Cristian,

The ">" is a formatting character; what needs to match is the string after the ">" in the genome file and the entries in the contig column of your GTF. Your GTF is quite different that your genome file; your genome file has 10 contigs labeled by number, but your GTF has many, many contig names labelled by numbers and names.

For Cufflinks to work, you can either (a) turn off bias correction or (b) restrict entries in your GTF to those that match your reference genome.

Finally, please reply all to emails so that all emails remain on list for archival and community purposes.

Thanks, J.

On Mar 30, 2011, at 12:02 PM, Cristian Rojas wrote:

Thanks Jeremy. But in genomes fasta files very often any chromosome represents a sequence followed by ">". Then, it is no possible match contig names in GTF with ">" names in Genome fasta. What must I do? Cristian

----- Mensaje original ---- De: Jeremy Goecks jeremy.goecks@emory.edu Para: Cristian Rojas cristianrojasbr@yahoo.com.ar CC: galaxy-user@lists.bx.psu.edu Enviado: miércoles, 30 de marzo, 2011 12:53:50 Asunto: Re: [galaxy-user] Trouble with RNAseq analysis

Cristian,

The contig names in your GTF file don't match those in your reference (fasta) file. In order for Cufflinks to use a reference GTF, its contigs names must match those in your reference genome.

Best, J.

On Mar 30, 2011, at 11:31 AM, Cristian Rojas wrote:

...

Thanks Jeremy. I did it. Cristian

----- Mensaje original ---- De: Jeremy Goecks jeremy.goecks@emory.edu Para: Cristian Rojas cristianrojasbr@yahoo.com.ar CC: galaxy-user@lists.bx.psu.edu Enviado: miércoles, 30 de marzo, 2011 12:02:47 Asunto: Re: [galaxy-user] Trouble with RNAseq analysis

Cristian,

Please share your history with me (History Options --> Share/Publish --> Share

...

with User --> my email) and I'll take a look.

Thanks, J.

On Mar 30, 2011, at 10:48 AM, Cristian Rojas wrote:

...

Hi everybody, I am trying to analyze the differential expression between two RNAseq samples. But I found many troubles aligning my reads. I will describe what I did. First I groomed the FastQ files (2). Then I uploaded the Sorghum genome and aligned the reads to it with Tophat. Aftter that, I tried to use Cufflink with the BAM file of Tophat, using as annotation file an uploaded GTF

...

file and the Sorghum genome, but I received an error message in the three outputs of Cufflink. I tried to align against new brand Maize genome (now at Galaxy), and the same messages. I also converted the BAM file to SAM, but the

...

...

same. Any advice? What was wrong? Thanks in advance. Cristian

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