Feasibility of MinION Nanopore Rapid Sequencing in the Detection of Common Diarrhea Pathogens in Fecal Specimen

• Published in: Analytical chemistry (Washington) 2022-12, Vol.94 (48), p.16658-16666
• Main Authors: Khan, Md Anik Ashfaq, Ghosh, Prakash, Chowdhury, Rajashree, Hossain, Faria, Mahmud, Araf, Faruque, Abu S. G., Ahmed, Tahmeed, Abd El Wahed, Ahmed, Mondal, Dinesh
• Format: Article
• Language: English
• Subjects: Abundance; Assaying; Bacteria - genetics; Chemistry; Deoxyribonucleic acid; Diagnostic systems; Diarrhea; Diarrhea - etiology; Diarrhea - microbiology; DNA; Epidemics; Feasibility Studies; High-Throughput Nucleotide Sequencing - methods; Humans; Infectious diseases; Microorganisms; Nanopore Sequencing; Nanopores; Nucleotide sequence; Nucleotides; Optimization; Parameter sensitivity; Pathogens; Sequence Analysis, DNA - methods; Species; Target detection
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.2c02771

The need for fast detection of etiological agents outside the narrow target range of pathogens that may cause an event of an infectious disease epidemic necessitates rapid sequencing technologies to be implemented in routine diagnostic procedures. We tested the performance of a PCR-free rapid nanopore barcoding assay to detect microbial species by analyzing genomic contents extracted from acute diarrheal case specimens. Sequenced reads were processed in an automated analysis module for species identification, whereas pathogenic subspecies detection was aided by a sequence similarity search against a gene-specific database. Evaluation of assay and analysis parameters (e.g., run-time, sequence length, and species hit abundance level) was carried out using a standard bacterial community for assessing detection accuracy. It was observed that longer sequence length (≥500 nucleotides) along with higher species abundance level (≥1%) can be critical for exclusion of false-negative outcomes, while increased sequencing run-time can affect the proportional abundance of true-positive species. Under optimal parameters, the sensitivity of the rapid assay remained 100% for the detection of a target species in a background of nontarget fecal (diarrheal) DNA that weighed up to 64 times the DNA of the target species. The method was applied to acute diarrheal samples. Among these, 62.5% (5/8) were in agreement with target-specific traditional diagnosis methods for the presence/absence of pathogenic agent(s), 12.5% (1/8) were in disagreement, and pathogenic agents that were not targeted by the traditional methods were revealed by sequencing for 25% (2/8) of samples. These observations suggest that further optimization and evaluation of the rapid nanopore sequencing method could potentiate the widening of the range of pathogens that can be detected in acute diarrheal samples in the context of regular diagnostic needs as well as epidemics.