cadherin domain%3B identified by match to protein family HMM
>> PF02789;Dbxref=Genbank:YP_**002296275.1,GeneID:7008893;**
>> gbkey=CDS;product=hypothetical
>> protein;protein_id=YP_**002296275.1;transl_table=11
>>
>>
>> I used this file for cufflink but all the FPKM values are 0.  I checked
>> out this link: 
>> http://cufflinks.cbcb.umd.edu/**gff.htmland
>>  thought that
>> maybe the problem is because I don't have any mRNA feature in my gff
>> file. Since I am dealing with a bacterial genome, there is no
>> exon/intron or UTR info needed. Therefore I modified my GFF file into
>> the following:
>>
>> ##gff-version 3
>> #!gff-spec-version 1.20
>> #!processor NCBI annotwriter
>> ##sequence-region NC_011420.2 1 4355543
>> ##species http://www.ncbi.nlm.nih.gov/**Taxonomy/Browser/wwwtax.cgi?**
>> id=414684
>> NC_011420.2 RefSeq  region  1   4355543 .   +   .
>> ID=id0;Dbxref=taxon:414684;Is_**circular=true;culture-**
>> collection=ATCC:51521;gb-**synonym=Rhodocista
>> centenaria SW;gbkey=Src;genome=**chromosome;mol_type=genomic
>> DNA;strain=SW%3B ATCC 51521
>> NC_011420.2 RefSeq  mRNA11  3343.   +   .
>> ID=mRNA0;Name=RC1_0011;Dbxref=**GeneID:7008893;gbkey=Gene;**
>> locus_tag=RC1_0011
>> NC_011420.2 RefSeq  CDS 11  3343.   +   0
>> ID=cds0;Name=YP_002296275.1;**Parent=mRNA0;Note=Contains a type I
>> secretion target ggxgxdxxx repeat %282 copies%29 domain%3B Contains a
>> Cadherin domain%3B identified by match to protein family HMM
>> PF02789;Dbxref=Genbank:YP_**002296275.1,GeneID:7008893;**
>> gbkey=CDS;product=hypothetical
>> protein;protein_id=YP_**002296275.1;transl_table=11
>>
>>
>>
>> I re-ran cufflink however this time there is error reported. I can only
>> tell from the report that there is a segmentation fault but not further
>> details. The report is as follows:
>>
>> Error running cufflinks.
>> return code = 139
>> Command line:
>> cufflinks -q --no-update-check -I 100 -F 0.10 -j 0.15 -p 4 -G
>> /galaxy/test_pool/pool5/files/**000/327/dataset_32.dat
>> /galaxy/test_database/files/**000/325/dataset_325086.dat
>> [19:41:41] Loading reference annotation.
>> Segmentation fault
>>
>> cp: cannot stat `/galaxy/test_pool/pool3/tmp/**
>> job_working_directory/000/170/**170197/global_model.txt': No such file
>> or directory
>> cp: cannot stat `/galaxy/te

Re: [galaxy-user] GFF not recognized in CUFFLINK - Re: adding new genome

2012-09-26 Thread Jennifer Jackson 

Hello,

Instructions for including a new genome indexed for tools, including how 
to download the same indexes used on the public Main Galaxy instance 
hosted at http://usegalaxy.org, are in these wikis:


http://wiki.g2.bx.psu.edu/Admin/Data%20Integration
http://wiki.g2.bx.psu.edu/Admin/NGS%20Local%20Setup

The "dbkey" that you will want to use is simply "hg19" for this genome, 
should you use the rsync service.


Going forward, the galaxy-...@bx.psu.edu mailing list would be a good 
choice for local install questions. The development community can help 
solve more complex problems as they come up and/or you might be 
interested in following discussions:

http://wiki.g2.bx.psu.edu/Support#Mailing_Lists
http://wiki.g2.bx.psu.edu/Mailing%20Lists

Hopefully this helps!

