Shin, Kenny2026-05-132026-05-132026-05-13http://hdl.handle.net/10393/51643https://doi.org/10.20381/ruor-3194116S rRNA gene (16S) sequencing is widely used for exploratory analysis of microbiomes which is less resource intensive than the shotgun whole metagenome (shotgun) sequencing. Currently, comprehensive comparisons between 16S and shotgun sequencing methods are lacking. Here, we evaluated various 16S-based sequencing and data processing methods against shotgun sequencing for similarity in analysis outcome. We processed human gut microbiome samples using five 16S sequencing methods: Illumina V6, IonTorrent V6, Nanopore 16S, PacBio 16S, and LoopSeq 16S, through various bioinformatic tools (DADA2, Emu) and reference databases (Silva, EmuDB) to generate taxonomic and differential abundance data. Nanopore demonstrated improved taxonomic detection metrics in mock and human samples at genus and species levels compared to conventional methods when using the Emu pipeline and database, for the closest approximation of the gold standard shotgun data. No single differential abundance analysis method was perfect, and multiple workflows should be assessed to minimize biases.en16S rRNANext-generation sequencingMetagenomeBacterial taxonomySpeciesThesis