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 <j...@bx.psu.edu
<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
<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 <tel:011420.2%201%204355543>
##species
http://www.ncbi.nlm.nih.gov/__Taxonomy/Browser/wwwtax.cgi?__id=414684
<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 gene 11 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
<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 <tel:011420.2%201%204355543>
##species
http://www.ncbi.nlm.nih.gov/__Taxonomy/Browser/wwwtax.cgi?__id=414684
<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 mRNA 11 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 0.100000 -j 0.150000 -p
4 -G /galaxy/test_pool/pool5/files/__000/327/dataset_327777.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 <mailto:email%3ado...@indiana.edu>
<mailto:Email%3Adong3@indiana.__edu
<mailto:email%253ado...@indiana.edu>>
Lab Phone:812-855-8443 <tel: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 <http://usegalaxy.org>. Please keep all
replies on the list by
using "reply all" in your mail client. For discussion of
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To manage your subscriptions to this and other Galaxy lists,
please use the interface at:
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--
Jennifer Jackson
http://galaxyproject.org
--
Qian Dong
Bauer Lab, MCBD
Simon Hall: 313-317
212 S. Hawthorne Dr.
Bloomington, IN 47405
Email:do...@indiana.edu <mailto:email%3ado...@indiana.edu>
Lab Phone:812-855-8443
--
Jennifer Jackson
http://galaxyproject.org
___________________________________________________________
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/