Jen
Galaxy team

On 9/26/12 12:47 PM, Kenneth Auerbach wrote:

Hi,

I'm getting this cuffmerge error on our local Galaxy instance. I assume
it is because it doesn't have the hg19 reference genome. Can you please
tell me how I would include into this Galaxy installation?






Thank you.


--
Jennifer Jackson
http://galaxyproject.org
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using "reply all" in your mail client.  For discussion of
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Re: [galaxy-user] GFF not recognized in CUFFLINK

2012-09-26 Thread Jennifer Jackson 

Hi Qian,

This is the source of the mismatch. In fasta format, any text between 
the leading ">" and the first whitespace (tab, space, etc) is considered 
the "identifier".

http://wiki.g2.bx.psu.edu/Support#Error_from_tools
http://wiki.g2.bx.psu.edu/Learn/Datatypes#Fasta

Which in your case is this:

   gi|289546492|ref|NC_011420.2|

The identifier must be changed to be only this:

   NC_011420.2

to be consistent with the GFF file. There are many ways to do this, one 
simple way is to use the tools in 'Text Manipulation'. Use 'Remove 
beginning of a file' to strip the first line. Then create a plain text 
file on your computer, one line, with just the identifier content like this:


   >NC_011420.2

Very important - do not make the file more than one line long - meaning 
do not add in an extra 'return' at the end of the first line. The plain 
text file just needs these characters - no more - including no 
whitespace characters (spaces, tabs, newlines). Upload this file to your 
instance as text and "Concatenate datasets" with the sequence file that 
had the first line removed, placing this identifier dataset on top. Then 
change the datatype back to ".fasta" on the final dataset, if necessary 
(click on the pencil icon).


Then re-run Bowtie to have the identifiers in your SAM file consistent 
with the GFF file.


After running Bowtie, be certain to sort the resulting dataset before 
running Cufflinks. Tophat produces sorted output, but Bowtie does not 
and Cufflinks requires sorted input.

http://main.g2.bx.psu.edu/u/jeremy/p/transcriptome-analysis-faq#faq2

Take care,

Jen
Galaxy team

On 9/26/12 12:54 PM, Qian Dong wrote:

Dear Jennifer,

My .fasta reference genome is like this:

'>gi|289546492|ref|NC_011420.2| Rhodospirillum centenum SW chromosome,
complete genome'

and in the SAM file generated by BOWTIE it says:


@SQ SN:gi|289546492|ref|NC_011420.2|LN:4355543

So I think they are all the same as "NC_011420.2". Is there anything else I can 
try?

Thank you,

Qian


On Wed, Sep 26, 2012 at 3:35 PM, Jennifer Jackson mailto:j...@bx.psu.edu>> wrote:

Hello,

The first thing to double check is that the chromosome identifier is
an exact match between the reference genome and the reference
annotation.

The GFF3 file is naming the chromosome "NC_011420.2".

The reference annotation chromosome should be named exactly the same
way. Check this in the input BAM/SAM datasets or the original .fasta
reference genome.

Hopefully this finds the problem. Correcting mismatched names (due
to various reasons) is the most common solution to this sort of issue:
'Tools on the Main server: Example', bullet item #2:
http://wiki.g2.bx.psu.edu/__Support#Interpreting___scientific_results 


Best,

Jen
Galaxy team



On 9/26/12 8:46 AM, Qian Dong wrote:

Dear Team,

I've been having a problem with cufflink regarding GFF files. I
tried
searching the mailing list first and failed to find an answer.
Could you
help me look at this?

I downloaded my genome annotation GFF file from NCBI (soon I
realized
NCBI format may be a problem) for my bacterial RNA-seq data
analysis. My
GFF file looks like the following:

'
##gff-version 3
#!gff-spec-version 1.20
#!processor NCBI annotwriter
##sequence-region NC_011420.2 1 4355543 
##species
http://www.ncbi.nlm.nih.gov/__Taxonomy/Browser/wwwtax.cgi?__id=414684

NC_011420.2 RefSeq  region  1   4355543 .   +   .

