For a study, it was determined that the area of metagenomics grew dramatically in the last two decades as sequencing technology improved. It allowed for a more thorough characterization of microbial populations. Applying these technologies resulted in a breakthrough in the researchers’ understanding of the human, animal, and environmental microbiomes, demonstrating that the gut microbiota can be compared to an organ that was intrinsically connected to a wide range of disorders. The use of next-generation sequencing-by-synthesis methods to characterize microbial communities found significant alterations in the microbiota linked with severe conditions such as Clostridium difficile infection. However, genus- and species-level classification had been difficult due to limits in sequencing read length, primer biases, and database quality. Third-generation technologies, such as Pacific Biosciences’ single-molecule, real-time (SMRT) method, enabled more precise and unbiased identification of species that were likely to be clinically relevant. For example, samples from patients treated for C. difficile infection were compared using Illumina next-generation characterization and SMRT sequencing revealed similar community composition at the species and family levels. Still, SMRT sequencing allowed for species-level description, allowing for a better understanding of the disease’s microbial ecology. As sequencing technology progress, new species-level insights in the outline of complex and clinically relevant microbial communities will become available.