Hello everyone,
I’m currently processing some samples using NGS with an HPV-STI kit (ChapterDx). The kit comes with its own analysis software and is quite straightforward — essentially it targets highly conserved amplicons for HPV genotypes and other STI-associated pathogens.
The final output for each run (192 multiplex samples per run) is a count table where I can see:
Total reads per sample
Total reads for the internal control (GAPDH)
Number of reads per pathogen
My impression is that this test is semi-quantitative, but mostly qualitative, as I haven’t found references where pathogen read counts are used as a statistically meaningful quantitative measure. So far, the kit seems to only support reporting pathogen presence/absence.
My question is:
Would it be viable or make sense to calculate something like a relative viral load from these data?
I’m not from a virology background, but I do know that true viral load experiments are usually more robust and account for multiple factors. My results currently look something like this:
I’d just like to get more out of the data beyond simply reporting presence or absence.
Thanks in advance for any insights.
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There may be a reason for that. Did you take a look at the methodology details. There may be some procedure in there (selective PCR/normalization across samples) that may prevent estimation of additional parameters you are thinking about.
A quick look at the kit shows that it simultaneously detects ~47 pathogens. It means that tube has primers for those. Perhaps there is not enough material in the master mix to allow for quantitative estimation.
No, not even semi-quant. Don't go down this path, it will lead to bad things.
edit: to clarify, don't use and abuse an assay for something it's not designed to do.