To date no studies have explored MinION technology in clinical gut metagenomic samples. Diagnostics in metagenomic samples is still challenging due to lower MinION sequence yields and accuracy, but essential since many clinical samples are complex. The real-time nature of data generation could provide users with a rapid screening platform however, this real-time functionality and a different error profile require development of methods and bioinformatics pipelines, particularly for the clinical arena.ĭespite technical challenges in metagenomic profiling and diagnostics 13, MinIONs have been successfully used in medical research on low-complexity samples including: outbreak surveillance 14 characterization of bacterial isolates 15 and low microbial biomass samples 16, 17. In contrast to many NGS platforms, which require large capital investments and numerous samples to be multiplexed, newer sequencing platforms such as the MinION by Oxford Nanopore Technologies (ONT) represent inexpensive portable sequencing devices capable of producing long reads 12. Optimization of metagenomic methodologies and bioinformatics tools could allow the identification of at-risk individuals, profiling of infectious agents and tailoring of treatments 11. Metagenomics also allows the identification of functional traits, for example, antibiotic resistance genes, which are important in light of the antimicrobial resistance (AMR) threat 8, 9, 10. The culture-independent, sensitive, data-rich nature of metagenomic sequencing, combined with powerful bioinformatics tools, have allowed researchers to differentiate patient groups from healthy individuals based on their microbial profiles 1, 2, 3, 4, 5, 6, including those with increased risk of pathogen overgrowth 7. Next-generation sequencing (NGS) has revolutionized the profiling of environmental and clinical microbial communities. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. The MinION sequencing platform offers near real-time analysis of DNA sequence this makes the tool attractive for deployment in fieldwork or clinical settings.
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