Identification of microbial community in the urban environment: The concordance between conventional culture and nanopore 16S rRNA sequencing

Abstract

IntroductionMicrobes in the built environment have been implicated as a source of infectious diseases. Bacterial culture is the standard method for assessing the risk of exposure to pathogens in urban environments, but this method only accounts for <1% of the diversity of bacteria. Recently, full-length 16S rRNA gene analysis using nanopore sequencing has been applied for microbial evaluations, resulting in a rise in the development of long-read taxonomic tools for species-level classification. Regarding their comparative performance, there is, however, a lack of information.MethodsHere, we aim to analyze the concordance of the microbial community in the urban environment inferred by multiple taxonomic classifiers, including ARGpore2, Emu, Kraken2/Bracken and NanoCLUST, using our 16S-nanopore dataset generated by MegaBLAST, as well as assess their abilities to identify culturable species based on the conventional culture results.ResultsAccording to our results, NanoCLUST was preferred for 16S microbial profiling because it had a high concordance of dominant species and a similar microbial profile to MegaBLAST, whereas Kraken2/Bracken, which had similar clustering results as NanoCLUST, was also desirable. Second, for culturable species identification, Emu with the highest accuracy (81.2%) and F1 score (29%) for the detection of culturable species was suggested.DiscussionIn addition to generating datasets in complex communities for future benchmarking studies, our comprehensive evaluation of the taxonomic classifiers offers recommendations for ongoing microbial community research, particularly for complex communities using nanopore 16S rRNA sequencing.

EnsembleSeq: A workflow towards real-time, rapid and simultaneous multi-kingdom amplicon sequencing for holistic and cost-effective microbiome research at scale

“…It allows a maximum of 24 unique samples in a single run using the proprietary barcoded-16S primers, limiting the utility in studies aiming for scale in MINION devices. Given that 16S barcoding has been the default and benchmarked choice, many prior published reports of microbiome research using nanopore sequencing are often observed to be limited by scale in individual runs (15, 18, 42–44). In order to increase the scale of the captured microbiome, we sought to adopt the Native Barcoding Kit – 96 (SQK-NBD114.96) instead to natively barcode the sample specific amplicons (total 96 samples) entailing end-repair/end-prep, PCR free barcoding and adapter ligation.…”

Who inhabits the built environment? A microbiological point of view on the principal bacteria colonizing our urban areas

“…The prompt development of HTS led to the sequencing of full-length 16S rRNA by nanopore technology that implemented the taxonomy based on culture-based methods, particularly for the pathogenic species ( Lee et al, 2023 ).…”

“…Regardless these meta-analysis studies and mock community assessments are valuable in defining the accuracy, sensitivity, and specificity of microbial classifiers, a complex BE microbial community could provide a more realistic understanding of biological classifiers and mechanistic relationships ( Lee et al, 2023 ). However, surveys regarding a genomic-functional reconstruction often lack information about gene transcription, translation, and protein affinity and activity could be limiting ( Hester et al, 2019 ).…”

Implication of Nanopore Sequencing Technology for Pathogenic Diagnostics: A Mini-Review

“…The pathogens are not only found in the host but also the environment. Therefore, the detection of pathogens in different environments such as soil, river, water, and air samples was conducted using ONT [45,[52][53][54][55]. The nanopore sequencing method was also successfully applied for the detection of antibiotic resistance genes in microbes [45,53].…”