bacterial transcriptomes

[sdantoft]

Hi all,

(If a similar question has already been posted, kindly direct me to the answer). We have just sequenced a bacterial transcriptome under different growth conditions and one of the problems we had was that out of 13million reads, around 12 million reads mapped to unannotated regions. If you blast the sequence, it is apparently rRNA. That left me with only 1 million reads that actually map to the genome, and I have used this to calculate gene expression values (RPKM) - this means that the coverage per gene is very low, and possibly we miss out on genes with low levels fo expression. The total RNA was enriched for mRNA using MicrobeExpress (Ambion) by the external sequencing facility. My question is whether anyone has faced a similar problem, and what can we do to improve mRNA enrichment?

Sorry for the long message and I look forward to your answers.

[MadsAlbertsen]

There are basically two possible problems:

1) The removal efficiency is highly dependant on intact rRNA. Make sure that rRNA is not degraded before applying MICROBExpress.

Working faster and including RNAse inhibitors or EDTA usually solves this.

2) Check that your bacteria is compatible with the MICROBExpress kit. MICROBExpress only removes 16S and 23S rRNA that are complementary to the included oligos. You can get an idea by looking at their homepage where they have a list of compatible bacteria.

Look at the follwing paper for a possible workaround if MICROBExpress is not compatible with your bacteria:
[Stewart et al,. 2010] Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics. The ISME Journal (2010) 4, 896–907; doi:10.1038/ismej.2010.18.

rgds
Mads Albertsen

[epibio]

You could also try the Ribo-Zero kit, which will work even with degraded RNA samples.

[sdantoft]

Thanks Mads .