A transcriptome constitutes the totality of RNA from a given defined species (better: defined strain) under a set of defined conditions (environment, time, etc.). So you cannot (!!!) map the data from one species/strain onto the other! It might be technically feasible, if you allow for enough missmatches (the ANI of different species should be below 96%), but your results would be meaningless because they do not reflect what would happen in  the other species/strain under the same set of conditions.

You can of course cultivate both species/strains under the same conditions and then compare the repsonse of orthologous genes. Keep in mind that you will then have to do quite a lot of tricky normalization steps with spike in controls (at the very least after harvesting, total RNA isolation and mRNA enrichment each) in order to prove that your observations are based on the same number of cells.

Dear Christian, thank you for your prompt reply. Can you refer to some resources that contain information regarding orthologous genes from differential bacterial species?

Dear Philipp, that is indeed my question. I am looking forward to your feedback.

I think with the mapping approach, assuming the strains are close enough and both have sequenced genomes, you'd want to do the mapping in both directions - i.e. map RNA-Seq data from both species to reference genome of species A, then map RNA-Seq data from both species to reference genome of species B.

Then there are 4 sets of reads - only map to genome A, only map to genome B, map to both genomes, map to neither genome. If enough reads map to both genomes, you could do some analysis just using these. The mapping might be of interest anyway, just to see what amount of overlap there is.

@Rohan: If you map reads from the mouse on a human reference, that tells you (at best) what genes are similar in the two species, NOT what the reaction of human cells would be upon the treatment. If you believe the latter, you are gravely mistaken. The human transcriptome might not react or react in a completely different manner. To use a visual metaphor: That would be analogous to watching the reaction of a house cat to a buffalo and then claiming how a lion would react in the same situation (as both species share fur, eyes, claws, and a number of other similarites). Sorry, but that approach is not scientifically sound.

Just consider the following possibility: If there is a regulator present in mice but not in humans that reacts to your drug (or does not react in case of an ortholog with changed specificity) and changes the transcription of one or more genes that are highly conserved between man and mice, your approach would tell you that the drug would have the same effect in both, which is, as should be self evident, not going to happen in such a case. If you want to analyze the reaction of the human transcriptome you have to use human cells...

Concerning the question(s) itself: Based on the fact that you want to map reads, I figure that you have the genomes of your two different species available. If you are then interested in orthologous genes, there are a number of ways to identify those. A simple approach would be a bidirectional best BLAST hit analysis: Use BLASTP to compare all protein sequences from species 1 to those of species 2, keeping only the best hit for each protein (list output for BLAST is your friend here). Do the same for all proteins from species 2 against those of species 1 and then check whether you get a reciprocal hit. You might want to apply additional criteria like at least 75% coverage of at least one candidate and/or minimal values for identical/positive amino acids (I usually use 33%/50% for this). If you want to make your life easier, you might use tools like EDGAR (see the attached links, you might have to request a private project if your species are not part of an open project) or databases like eggNOG to do that in a more standardized and automated way. Is that what you are looking for?

But to then compare the response to the respective transcriptomes is a bit more tricky. If that is what you want to do, let me know.

https://edgar.computational.bio.uni-giessen.de/cgi-bin/edgar_login.cgi?cookie_test=1&open=1

http://www.biomedcentral.com/1471-2105/10/154

http://nar.oxfordjournals.org/content/42/D1/D231.long

Dear Chistian, Philipp, Catriona, Rohan,

Thank you for your useful feedback. Using this information, I have decided to apply the following approach:

http://www.pathogenomics.sfu.ca/ortholugedb/

Hi guys,

I was going through your conversations, which was very useful. I just wanna begin preparing samples (two strains of Mycoplasma) for sending RNAseq. I appreciate if any of you can help me how to prepare RNA from the scratch? which criteria should be considered before and during the RNA extraction?