ID=id0;Dbxref=taxon:414684;Is___circular=true;culture-__collection=ATCC:51521;gb-__synonym=Rhodocista
centenaria SW;gbkey=Src;genome=__chromosome;mol_type=genomic
DNA;strain=SW%3B ATCC 51521
NC_011420.2 RefSeq  gene11  3343.   +   .

ID=gene0;Name=RC1_0011;Dbxref=__GeneID:7008893;gbkey=Gene;__locus_tag=RC1_0011
NC_011420.2 RefSeq  CDS 11  3343.   +   0
ID=cds0;Name=YP_002296275.1;__Parent=gene0;Note=Contains a type I
secretion target ggxgxdxxx repeat %282 copies%29 domain%3B
Contains a
Cadherin domain%3B identified by match to protein family HMM

PF02789;Dbxref=Genbank:YP___002296275.1,GeneID:7008893;__gbkey=CDS;product=hypothetical
protein;protein_id=YP___002296275.1;transl_table=11


I used this file for cufflink but all the FPKM values are 0.  I
checked
out this link: http://cufflinks.cbcb.umd.edu/__gff.html
 and thought that
maybe the problem is because I don't have any mRNA feature in my gff
file. Since I am dealing with a bacterial genome, there is no
exon/intron or U

Re: [galaxy-user] GFF not recognized in CUFFLINK

2012-09-26 Thread Kenneth Auerbach 

  
  
Hi,

I'm getting this cuffmerge error on our local Galaxy instance. I
assume it is because it doesn't have the hg19 reference genome. Can
you please tell me how I would include into this Galaxy
installation?






Thank you.
  

___
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/

Re: [galaxy-user] GFF not recognized in CUFFLINK

2012-09-26 Thread Jennifer Jackson 

Hello,

The first thing to double check is that the chromosome identifier is an 
exact match between the reference genome and the reference annotation.


The GFF3 file is naming the chromosome "NC_011420.2".

The reference annotation chromosome should be named exactly the same 
way. Check this in the input BAM/SAM datasets or the original .fasta 
reference genome.


Hopefully this finds the problem. Correcting mismatched names (due to 
various reasons) is the most common solution to this sort of issue:

'Tools on the Main server: Example', bullet item #2:
http://wiki.g2.bx.psu.edu/Support#Interpreting_scientific_results

Best,

Jen
Galaxy team


On 9/26/12 8:46 AM, Qian Dong wrote:

Dear Team,

I've been having a problem with cufflink regarding GFF files. I tried
searching the mailing list first and failed to find an answer. Could you
help me look at this?

I downloaded my genome annotation GFF file from NCBI (soon I realized
NCBI format may be a problem) for my bacterial RNA-seq data analysis. My
GFF file looks like the following:

'
##gff-version 3
#!gff-spec-version 1.20
#!processor NCBI annotwriter
##sequence-region NC_011420.2 1 4355543
##species http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=414684
NC_011420.2 RefSeq  region  1   4355543 .   +   .
ID=id0;Dbxref=taxon:414684;Is_circular=true;culture-collection=ATCC:51521;gb-synonym=Rhodocista
centenaria SW;gbkey=Src;genome=chromosome;mol_type=genomic
DNA;strain=SW%3B ATCC 51521
NC_011420.2 RefSeq  gene11  3343.   +   .
ID=gene0;Name=RC1_0011;Dbxref=GeneID:7008893;gbkey=Gene;locus_tag=RC1_0011
NC_011420.2 RefSeq  CDS 11  3343.   +   0
ID=cds0;Name=YP_002296275.1;Parent=gene0;Note=Contains a type I
secretion target ggxgxdxxx repeat %282 copies%29 domain%3B Contains a
Cadherin domain%3B identified by match to protein family HMM
PF02789;Dbxref=Genbank:YP_002296275.1,GeneID:7008893;gbkey=CDS;product=hypothetical
protein;protein_id=YP_002296275.1;transl_table=11


