Integrate fungal qPCR and composition results

[qwcheng]

Hi all,

I'm planning to analyze fungal microbiome composition in my samples but was wondering if I should sequence 18S or ITS. We have run a prelim qPCR test targeting 18S to quantify total fungi, using the method here (FungiQuant: A broad-coverage fungal quantitative real-time PCR assay | BMC Microbiology | Full Text). I would like to integrate the qPCR and the microbiome composition results so that I can have a rough (not precise) estimate of the total abundance of each fungal taxon. To my knowledge (please correct me if I'm wrong) ITS is better for fungal classification than 18S, but if I sequence ITS and compare the results with 18S qPCR, will I be comparing apples and pears? I'm very new to qPCR and fungal microbiome, and I'm not sure if it's reasonable to say that fungal 18S:ITS = 1:1. I'd really appreciate it if someone could offer suggestions on this!

[colinbrislawn (Colin J Brislawn)]

Hello @qwcheng,

This sounds like a cool technique. Using qPCR to estimate 'total abundance' of taxa is highly complementary to amplicon techniques which only show the composition of samples.

@qwcheng

To my knowledge (please correct me if I'm wrong) ITS is better for fungal classification than 18S

Yes? Here's the best citation I could find compairing ITS and 18S coverage. Looks like ITS is more variable, but I'm not sure if this means it covers more the fungal tree...

@qwcheng

To my knowledge (please correct me if I'm wrong) ITS is better for fungal classification than 18S, but if I sequence ITS and compare the results with 18S qPCR, will I be comparing apples and pears?

This is correct. The goal is to use amplicon sequencing for taxonomy and qPCR for absolute abundance, so you want to use the same primers for both.

@qwcheng

I'm very new to qPCR and fungal microbiome, and I'm not sure if it's reasonable to say that fungal 18S:ITS = 1:1.

I like your analogy; 18S and ITS are and , but they are both fruit!