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
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
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.
We try to have an outside total abundance determination (i.e. copies of 16S gene per dry weight of soil using same primers as in the sequencing) then combine that to the relative abundance numbers (compositional data) lots of issues with copies per genome, extraction efficiency etc. but still better than not doing anything. try this paper https://doi.org/10.1007/s00248-023-02273-z
Welcome to the forums, @jennyn!
Thank you for mentioning this! This is what I was thinking of, so I tracked down a citation:
Final gene copies per extract were calculated by multiplying elution volume by gene abundance and were then normalized to grams of dry sediment extracted. Absolute concentrations (referred to as “abundance” throughout) of specific Operational taxonomic units (OTUs) were calculated by multiplying percent abundance from 16S rRNA gene and ITS sequencing by the sample’s total bacterial, archaeal, or fungal concentration (ng μL^-1 g dry sed^-1) from qPCR.