Evaluation of 16S RNA gene amplicon sequencing analyses

We adopt the direct PCR strategy amplifying 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results showed that differences in cell wall morphology significantly affected the direct PCR efficiency and the sequencing data. Notably, mechanical cell disruption preceding the direct PCR was indispensable to obtain an accurate representation of the bacterial composition in the specimen. Furthermore, a 16S analysis of the mock polymicrobial samples revealed that optimization of the primer sequences would be required to avoid preferential detection of particular taxa and cover a broad range of bacterial species. This study indicates that establishment of the simple work flow for rapid bacterial identification via MinION sequencing would reduce the turnaround time from sample to result, and also provide a reliable measure, not requiring trained personnel and specialized techniques.