Novice at meta-transcriptomics

[nabilquraishi]

Dear all,
I am a research fellow at Birmingham UK, looking to study meta-transcriptomics from colon biopsies in patients with colitis. I am very new to this but have had some sequencing experience with 16s and metagenomics (including library preparation). I am still trying to get my head around the different steps in order to successfully prepare cDNA libraries and I would be grateful for any help anyone can provide. So far I am able to successfully get RNA out of colon biopsies with a RIN > 8 and around 300ng/ul (total volume 50ul) with the current protocol that I am using. I have a few questions please:

1) The next step from what I understand is to remove host RNA and then enrich bacterial mRNA by removing rRNA. I have got two kits for these - Ambion MicrobeEnrich and MicrobeExpress for these steps respectfully, but havent yet used them.
2) As I don't expect there to be a lot of bacterial mRNA should I use the Smarter standed RNA-Seq kit to make cDNA libraries?
3) How many samples can I multiplex in a run? - We have a Hiseq and Miseq sequencing platforms. Is it just a matter of sequencing a few and seeing how much depth I have got?

Thank you again for any help you can provide!

[Genetic Librarian]

Try to crunch some numbers.
Assume X bacterial cells per host cell (e.g. 10, check literature) and then assume the RNA content of each cell (based on genome sizes).
Then assume how good your kits are in removing host RNA and rRNAs (e.g. 99%)
You can check in the Illumina specs how much read output each sequencer has. By using a simple excel file with above considerations (and modifying them), you will get a feeling for how many bacterial transcript reads your experiment will result under given circumstances.
It really helps.

I did it that way once (after I got ****ty sequencing results) to explain to myself why only a tiny fraction of reads was of bacterial origin. The bacterial mRNA fraction is tiny and even after 99% enrichment it still might be tiny (as in my case). The important number here seems to be the ratio of bacterial cells to host cells (as in metatranscriptomics works well in infected tissue that is crawling with bacteria etc.). Be aware that my failure is 5 years old, so enrichment kits might have gotten better etc. Still, calculate it through.