Hello,

I have 16S data where a patented spike-in method was used for standardized nucleic acid preparation from stool samples. I’d like to understand how best to normalize this spike-in during the analysis.

In my search, I came across the "SCML method," which is spike-in-based calibration to total microbial load and is used to determine the absolute frequencies of identified bacterial species. I also found that DESeq2 and DiffBind could be applied for normalization.

Could you please advise on the best approach, or if there are any other methods I should consider for normalization?

Thank you in advance!