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The potential challenges in deploying MinION sequencing include the need to pool a number of 1D ligation data reads together to generate a full 2D library, and a low yield of nanopore sequencing reads (which can be as low as 30%, or 20%, or 10%)128, which would pose a challenge for species identification in case of negative results. Sequencing reads are not available in a standard FASTQ format, but rather in FASTA format, and require conversion to a new FASTQ format (see accompanying article by Fothergill et al. in this issue) and analysis software130. The MinION 1D technology also has a higher error rate compared to second generation sequencers. The Illumina MiSeq has a base error rate of less than 0.1% (comparable to second generation sequencers), but the error rate of MinION reads is higher, estimated to be 0.5% of nucleotides131. However, a recent analysis of human ribosomal RNA (rRNA) sequences found that Illumina MiSeq datasets are more likely to contain erroneous rRNA sequences compared to MinION data. This is because MiSeq sequencers generate longer reads (which can span repeat regions) compared to MinION. The pathogenicity of the ribosomal RNA sequences is not significantly affected by the higher error rate of MinION sequencing because, apart from a single base difference for >99% of the sequences, the presence of errors does not affect the ability to identify a pathogenic isolate (P. Wei, personal communication). These results highlight the fact that errors generated by MinION sequencing might be reduced with improvements in the chemistry and analysis pipeline of the MinION sequencer. Additionally, the use of an Oxford Nanopore MinION sequencer for routine diagnosis in Australia may be limited to the use of single sites where longitudinal data could be stored in a public database. As more clinics are using MinION sequencers for routine diagnostics, the data will need to be deposited in public databases for public access, which could lead to concerns around privacy and confidentiality. Information technology, national and international guidelines, and legislation which may impact and regulate this area will need to be reviewed and discussed.
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