Applications of the Third-Generation DNA Sequencing Technology to the Detection of Hop Tolerance Genes and Discrimination of <i>Saccharomyces</i> Yeast Strains

To expand the applicability of the third-generation DNA sequencing technologies for the brewing industry, the new experimental protocols for quality control (QC) brewery laboratories have been developed, using the MinION system. Firstly, the hop tolerance genes, <i>horA</i> and <i>horC</i>, were amplified with multiplex PCR, and the PCR amplicons were sequenced by the MinION system. As a result, all of the beer spoilage lactic acid bacteria (LAB) strains carrying <i>horA</i> and/or <i>horC</i> were positively identified. Secondly, <i>STA1</i>, <i>SbKEX2</i> and <i>GEX1</i>, were evaluated as diagnostic marker genes (DMGs) for discriminating <i>Saccharomyces cerevisiae</i> and <i>S. pastorianus</i> strains that possess essentially indistinguishable nucleotide sequences of the rDNA and internal transcribed spacer (ITS) regions. As a consequence of multiplex PCR specific to <i>STA1</i>, <i>SbKEX2</i> and <i>GEX1</i>, these DMGs were shown to separate these hard-to-discriminate strains into four groups, namely top-fermenting ale yeast strains (<i>S. cerevisiae</i>), bottom-fermenting lager yeast strains (<i>S. pastorianus</i>), non-brewing <i>S. cerevisiae</i> wild yeast strains, and <i>S. cerevisiae</i> var. <i>diastaticus</i>. This study also demonstrated that the multiplexed PCR amplicons are independently recognized and correctly identified by the MinION system. Taken together, the newly developed methods using the MinION system are applicable to the identification and discrimination of important brewery microorganisms for microbiological QC. It was also shown that the concurrent applications of the rDNA-sequencing-based species identification and DMG approaches by the MinION system are useful for the breweries that now manufacture both traditional beers and non-traditional beverages. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.1939606 .