How To Annotate A Previously Unknown Bacterial Organism With Glimmer3?

Question

[hugh.winchester]

Hi there!

Firstly apologies it this seems really basic, but I've been set an assignment to annotate an previously unknown bacterial organism for my course.

I am trying, and mostly failing to use Glimmer3 correctly, and after doing some Google searching I'm even more confused. I've installed it and everything and even managed to get some output using the glimmer-from-scratch.csh command, but none of it seems very useful. I've tried to using the 'extract' command to pull out some long ORFs but when blasted they don't match up with anything. Was glimmer meant to be trained against something before being run? I was under the impression the from scratch script did everything that you needed, but I'm not too sure.

Also, as a slight aside, how would you go from a working glimmer to identify the 16s rRNA? I tried running my assembly (from velvet) through RNAmmer, but it only identified the 23s rRNA. Is there anyway to easily do this?

Answer 1

[Neilfws]

I assume that you are using g3-from-scratch.csh - there is no script named glimmer-from-scratch.csh.

If you have installed the software correctly, run the script without seeing any error messages and obtained output, then your problem may simply be understanding the limitations of the process, rather than a problem with the software itself.

First: it's entirely possible that the few ORFs you examined do not have significant BLAST hits. A large percentage of ORFs in newly-sequenced genomes do not.

Second: you do not go from a "working glimmer" to 16S rRNA, since glimmer identifies protein-coding ORFs. As you mention, RNAmmer is the software used here for RNA prediction. It may be that your assembly is incomplete and does not include a 16S rRNA sequence. You could try a simple pairwise search of your contigs using a known 16S rRNA gene as a query.

I'm sure your course instructors will be happy to discuss your results with you. Bioinformatics is never as simple as running the program and getting the "right answer".

[rosarylimyt]

Sorry for this side note, but I'm also confused by the workings of GLIMMER3. I tried experimenting with the sample files that came with the download to see if I fully understand how to operate GLIMMER3 by reproducing the given results. The thing is I'm unsure if the scripts given (namely g3-from-scratch, g3-from-training, g3-iterated) are to be used in a consecutive manner. I'd assumed these scripts had to be used in order starting with g3-from-scratch, and I've generated a number of files. Thereafter I'm stuck at g3-from-training. From the tutorial I downloaded, it says to type in the form: g3-from-training.csh [yourgenom.seq] train.coords run2 I can't find a .coords file generated from the previous g3-from-scratch run and so I can't proceed. Please advise me on what I should do.