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. 2023 Apr 20;61(4):e0163122.
doi: 10.1128/jcm.01631-22. Epub 2023 Mar 29.

Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance

Gabriel E Wagner  1 , Johanna Dabernig-Heinz #  1 , Michaela Lipp #  1 , Adriana Cabal  2 , Jonathan Simantzik  3 , Matthias Kohl  3 , Martina Scheiber  1 , Sabine Lichtenegger  1 , Ralf Ehricht  4   5   6 , Eva Leitner  1 , Werner Ruppitsch  2 , Ivo Steinmetz  1
Affiliations

Real-Time Nanopore Q20+ Sequencing Enables Extremely Fast and Accurate Core Genome MLST Typing and Democratizes Access to High-Resolution Bacterial Pathogen Surveillance

Gabriel E Wagner et al. J Clin Microbiol. .

Abstract

Next-generation whole-genome sequencing is essential for high-resolution surveillance of bacterial pathogens, for example, during outbreak investigations or for source tracking and escape variant analysis. However, current global sequencing and bioinformatic bottlenecks and a long time to result with standard technologies demand new approaches. In this study, we investigated whether novel nanopore Q20+ long-read chemistry enables standardized and easily accessible high-resolution typing combined with core genome multilocus sequence typing (cgMLST). We set high requirements for discriminatory power by using the slowly evolving bacterium Bordetella pertussis as a model pathogen. Our results show that the increased raw read accuracy enables the description of epidemiological scenarios and phylogenetic linkages at the level of gold-standard short reads. The same was true for our variant analysis of vaccine antigens, resistance genes, and virulence factors, demonstrating that nanopore sequencing is a legitimate competitor in the area of next-generation sequencing (NGS)-based high-resolution bacterial typing. Furthermore, we evaluated the parameters for the fastest possible analysis of the data. By combining the optimized processing pipeline with real-time basecalling, we established a workflow that allows for highly accurate and extremely fast high-resolution typing of bacterial pathogens while sequencing is still in progress. Along with advantages such as low costs and portability, the approach suggested here might democratize modern bacterial typing, enabling more efficient infection control globally.

Keywords: Bordetella pertussis; bacterial typing; molecular surveillance; next-generation sequencing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
B. pertussis cgMLST (2983 loci) allelic differences between the Q20+ LR-AS and gold-standard SR-AS serving as a reference. Racon polishing was evaluated as an optional step in addition to Medaka polishing. Green arrows represent a decrease and red arrows an increase in the allelic differences if optional Racon polishing was applied.
FIG 2
FIG 2
Comparison of cgMLST-based minimum spanning trees (MSTs) of LR-AS (A) and reference SR-AS (B), each comprising the same 40 B. pertussis strains, shows the great performance of LR-AS for high-resolution molecular typing. The respective clusters are preserved independent of the sequencing technology used. The same is observed for allelic differences between isolates: minimal differences between the trees are not a result of sequencing errors but can be attributed to differences in the number of missing targets and one base ambiguity. Numbers on lines indicate the number of allelic differences between the respective strains. The cluster threshold was set to six. Replicates are indicated by the red font color. *, new strains; bacterial populations contain “Pop.” Nodes/strains are colored by genetic profile, whereas only differences in the genotype are denoted (Loci ptxS1-A, ptxP-3, and fim2-1 are conserved in the strains presented here).
FIG 3
FIG 3
Neighbor-joining tree of 40 B. pertussis strains shows the high quality of nanopore assemblies, which cluster directly with their short-read pendants and the polished hybrid assemblies. The tree nicely reflects the B. pertussis phylogeny of Austrian strains (52) and underlines the applicability of nanopore data for cgMLST-based phylogenetic analysis. The tree was rooted in the vaccine strain Tohama I. Strains can be distinguished by the color of the symbols. LR-AS are indicated in black, SR-AS in dark red, and HYB-AS in a violet font color. Technical replicates are marked in a red font color and bacterial populations by “Pop.”
FIG 4
FIG 4
Q20+ nanopore sequencing cgMLST workflow. The time of each step is given for the number of samples denoted. Live basecalling on a dedicated GPU was used to circumvent any additional time-consuming postrun basecalling, which is absolutely essential for fast analysis. The duration of all other bioinformatic steps are rough estimates for standard desktop PCs (e.g., 8-Core CPU, 16 GB RAM, SSD drive). *, sequencing time varies greatly depending on the number of samples, genome size of species under investigation, the sequencing coverage desired, and also the pore count of the flow cell (see Materials and Methods for details). The time given is an estimate for 12 B. pertussis strains in our study to obtain 30 to 100× coverage (see paragraph on real-time molecular typing for detailed information regarding sequencing time and coverage). Created with BioRender.com.
FIG 5
FIG 5
B. pertussis cgMLST (2,983 loci) allelic differences between the assemblies of live sequencing data at the time denoted (to achieve a certain coverage) and gold-standard SR-AS serving as a reference. Racon polishing was evaluated as an optional step in addition to Medaka polishing. Green arrows represent a decrease, and red arrows an increase in the allele differences if additional Racon polishing was applied.
FIG 6
FIG 6
MSTs generated from a live basecalling run and cyclic analysis. The preliminary LR-AS trees are shown in blue, the final LR-AS tree (at least 100× coverage for all isolates) in green, and an SR-AS tree in red as reference. Although the tree at 20× coverage still has several errors, close isolates already indicate potential transmissions demanding further investigations based on epidemiological data. The 30× MST almost resembles the final one. No differences were observed between the other LR-AS-based trees. The differences from the SR-AS MST result from differences in missing targets and are not due to incorrect allele assignment. In agreement with our processing suggestion in the main text, optional Racon polishing (blue trees) was performed for assemblies of coverage of ≤50. The indicated time corresponds to the time at which the last sample reaches the respective coverage.
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