Evaluation of bacterial consortia associated with dairy fermentation by ribosomal RNA (rrn) operon metabarcoding strategy using MinION device

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The ability of the third generation sequencing technologies to provide longer sequence reads contributes to the use of the longest possible amplicons as specific bacterial markers for metabarcoding studies. Nanopore sequencing technologies are increasingly used worldwide to profile microbiomes in environmental and food samples. The identification of beneficial or pathogenic bacteria in dairy fermented foods is related to their valuable health properties and also contributes to food safety issues. Here we described and optimised a PCR-based methodology of almost the entire ribosomal operon sequences (16S-ITS-23S) and their subsequent sequencing by MinION device. We used three different sequencing data processing and analysis strategies. Two of those utilized user-friendly software without the need of being conversant with any programming language. We tested all workflows on a simple mock community composed of a mixture of 7 bacterial DNA. Our scripted bioinformatics pipeline denoted as “AEROS”, representing an approach based on taxonomic classification with our reference database called AEROS-DB (Almost Entire Ribosomal Operon Sequences), was applied to traditional Slovak sheep cheese made from unpasteurized milk. All bacterial genera included in the mock community were detected with relatively small differences compared to the expected relative abundance using each of the three approaches. The AEROS approach provided more accurate composition data on this community at the species level as well. The results suggested that the use of almost entire rrn operon sequences in metabarcoding studies is suitable to analyze the bacterial consortia in cheeses and related dairy fermented products.