I'm going to be using some spike-ins when performing my amplicon sequencing to allow me to better perform microbial quantification. I'm using the Zymo Spike-in Control II and a yeast strain for my 16S and ITS sequencing, respectively. I understand that I want my spike-in to account for 1-10% of the total reads, so I'm trying to understand how much of each to add to my samples prior to DNA extraction.
I had an idea to perform extractions with and without the spike-ins and then perform some RT-qPCRs to get a rough idea of the quantity of amplicons from each spike-in.
However, I'm unsure of the best way to develop qPCR primers for the ITS2 region which I'm planning to sequence using the ITS4 and fITS7 primer pair.
If anyone has any advice on the subject, please let me know. Many thanks 
Hello @Caleb_Sheff,
Developing and validating a new qPCR assay will be a lot of work. I would search for any viable existing assays that you can use as-is, if you haven't already.
Given that you already have an amplicon of interest, you could make a quick and dirty SYBR green assay, though these aren't always the most accurate.
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