standard operating procedure 454 analysis

[neoanderson]

Hello all,
Im new to 454 data analysis. I have a 6mbp genome that has been sequenced at>10X. this is an emerging bacterial pathogen.
what sort of post 454 data analysis should one do? does anyone have any standard operating procedures?
As of now, I have a broad idea of what I would like to do. I have listed some of them below. I would appreciate any comments/suggestions.
1)with the 400 or so contigs, I intend to align it with reference genomes(mauve? - will this help me in judging the quality of assembly?).
2)carry out a rast server based annotation
3)extract nucleotide and protein sequences from the gbk output of rast. (what is the best way to do it such that protein sequences are obtained as fastA with the product name as header rather than contig number?)
4)carry out multiway blast searches against related genomes
5)look out for presence of plasmids in the contigs by searching for regions of high coverage?
6)look for regions of atypical nucleotide composition ?

I would really appreciate your thoughts on this post.
Thanks very much !

[Loris]

I think I can plug xBASE services here.

I suggest you use my-xBASE to align your data to a reference, also try de-novo assembly. Then use the annotation service to generate a preliminary annotation.

xBASE main page - my-xBASE and annotation services linked in the right column.

for (5), I plot a graph of contig length vs. reads/length. Plasmid contigs stick out from the vast majority of large contigs at a non-integral multiple of the read-density.