A sample-to-report solution for taxonomic identification of cultured bacteria in the clinical setting based on nanopore sequencing

Abstract

Amplicon sequencing of 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories, and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations with the most significant being the inability to resolve mixed amplicons when closely related species are co-amplified from a mixed culture. This often leads to either increased turnover time or absence of usable sequence data. Short-read NGS technologies could address the mixed amplicon issue, but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling flexible number of samples per run and adjustable sequencing time. Here we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (Long Read Consensus ANalysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, artificial read mixtures of closely related species were used to assess LORCAN's behaviour when dealing with samples with known cross-contamination level. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability and availability of amplicon sequencing in diagnostic settings. 3/22

The application of nanopore targeted sequencing in the diagnosis and antimicrobial treatment guidance of bloodstream infection of febrile neutropenia patients with hematologic disease

“…All DNA extraction and DNA amplification methods were performed based on previously reported method with some modifications 5,17,18 . Briefly, 1.5 ml of EDTA‐whole blood samples were centrifuged at 800 × g for 10 min at room temperature.…”

“…All DNA extraction and DNA amplification methods were performed based on previously reported method with some modifications. 5,17,18 Briefly, 1.5 ml of EDTA-whole blood samples were centrifuged at 800 × g for 10 min at room temperature. Then, the lower part of red blood cells was discarded and about 600 μl of supernatant including leukocytes and plasma were separated and transferred to a new Eppendorf tube.…”

Investigating the biological and technical origins of unknown bases in the S region of the SARS-CoV-2 Delta variant genome sequences

“…Nanopore library preparation was performed with SQK-LSK109 (Oxford Nanopore Technologies, Oxford, UK) according to the ONT "PCR tiling of COVID-19 virus" (version: PTC_9096_v109_revE_06Feb2020, last update: 26/03/2020). Reagents, quality control and flow cell preparation were done as described previously [13,14]. Sequencing was performed on GridION X5 (Oxford Nanopore Technologies) with realtime basecalling enabled (ont-guppy-for-gridion v.4.2.3; fast basecalling mode).…”