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  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 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  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>
Lab Phone:812-855-8443



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--
Jennifer Jackson
http://galaxyproject.org
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