Manual Reference Method Versus Commercial Automated Software for Data Analysis and Result Interpretation of 16S Bacterial Sequences

Currently, 16S ribosomal DNA (rDNA)-based sequencing is the “gold-standard” for identifying environmental microorganisms. While it is the most accurate bacterial identification method, the overall performances of current 16S rDNA identification systems are not uniform. By comparing the performance of Accugenix’s 16S rDNA identification method to another commercially available system, we pinpoint several key factors contributing to variation in 16S rDNA identification systems. First and foremost, the breadth of the microbial reference library greatly impacts the accuracy of identifications – Accugenix’s larger library, which exhibits thorough coverage of isolates relevant to the pharmaceutical and biotechnology industries, outperformed all other commercially available databases. Moreover, we reveal that the software used to analyze 16S sequences also affects the accuracy of microbial identifications – Accugenix’s manual reference method, which enables editing of base-caller errors that typically occur near the end of sequences or polymorphic 16S sequences, is more precise than fully automated sequence analysis methods. Additionally, the direct DNA distance measurements used by Accugenix are also more accurate than quality score methods used by others. Details regarding how library coverage and software parameters affect identification accuracy are reviewed.