I used this file for cufflink but all the FPKM values are 0.  I checked
out this link: http://cufflinks.cbcb.umd.edu/gff.html and thought that
maybe the problem is because I don't have any mRNA feature in my gff
file. Since I am dealing with a bacterial genome, there is no
exon/intron or UTR info needed. Therefore I modified my GFF file into
the following:

##gff-version 3
#!gff-spec-version 1.20
#!processor NCBI annotwriter
##sequence-region NC_011420.2 1 4355543
##species http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=414684
NC_011420.2 RefSeq  region  1   4355543 .   +   .
ID=id0;Dbxref=taxon:414684;Is_circular=true;culture-collection=ATCC:51521;gb-synonym=Rhodocista
centenaria SW;gbkey=Src;genome=chromosome;mol_type=genomic
DNA;strain=SW%3B ATCC 51521
NC_011420.2 RefSeq  mRNA11  3343.   +   .
ID=mRNA0;Name=RC1_0011;Dbxref=GeneID:7008893;gbkey=Gene;locus_tag=RC1_0011
NC_011420.2 RefSeq  CDS 11  3343.   +   0
ID=cds0;Name=YP_002296275.1;Parent=mRNA0;Note=Contains a type I
secretion target ggxgxdxxx repeat %282 copies%29 domain%3B Contains a
Cadherin domain%3B identified by match to protein family HMM
PF02789;Dbxref=Genbank:YP_002296275.1,GeneID:7008893;gbkey=CDS;product=hypothetical
protein;protein_id=YP_002296275.1;transl_table=11



I re-ran cufflink however this time there is error reported. I can only
tell from the report that there is a segmentation fault but not further
details. The report is as follows:

Error running cufflinks.
return code = 139
Command line:
cufflinks -q --no-update-check -I 100 -F 0.10 -j 0.15 -p 4 -G 
/galaxy/test_pool/pool5/files/000/327/dataset_32.dat 
/galaxy/test_database/files/000/325/dataset_325086.dat
[19:41:41] Loading reference annotation.
Segmentation fault

cp: cannot stat 
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/global_model.txt':
 No such file or directory
cp: cannot stat 
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/isoforms.fpkm_tracking':
 No such file or directory
cp: cannot stat 
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/genes.fpkm_tracking':
 No such file or directory


My questions will be:

1. Is there any way to modify a NCBI bacterial genome annotation GFF
file to make it usable for cufflink? Our genome annotation is only
available in NCBI, not ensemble or USDC so this is pretty much my only
choice..

2. Should I proceed with modifying the GFF file or should I convert it
into GTF and use the GTF instead in cufflink?

I am a biochemist and really new to the computer world so any advice
will help!

Thanks a lot,

Qian
--
Qian Dong
Bauer Lab, MCBD
Simon Hall: 313-317
212 S. Hawthorne Dr.
Bloomington, IN 47405
Email:do...@indiana.edu 
Lab Phone:812-855-8443



___
The Galaxy User list should be used for the discussion of
Galaxy a

[galaxy-user] GFF not recognized in CUFFLINK

2012-09-26 Thread Qian Dong 
Dear Team,

I've been having a problem with cufflink regarding GFF files. I tried
searching the mailing list first and failed to find an answer. Could you
help me look at this?

