Multilocus sequence typing (MLST) is one of the most commonly used methods for studying microbial lineage worldwide. However, the traditional MLST process using Sanger sequencing is time-consuming and expensive. We have designed a workflow that simultaneously sequenced seven full-length housekeeping genes of 96 meticillin-resistant Staphylococcus aureus isolates with dual-barcode multiplexing using just a single flow cell of an Oxford Nanopore Technologies MinION system, and then we performed bioinformatic analysis for strain typing. Fifty-one of the isolates comprising 34 sequence types had been characterized using Sanger sequencing. We demonstrate that the allele assignments obtained by our nanopore workflow (nanoMLST, available at https://github.com/jade-nhri/nanoMLST) were identical to those obtained by Sanger sequencing (359/359, with 100 % agreement rate). In addition, we estimate that our multiplex system is able to perform MLST for up to 1000 samples simultaneously; thus, providing a rapid and cost-effective solution for molecular typing.
Keywords: MinION; dual-barcode multiplexing; meticillin-resistant Staphylococcus aureus (MRSA); molecular typing; multilocus sequence typing (MLST); nanopore.