Accu16S/AccuITS: Accurate and broadly applicable amplicon sequencing for absolute microbiome quantification

Traditional 16S rRNA gene and Internal Transcribed Spacer region amplicon sequencing provides only relative abundance, often leading to biased ecological interpretations. To overcome this limitation, we developed Accu16S/AccuITS, an absolute quantification method for bacterial and fungal amplicons based on synthetic internal spike-in DNA with known copy numbers. By adding internal standards prior to Polymerase Chain Reaction and sequencing, absolute microbial abundances can be calculated using standard curve regression. Accu16S/AccuITS exhibits sensitivity and consistency comparable to quantitative Polymerase Chain Reaction and is applicable to diverse sample types. A single sequencing run simultaneously yields relative abundance, total absolute abundance, and taxon-specific absolute abundance. Case studies across diverse ecosystems demonstrate that absolute quantification provides ecologically and functionally meaningful insights beyond those obtained from relative abundance analyses.