Rapid identification of pathogens from positive blood culture bottles with the MinION nanopore sequencer

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Abstract
Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. Methodology. 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. Results. The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The ‘pass’ reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. Conclusion. We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.

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