I downloaded my genome annotation GFF file from NCBI (soon I realized NCBI
format may be a problem) for my bacterial RNA-seq data analysis. My GFF
file looks like the following:

'##gff-version 3#!gff-spec-version 1.20#!processor NCBI
annotwriter##sequence-region
NC_011420.2 1 4355543##species
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=414684NC_011420.2
RefSeqregion14355543.+.ID=id0;Dbxref=taxon:414684;Is_circular=true;culture-collection=ATCC:51521;gb-synonym=Rhodocista
centenaria SW;gbkey=Src;genome=chromosome;mol_type=genomic DNA;strain=SW%3B
ATCC 51521NC_011420.2RefSeqgene113343.+.
ID=gene0;Name=RC1_0011;Dbxref=GeneID:7008893;gbkey=Gene;locus_tag=RC1_0011
NC_011420.2RefSeqCDS113343.+0ID=cds0;Name=YP_002296275.1;Parent=gene0;Note=Contains
a type I secretion target ggxgxdxxx repeat %282 copies%29 domain%3B
Contains a Cadherin domain%3B identified by match to protein family HMM
PF02789;Dbxref=Genbank:YP_002296275.1,GeneID:7008893;gbkey=CDS;product=hypothetical
protein;protein_id=YP_002296275.1;transl_table=11
I used this file for cufflink but all the FPKM values are 0.  I checked out
this link: http://cufflinks.cbcb.umd.edu/gff.html and thought that maybe
the problem is because I don't have any mRNA feature in my gff file. Since
I am dealing with a bacterial genome, there is no exon/intron or UTR info
needed. Therefore I modified my GFF file into the following:

##gff-version 3#!gff-spec-version 1.20#!processor NCBI
annotwriter##sequence-region
NC_011420.2 1 4355543##species
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=414684NC_011420.2
RefSeqregion14355543.+.ID=id0;Dbxref=taxon:414684;Is_circular=true;culture-collection=ATCC:51521;gb-synonym=Rhodocista
centenaria SW;gbkey=Src;genome=chromosome;mol_type=genomic DNA;strain=SW%3B
ATCC 51521NC_011420.2RefSeqmRNA113343.+.
ID=mRNA0;Name=RC1_0011;Dbxref=GeneID:7008893;gbkey=Gene;locus_tag=RC1_0011
NC_011420.2RefSeqCDS113343.+0ID=cds0;Name=YP_002296275.1;Parent=mRNA0;Note=Contains
a type I secretion target ggxgxdxxx repeat %282 copies%29 domain%3B
Contains a Cadherin domain%3B identified by match to protein family HMM
PF02789;Dbxref=Genbank:YP_002296275.1,GeneID:7008893;gbkey=CDS;product=hypothetical
protein;protein_id=YP_002296275.1;transl_table=11

I re-ran cufflink however this time there is error reported. I can only
tell from the report that there is a segmentation fault but not further
details. The report is as follows:

Error running cufflinks.
return code = 139
Command line:
cufflinks -q --no-update-check -I 100 -F 0.10 -j 0.15 -p 4 -G
/galaxy/test_pool/pool5/files/000/327/dataset_32.dat
/galaxy/test_database/files/000/325/dataset_325086.dat
[19:41:41] Loading reference annotation.
Segmentation fault

cp: cannot stat
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/global_model.txt':
No such file or directory
cp: cannot stat
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/isoforms.fpkm_tracking':
No such file or directory
cp: cannot stat
`/galaxy/test_pool/pool3/tmp/job_working_directory/000/170/170197/genes.fpkm_tracking':
No such file or directory


My questions will be:

1. Is there any way to modify a NCBI bacterial genome annotation GFF file
to make it usable for cufflink? Our genome annotation is only available in
NCBI, not ensemble or USDC so this is pretty much my only choice..

2. Should I proceed with modifying the GFF file or should I convert it into
GTF and use the GTF instead in cufflink?

I am a biochemist and really new to the computer world so any advice will
help!

Thanks a lot,

Qian
-- 
Qian Dong
Bauer Lab, MCBD
Simon Hall: 313-317
212 S. Hawthorne Dr.
Bloomington, IN 47405
Email:do...@indiana.edu
Lab Phone:812-855-8443
___
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/