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I am attempting to perform metatranscriptomics analysis of a cleanroom. The DNA input is known to be rather low (<1pg). Despite this, I want to still attempt to try it. I am thinking it might be best to perform extraction trials before hand where certain parameters are manipulated. I can then determine from these extraction trials which values for which parameters are best.

As an example, I am reading a peer-reviewed paper called "Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics" where they used the Illumina Nextera XT DNA library preparation kit on low biomass samples. Two of the parameters they manipulated were:

1) PCR cycle numbers (Values = 12, 14, 16, 18, 20) 2) ATM dilution (Values = undiluted, 1:5, 1:10, 1:20, 1:50)

I am hoping to use the same type of extraction trials to determine which combination of values from these two parameters is best for a clean room. My first question is: How can I determine which combination is best (as there are 25 combinations)? I have two ideas (but am open to others):

1) Check the amount of RNA derived 2) Check some quality metric on RNA

As I hope to do triplets of each of the 25 combinations, I will have 75 samples. I am hoping not to have to do sequencing as it is time consuming and possibly expensive in order to chose the best combination. Is there a way to chose which combination works best without having to sequence?

My second question is: Are there any other parameters in this workflow that I can also test for if I am not getting decent results in these low-biomass cleanrooms?

(As you can tell, I do not have too much direct molecular biology experience, so feel free to correct or question any of my post and/or "dumb down" your response). Thank you for sharing advice (for either question)!

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  1. Consider doing triplicates of just 4 combinations of cycle/dilution: low+low, low+high, high+ low, high+high. Simpler.

  2. Most important: prove you can extract DNA from a clean room. If you extract from a real clean room and get no result from the PCR, maybe it is because you failed to collect anything or maybe nothing was there. Idea - spray a known quantity of DNA into a room that will serve as your sham clean room. Then do your extraction protocol. You can test the cycle / dilution parameters with the sham clean room extraction and have those variables optimized for when you collect from a real "wild" clean room you did not dope with DNA.

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