FORUMhuman and bacterial ncRNA fucntional analysis tools and functional pathway enrichment tools for bacterial RNA-seq datasets
Zakhar Krekhno asked 2 years ago

Hi,   I have processed an RNA-seq experiment of bacterial infection of human cells. I\’ve done all the analyses (clean-up, count reads with Salmon, do differential expression with DESeq2). Now I have a list of ncRNA that are differentially expressed upon this infection (compared to non-infectious control) and a list of mRNA. My question is how would I analyze the ncRNA (as in how do I assess whether it is exosomally associated, their predicted targets, and functions, for both human and bacterial ncRNA). Also, how would I analyze bacterial mRNA (as in perform functional enrichment – to see which pathways are up- or down-regulated), I have tried using Blast2go (using it on the protein sequences associated with differentially expressed mRNA genes) but it seems extremely slow – taking around 2 days per job. Is there a faster way to do this, maybe a better tool?   Thank you,

jrosner Staff replied 2 years ago

Hi Zakhar, sorry about the slow response, not many of us are around over the holidays. But please be patient and we’ll see if we can get you a response shortly.

1 Answers
jhgalvez Staff answered 2 years ago

Hello Zakhar,


Apologies for the late reply, as was mentioned, we were on holidays and only just got back. In any case, to reply to your question, there are several possible approaches, but one thing that is not clear to me is how exactly you obtained your samples. Specifically, I did not understand if the RNA you extracted was from the infectious agent (the bacteria) or the human cells, or a combination of both. That would make a big difference since a combined dataset would mean you need to search for the annotation across bacterial and human databases at the same time, which could complicate matters. In this case, I will just assume your samples contain human RNA only and therefore it is a relatively straightforward analysis.


For the ncRNA, the information you are looking for would come from one or more databases. I would specifically recommend something like which has information on all types of non-coding RNA from human and several other species. You can download the data from the website and then annotate any differentially expressed ncRNA using the ID number.


As for the functional analysis of differentially expressed mRNA, I usually direct people to use GSEA (, because it has a relatively intuitive user interface and the results can then be exported into several other packages for visualization, including Cytoskape. It also incorporates information from many other sources such as KEGG pathways and GO terms. For a more automated, but less flexible way of doing this analysis, you can use the fGSEA package on Bioconductor (


All of those websites have more information on how to carry out analyses, including tutorials and examples. If you run into any issues, you can always ask us a follow-up question about your specific issue and we would be happy to help.

Good luck!