Evaluation of the Oxford Nanopore Minion for the identification and differentiation of MRSA and non-MRSA isolates

Detta är en Master-uppsats från Högskolan i Skövde/Institutionen för biovetenskap

Författare: Kristoffer Nilsson Grimstad; [2020]

Nyckelord: ;

Sammanfattning: Staphylococcus aureus are bacterial pathogens causing infectious diseases. Methicillin-resistant S. aureus, or MRSA, carry the mecA or mecC genes generating resistance to β-lactam antibiotics. MRSA is problematic to treat and crucial to rapidly detect while current diagnostic workflows are time-consuming. DNA sequencing offers an alternative to existing methods. Illumina, the leading sequencing platform, is time-consuming and generates short reads. The Oxford Nanopore Technologies MinION presents rapid sequencing protocols, generating long reads at reduced accuracy. Here, the MinION device was evaluated to rapidly identify and differentiate between MRSA and non-MRSA isolates, with estimations on turn-around-times for clinical implementation.DNA extracted from 12 bacterial isolates were sequenced on the MinION. The automated workflow for raw MinION reads EPI2ME called taxonomy and resistance genes. MinION reads and corresponding Illumina reads were also assembled with Unicycler with short-read, long-read and hybrid assembly, and analyzed with the Center for Genomic Epidemiology bioinformatic tools. MinION and Illumina reads analyzed with the 1928 Diagnostics S. aureus pipelines were also compared. All tools correctly identified mecA in confirmed MRSA isolates, and mecC in an unconfirmed isolate. Similar results in terms of species, virulence and resistance genes were observed. Strain typing was problematic for MinION reads compared to Illumina, due to increased error rates. The minimum estimated turn-around-time for the MinION in this project, from library preparation, was approximately 6.5 hours to 7.5 hours per sample. Although further investigations are required, the MinION offers an intriguing alternative to current methods for identifying MRSA, aiding in rapid diagnosis and treatment.

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