16S sequencing from total RNA?

[emily202]

I'm working with a researcher that wants to identify the bacterial communities present in human dental pulp tissue, blood, and saliva from isolated RNA. What is the advantage (or disadvantage) from starting with RNA versus DNA? Typical 16S workflows use DNA as input with targeted 16S rRNA V3/V4 region amplification for library generation and long-read sequencing. This researcher has already performed DNA-Seq in a previous study but now "wants to confirm results through RNA"...

[Genetic Librarian]

Well, on DNA level you get everything, dead or alive. On RNA level, you get the viable bacterial community, which can be very different in sites of active immune response for example.

[emily202]

Thanks for your reply.

Yes, RNA-seq can provide what's transcriptionally active at the time of sampling. I'm wondering if there is an RNA workflow similar to 16S metagenomic sequencing for taxonomic classification. Perhaps this is more of an analysis tool question than methodology, however, I'm proposing total RNA isolation, host depletion and bacterial enrichment prior to library generation since these samples are suspected to be of low bacterial load.

Does anyone have experience or suggestions for bacterial taxonomic characterization using RNA-seq?

[Genetic Librarian]

No, the process is much simpler as you think. You simply isolate total RNA and reverse transcribe that into cDNA using random primers. Than you do your normal 16S amplicon workflow on that cDNA. You just use cDNA instead of genomic DNA as input. That way you get a 16S taxonomic dataset of the active microbiota.
What you suggest to do is a metatranscriptome which is a whole different approach.

[emily202]

Should I still use a host depletion and bacterial enrichment method prior to reverse transcription if I only want to target bacteria for sequencing? I’m concerned that the sequencing reads will largely be human if not. Thermo has a MICROBEnrich and MICROBExpress line for host separation and bacteria mRNA enrichment. Illumina has a 16S rRNA workflow on the MiSeq using V3/V4 primer pair for amplification that I would like to use for this project if compatible.

[Genetic Librarian]

If you use an amplicon approach (16S v3v4) you do not need any form of depletion, because you just amplify bacterial 16S sequences . The primers are specific, so that it does not matter how much of host RNA is present. The same reason why you do not get host reads in your 16S datasets from DNA.
Host depletion and bacterial enrichment is something you need for metatranscriptomes, not for 16S.

[emily202]

Of course - makes sense! I think I'm overthinking this one

Thanks for your input!