A Comparative Analysis of Denoising Methods and Nucleic Acid Templates for 16S rRNA Amplicon Sequencing of the Gut Microbiome

Of the three bioinformatics pipelines (DADA2, Deblur, VSEARCH) evaluated for analyzing paired 16S rRNA DNA and RNA amplicons from human fecal samples, DADA2 demonstrated superior performance by producing more biologically coherent community profiles and a better-balanced feature table. Employing this optimized DADA2 workflow, we found no significant differences between DNA and RNA-derived microbial communities across composition (phylum and genus levels), alpha and beta diversity, or predicted metagenomic function, with only sporadic genus-level biomarkers and functional differences detected. Based on these findings and the practical advantages of DNA regarding cost and stability, we recommend a standardized DADA2-based DNA amplicon sequencing workflow for gut microbiome profiling to enhance reproducibility and cross-study comparability.