Empirical methods for differential absolute abundance inference

Cytometry of bulk cell concentration

Done with flow cytometry or microscopy

qPCR measurement of bulk DNA concentration

• Pro: Shares extraction and copy-number bias with 16S sequencing, which might greatly reduce the error in DA when used with 16S seq.
• Pro: Available from some sequencing companies
• Con: Unclear if/when it is actually proportional to input microbial biomass or cell density. Saturation in DNA extraction seems likely at fecal levels - something we can explicitly test in our VV follow-up
• Con: Some have suggested/found that the results are very noisy (and seems too labor intensive/expensive to do replicates) - I’m not sure if noise is in the extraction yield or in the qPCR measurement though guess in yield

DNA yield / concentration of extracted DNA

• Pro: Usually get this for free as a normal part of the sequencing experiment

Spike-ins

Cells

DNA, before extraction

DNA, post extraction

Housekeeping taxa

Host reads (shotgun sequencing)

Organelle reads (amplicon sequencing)

Other housekeeping taxa

Either using prior knowledge, or determining from the data

Targeted qPCR or ddPCR on DNA

Based on DNA so has same cons as bulk qPCR. If there is significant saturation during extraction, get competition and so the same problem arises (though prob to a lesser degree)

Targeted ddPCR directly on cells

I think this can be multiplexed to multiple taxa; can do total and individual taxa in one measurement; probes expensive?

Equivolumetric protocol

Approach of cruz2021equi

Other methods

• Flow cytometry with taxon-specific probes; can be used to get bulk and targeted measurement
• Other methods in zhang2017soil: adenosine tri-phosphate (ATP), phospholipid fatty acids (PLFA), microbial biomass Carbon (MBC)