SUMMARYResearch backgroundSourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity of the product of interest be elucidated.Experimental approachUsing the opportunities of next-generation sequencing (NGS) of the V1-V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made ofTriticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity.Results and conclusionsAll three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genusLactobacillus(relative abundance of 63.11–82.28%) and family Leuconostocaceae, genusWeissella(relative abundance of 3.67–36.31%) was observed.Lactiplantibacillus plantarumandLevilactobacillus breviswith relative abundance of 16.15–31.24% and 6.21−16.29% respectively, were the two dominant species identified in all three DNA isolates.Novelty and scientific contributionThe presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.