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Turk Hijyen ve Deneysel Biyoloji Dergisi, vol.82, no.2, pp.311-322, 2025 (Scopus)
Objective: This study aims to evaluate and compare Sanger sequencing and third-generation 16S rRNA sequencing methods in terms of their ability to accurately identify various bacteria under identical primer sets and specific PCR conditions using four American Type Culture Collection (ATCC) strains. In this context, the capacity of each method was assessed to accurately and comprehensively identify bacteria from pure cultures, where the genus and species of the organisms are confirmed and known. Methods: In the study, identical primer sets and PCR conditions were used to enable a comparison between the two sequencing methods. While eight primers targeting the 16S rRNA region were used in the Sanger sequencing method, only two primers were used for the same region in the third-generation 16S rRNA sequencing method. The raw data were analyzed using GeneStudio software for Sanger sequencing and an appropriate bioinformatics workflow for the third-generation 16S rRNAsequencing method. Results: The analysis demonstrated that third-generation 16S rRNA sequencing outperforms Sanger sequencing in achieving comprehensive and efficient profiling of the full 16S rRNA region. Third-generation 16S sequencing reduces primer requirements, minimizes sequence loss in primer-binding regions, and significantly decreases processing time from days to hours, enabling rapid, high-throughput sequencing in a matter of minutes. In contrast, Sanger sequencing provides high single-read accuracy but falls short in terms of speed and efficiency, making it less suitable for broader applications. Conclusion: This study highlights third-generation 16S sequencing as a faster, more comprehensive, and more effective option for high-resolution microbial investigations. While Sanger sequencing remains a valuable tool for certain scenarios due to its high single-read accuracy, its speed and depth limitations render it inadequate for broader applications. These findings emphasize the advantages of third-generation 16S sequencing in providing scientists with a reliable and thorough approach to characterizing microorganisms for various applications.