best strategy for rRNA from shotgun?

[idoerg]

Hi,

I have a 454 metagenomic sample, from which I would like to extract species frequency & diversity, among other things. I am currently using MG-RAST, with GreenGenes as my SSUrRNA reference database, 96% identity cutoff, 100 bp minimum alignment length and a e-value of 10^-5. I was wondering if anyone has some experience with doing this, and whether better parameters can be used to increase sensitivity/specificity of finding SSUrRNA in such a sample?

Thanks,

Iddo

[Chuckytah]

Hi,

I have a 454 metagenomic sample, from which I would like to extract species frequency & diversity, among other things. I am currently using MG-RAST, with GreenGenes as my SSUrRNA reference database, 96% identity cutoff, 100 bp minimum alignment length and a e-value of 10^-5. I was wondering if anyone has some experience with doing this, and whether better parameters can be used to increase sensitivity/specificity of finding SSUrRNA in such a sample?

Thanks,

Iddo

Hello,

i'm doing a work similar, i think... but i used 80%identity cutoff, isn't right? I think if I use 96% a lot of sequences will not appear, right? 80% its good? or some will miss too?

[idoerg]

Hello,

i'm doing a work similar, i think... but i used 80%identity cutoff, isn't right? I think if I use 96% a lot of sequences will not appear, right? 80% its good? or some will miss too?

80% for SSUrRNA is not specific enough, and basically you will be grouping all bacteria together as a single OTU

[Chuckytah]

80% for SSUrRNA is not specific enough, and basically you will be grouping all bacteria together as a single OTU

it is not form SSurRNA it's DNA samples, from root-soil, plant. and i need to separate from plant and fungi only.