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

摘要

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. Łukasz Krych et al. uses MinION portable sequencing platform to develop a fast and cost-effective method for bacterial DNA enrichment and tagmentation (ON-rep-seq). Their method allows species-level identification and strain-level differentiation of 288 isolates on a single flow for less than $2 per sample at very high speed.