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DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq


Despite the massive developments within culture-independent methods for detection of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. Yet, the problem of rapid, accurate and inexpensive identification of bacterial isolates down to species/strain level remains unresolved. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (<24 h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation for 96 isolates takes less than 5 h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting reads error within peaks of LCp generates a set of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation.

Authors: Łukasz Krych, Josué L. Castro-Mejía, Laura M. Forero-Junco, Daniel N. Moesby, Morten B. Mikkelsen, Morten A. Rasmussen, Maciej Sykulski, Dennis S. Nielsen

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