Despite the massive developments within culture-independent methods for detection and quantification of microorganisms during the last decade culture-based methods remain a cornerstone in microbiology. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (< 24h) and cost-effective species level identification and strain level differentiation using MinION portable sequencing platform (ON-rep-seq). DNA library preparation takes less than 5h and ensures highly reproducible distribution of reads that can be used to generate strain level specific peak profiles (flowgrams). We have developed a pipeline that by correcting the random error of reads within peaks generates a set (~10 contigs per sample 300bp - 3Kb) of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of flowgrams allows for strain level differentiation. With benchmarked 288 isolates identified on a single flow cell and a theoretical throughput to evaluate about 1,200 isolates, our method allows for detailed bacterial identification for less than 2$ per sample at very high speed.