Standardization of 16S rRNA gene sequencing using nanopore long read sequencing technology for clinical diagnosis of culture negative infections

Abstract

The integration of long-read sequencing technology, such as nanopore sequencing technology [Oxford Nanopore Technologies (ONT)], into routine diagnostic laboratories has the potential to transform bacterial infection diagnostics and improve patient management. Analysis of amplicons from long-read sequencing of the 16S rRNA gene generates a comprehensive view of the microbial community within clinical samples, significantly enhancing sensitivity and capacity to analyse mixed bacterial populations compared to short read sequencing approaches. This study evaluates various ONT sequencing approaches and library preparation kits to establish a reliable testing and quality framework for clinical implementation. This study highlights the critical importance of using well-characterized reference materials in validating and revalidating long-read sequencing methods, leveraging a combination of standardized reference materials and clinical samples to navigate the evolving landscape of microbial diagnostics. It presents a robust validation framework for laboratory accreditation and outlines a methodology for comparing the performance of newer ONT chemistries with earlier versions. Additionally, the study details the methods and quality control measures necessary for achieving more accurate and efficient diagnoses of bacterial infections, ultimately reducing time to treatment and enhancing patient outcomes.

Keywords: 16S rRNA; Oxford nanopore; culture negative; long read sequencing; standardization.

Figure 1

Average relative abundance (%) of bacterial organisms present in MCM2α and MCM2β obtained when by ONT sequencing using either the R9 MinION flow cell or the R9 Flongle flow cell. Three concentrations of the MCM2α and MCM2β materials were tested in triplicate (neat, 1:10 and 1:100) and run in parallel on each of the ONT sequencing approaches. The species abundance (%) of each dilution is reported, including the nominal and dPCR reported composition for the two materials ( Supplementary Data 3 ).

Figure 2

Average relative abundance (%) of bacterial organisms present in MCM2α and MCM2β obtained by ONT sequencing using either the R10.4.1 MinION flow cell or the R10 Flongle flow cell and version 14 kit chemistry. Three concentrations of the MCM2α and MCM2β materials were tested in triplicate (neat, 1:10 and 1:100) and run in parallel on each of the ONT sequencing approaches. The species abundance (%) of each dilution is reported, including the nominal and dPCR reported composition for the two materials ( Supplementary Data 3 ).

Figure 3

Average relative abundance (%) of bacterial organisms present in MCM2α and MCM2β obtained by ONT sequencing using the R10.4.1 MinION flow cell and ONT 16S Barcoding all-in-one kit (v14). Three concentrations of the MCM2α and MCM2β materials were tested in triplicate (neat, 1:10 and 1:100) and analysis by EPI2ME Desktop agent. The species abundance (%) of each dilution is reported, including the dPCR reported composition for the two materials ( Supplementary Data 3 ).

Figure 4

Average relative abundance (%) of bacterial organisms present in MCM2α and MCM2β obtained by ONT sequencing using the R10.4.1 MinION flow cell and the in-house developed 16S ONT RBK method. Three concentrations of the MCM2α and MCM2β materials were tested in triplicate (neat, 1:10 and 1:100) and amplified in two PCRs targeting the V1-V2 and V1-V9 genomic regions of the 16S rRNA gene; analysis by EPI2ME Desktop agent. The species abundance (%) of each dilution is reported, including the dPCR reported composition for the two materials ( Supplementary data 3 ).