Metatranscriptomics in sterile rooms

Question

[GreenDiamond]

I am trying to determine state-of-the-art methods to conducting metatranscriptomic RNA-seq analysis of microbial ecology in sterile environments (like industrial clean rooms). Given the harshness and lack of nutrients in sterile indoor clean environments, the amount of DNA and RNA that can be obtained is smaller than usual. This can make it difficult to successfully conduct a metatranscriptomic analysis on such small biomass.

As a result, I am looking for methods that have successfully attacked this problem, but am having great difficulty finding any publications or references on the matter. Specifically, what are the newest and best approaches to trying to conduct metatranscriptomics RNA-seq analysis with low biomass (picogram level or even lower)? Any advice or introduction to such references would be so greatly appreciated. Thank you!

[WouterDeCoster]

Hi GreenDiamond,

I'm not sure if you are asking for bioinformatics or library prep solutions, can you clarify? If the latter is the case then your question is off topic for this website.

Don't forget to do appropriate negative controls.

Wouter

[GreenDiamond]

Thank you, WouterDeCoster. I realize I did not state that very clear, so I just updated it in bold to show that I am looking for bioinformatics approaches (I am also looking for library-prep elsewhere). Thank you.

[Michael]

Hi, not sure either if that is bioinformatics, but consider one important thought:

• if the "sterile" environment is "cleaner" than the sequencing and library prep environment you will pick up solely contamination
• meta-genomics/transcriptomics is laden with contamination already and therefore (near) sterile environments are possibly below its detection limit

Answer 1

[GenoMax]

This recent paper explored microbiomes in NASA clean rooms. Methods therein should be of interest. One additional publication. I do agree with other mods that this is not a bioinformatics question.

Answer 2

[Joe]

You may want to take a look at the Ancient DNA field. They've developed many protocols and approaches recently to deal with low abundance of DNA, and DNA which is highly degraded.

It's been picking up a lot of steam in the last ~5 years or so in particular so you should find some recent papers.

AFAIK, the solution is pretty much sequence the absolute shit out of it (NextSeqs/HiSeqs), and run lots of controls for: you, your environment, the 'kit-ome' and pretty much anything else you can think of.