CLC Genomic workbench -ANNOTATIONS-

[vanale]

Hi mates!
How is it going? For me not too good!!
I don't know if anybody is confdent with RNA-seq analysis by clc genomic workbench... Actually i'm trying to look at the transcriptome expression of several semples of treated grapevine leaves against untreated using a 36bp paired ends approach with illumina technology. I never used clc before and i'm not a bioinformatic, so i'm sorry if i'm gonna be a bit generic and unprecise.
To allign all my reads against the 8X reference genome the first think i've to do is to upload my genome on clc and to annotate it. The reference genome is a FASTA file, while annotations are in a GFF format. To annotate the reference genome i use the tool "annotate with a GFF/GTF file". It seems to work, as i can see the annotations on the reference genome in the view window. The problem is that when i try to look at the annotation table i can't do it. The only table i can see show me a list of all different chromosomes on my FASTA file. I point out this becouse, when i make the RNA-seq analysis, despite i can see the reads alligning on the annotations, i can't get any expression value for my genes. Ant i can't see any value in all the other column of the RNA-seq analyzed sample table. I guess there is a problem with the GFF file. But i'm really ignorant about that...Is anybody confident with this kind of analysis?? Does anybody have any suggestion?? I've a LOT of data and i can't analyze it...IT'S SO FROUSTRATING!!!!!!!
Cheers!!
Ale

[rglover]

If you've bought the software, you probably also pay the hefty support fee each year for the use of this email address: [email protected]. They're very helpful

[NextGenSeq]

Actually I don't find their support that great.

You usually have to edit the GFF file before using the plugin. Also, you have to select "show all annotations" in order to see them on the contigs.

I have a slightly amateurish word file that describes how to annotate your reference genome that I got from CLC Bio.

[vanale]

I'm just trying to communicate with the support and i'm gonna upload my data (ref-gen and gff file) in their ftp server to make them to have a look. They told me there is a new plugin is gonna be available at the end of April that should correct some bugs releted to annotating gff files. I want to believe!! The worse think is that i'm even trying to cope with the Genomatix platform as well, but it seems to me they upload only the 8X coverage of Vitis genome (not to smart considering that the 12X is now available) and i can't find my genes of interest blasting them with El dorado....strange!!!
At the end of the day thay're both driving me nuts!!!

[vanale]

Would you have a chance to send it to me?? I got a PDF explaining how to annotate a gff file on a reference genome with CLC, but i don't think this is the real problem... I think the real problem is in the GFF file.. I now there are different kind of GFF (GFF 1, 2 and 3 ??? ) may be the format of my data is not good??
Thanks!
Ale

[NextGenSeq]

I attached there guide to this message.

[vanale]

Thank you very much!!
Actually i exately did what's written in this PDF, and actually i figured out that my annotations are right: when i look at the entire reference sequence i can only see the chromosomes. On the countrary, if i double clich on a single chromosome obtained with the option "shoe table", i get all the annotations on that sigle chromosome. What i can't understand is why, when i perform my RNA-seq analysis, using default parameters i can't get any expression value. The only numbers i can obtain in the list of Features ID are the number of unique gene reads and the number of total gene reads. All the other column are wito value 0.00
I'm wondering if this is a problem with parameters or with mine annotations. I underline that in the gff file i used to annotate the ref-seq there are all this features: Gene, mRNA, CDS, UTR. May be i should annotate judst with Gene and mRNA features??
I miss the old good simple PCR

[etwatson]

I'm trying to build a reference sequence and I have gff3 files for all contigs. It seems like CLC doesn't like the gff3 files, because I don't see any annotations when the plugin has run. Do I need to convert the gff3 files to something else?

[boetsie]

If the plugin has finished attaching the .gff file to your .fasta, does it show you information that there are annotations attached?