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benjjneb / decontam

Simple statistical identification and removal of contaminants in marker-gene and metagenomics sequencing data
https://benjjneb.github.io/decontam/
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Regarding DNA quantitation data #43

Open kotaarima opened 5 years ago

kotaarima commented 5 years ago

Thank you very much for your great work. We are going to perform metagenomic analyses by 16S using human tissue samples. I would like to make sure about "DNA quantitation data". Is this "pre-PCR concentration" or "post-PCR concentration"? If it is pre-PCR, can we use "frequency-based contaminant identification" when we standardize samples at same pre-PCR concentration?

I really appreciate it if you could tell me.

Best regards, Kota

benjjneb commented 5 years ago

Please see this comment on an earlier issue: https://github.com/benjjneb/decontam/issues/33#issuecomment-436658460

Does that answer your question?

kotaarima commented 5 years ago

Thank you very much for your answer, but I have a couple of things to make sure. Does the "16S qPCR measurements of the original DNA samples" in #33 mean pre-PCR DNA concentration? Besides, even if the concentration were completely same, can we use frequency-based method? I thought that the DNA quantitation data need variety to assess frequency.

I appreciate all your help

benjjneb commented 5 years ago

Does the "16S qPCR measurements of the original DNA samples" in #33 mean pre-PCR DNA concentration?

Yes.

even if the concentration were completely same, can we use frequency-based method? I thought that the DNA quantitation data need variety to assess frequency.

That is correct. If all the DNA concentrations were somehow exactly the same then the frequency method will not work.

kotaarima commented 5 years ago

Thank you very much!

kotaarima commented 5 years ago

I'm sorry to keep asking, but please let me ask one question. How range does the distribution of DNA concentration need when we use Frequency-based identification? I think it would be better to be wide distribution, but how range is it enough to work?

benjjneb commented 5 years ago

We don't have strong evidence-based guidance on an exact minimum range of concentrations needed at this time. I'd say that less than a two-fold range of concentrations is probably too little, and that a ten-fold range is definitely enough.

kotaarima commented 5 years ago

Thank you so much. That information would be very helpful for me.