Converting 16S V3–V4 ASV reads to absolute copy numbers (qPCR / spike-in calibration?)

General Discussion
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Hi, good day Qiime2 team.
I’m working with 16S rRNA V3–V4 amplicons from bacterial strains using universal primers. After processing the raw .fastq.gz files with DADA2 in QIIME 2, I have an ASV table (samples × ASVs, reads?) and representative sequences.
I’d like to understand the relationship between ASV reads and the absolute copy number of the V3–V4 region present in the actual sample, Question1:more practically—how to convert reads into absolute copies per ASV.
Alternatively, if a set of spike-in sequences at known concentrations is mixed with the sample DNA, amplified with 16S rRNA V3–V4 primers, and sequenced, Question 2: can the spike-in copy numbers and reads be used to calibrate ASV reads in the sample to obtain absolute reads or copy numbers

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Did you do a lit review? There are a bunch of methods to do this so I'm not sure what you have already tried...

Robert Edgar tried this in 2017 to mixed success: https://www.biorxiv.org/content/10.1101/124149v1

Apparently, 16S rRNA Gene Copy Number Normalization Does Not Provide More Reliable Conclusions in Metataxonomic Surveys - PMC

Yes! But only for the same microbes that you spiked in of course.

I don't think there's a method for doing this within Qiime2 right now, but I've done it before in R.

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