Unravelling the enigma of the human microbiome: Evolution and selection of sequencing technologies

The human microbiome plays a crucial role in maintaining health, with advances in high‐throughput sequencing technology and reduced sequencing costs triggering a surge in microbiome research. Microbiome studies generally incorporate five key phases: design, sampling, sequencing, analysis, and reporting, with sequencing strategy being a crucial step offering numerous options. Present mainstream sequencing strategies include Amplicon sequencing, Metagenomic Next‐Generation Sequencing (mNGS), and Targeted Next‐Generation Sequencing (tNGS). Two innovative technologies recently emerged, namely MobiMicrobe high‐throughput microbial single‐cell genome sequencing technology and 2bRAD‐M simplified metagenomic sequencing technology, compensate for the limitations of mainstream technologies, each boasting unique core strengths. This paper reviews the basic principles and processes of these three mainstream and two novel microbiological technologies, aiding readers in understanding the benefits and drawbacks of different technologies, thereby guiding the selection of the most suitable method for their research endeavours. Current prevailing sequencing methodologies encompass Amplicon sequencing, Metagenomic Next‐Generation Sequencing (mNGS), and Targeted Next‐Generation Sequencing (tNGS). Recently introduced innovative platforms, MobiMicrobe high‐throughput microbial single‐cell genome sequencing technology and 2bRAD‐M simplified metagenomic sequencing technology, serve to ameliorate the constraints inherent to established techniques, each offering distinct core advantages. This manuscript delineates the foundational principles and operational protocols of both the mainstream and emergent microbiological sequencing technologies, The objective is to elucidate the merits and limitations of each approach, thereby providing informed guidance for researchers in selecting the most pertinent sequencing methodology for their scientific inquiries.