decontam Frequency method - Using cead counts vs. potential DNA concentration data

[Seppevdb12]

Hello,

I'm a master's student working on my thesis, which focuses on fungal communities using amplicon sequencing data.
I'm interested in applying the frequency method in the decontam R package to identify contaminants.
For each 96-well plate used during DNA extraction and library preparation, we included one well as a negative control (no biological sample).

I am currently unsure if I have direct DNA quantification data available for my samples.
This information is being checked by a lab assistant in another country and should be confirmed soon.

In the meantime, I was wondering, assuming I don't have direct DNA quantification data:

Is it generally valid to use total read count per sample as a stand-in for DNA concentration in decontam's frequency method?
Under what conditions would you advise against using total reads in this way? What are the potential pitfalls?
Given that I have only one negative control per plate and might not have DNA quantification data, would you recommend switching to the prevalence method in decontam instead?
If it turns out that I do have DNA concentration data, would your advice change significantly regarding the use of the frequency method?

Thanks in advance!

[benjjneb]

Is it generally valid to use total read count per sample as a stand-in for DNA concentration in decontam's frequency method?
Under what conditions would you advise against using total reads in this way?

Not is it not. The issue is that in almost all cases, there is a step in preparing the libraries in which lower concentration libraries are included at higher volumes in order to attempt to make the number of reads across samples equal. As a result, there is no longer a linear relationship between read counts and input sample DNA concentration. Thus, read counts can't be used a proxy for DNA concentration.

Given that I have only one negative control per plate and might not have DNA quantification data, would you recommend switching to the prevalence method in decontam instead?

One negative control is not enough to use the prevalence method. I suppose if you have many plates the total of the negative controls across plates could become sufficient (we recommend 5+).