Comparison of Illumina and Oxford Nanopore sequencing data quality for Clostridioides difficile genome analysis and their application for epidemiological surveillance

Abstract

Background: The burden of Clostridioides difficile as a nosocomial- and community-acquired pathogen has been increasing over the recent decades, including reports of severe outbreaks. Molecular and virulence genotyping are central for the epidemiological surveillance of this pathogen, but need to balance accuracy and rapid turnaround time of the results. While Illumina short-read sequencing has been adopted as the gold standard to investigate C. difficile virulence and transmission routes, little is known about the potential of Nanopore long-read sequencing in this field. The goal of our study was to compare sequencing and assembly quality of 37 C. difficile isolates using Illumina (SPAdes assembled) and Nanopore (Flye and Unicycler assembled) data alone, along with hybrid assemblies obtained with short-read polishing of long reads.

Results: Illumina sequencing produced reads with an average quality of 99.68% (Q25), while Nanopore sequencing produced reads reaching an average quality of 96.84% (Q15), showing a tenfold difference in quality. Sequence type (ST) designation from Nanopore assemblies failed to detect ST5, ST7, ST8, ST13 and ST49, while ST designation based on unpolished Nanopore reads using Krocus was successful for all STs. Nanopore sequences exhibited an average of 640 base errors per genome (~ 0.015% substitution rate), which was reflected by the incorrect assignment of over 180 alleles in core genome multilocus sequence typing (cgMLST) analysis. As a result, Nanopore-derived phylogenies were not as accurate as the Illumina reference, and therefore inadequate for precise investigation of transmission events. Both sequencing platforms provided comparable, satisfactory results for the detection of virulence genes tcdA, tcdB, cdtAB and in-frame deletions in tcdC.

Conclusion: Compared to Illumina, Nanopore has higher error rate, which limits its application for high-resolution epidemiological surveillance. However, the short analysis time, lower cost and more simple procedure combined with correctly identified STs and virulence genes, makes it an alternative when fast and less detailed analyses are preferred.

Keywords: Clostridioides difficile; Epidemiological surveillance; Genotyping; Illumina; Oxford Nanopore; Whole genome sequencing (WGS).

Fig. 1

Experimental workflow. Created using BioRender.com

Fig. 2

Phylogenetic tree (cgMLST) showing the allelic distances (scaling factor 10) between all 37 isolates and the four different assembly methods; Illumina, SPAdes (ILL), Nanopore, Flye (NPF), Nanopore, Unicycler (NPU) and hybrid assemblies (HYB). A) Distance tree including Illumina, hybrid and Nanopore isolates sequenced with R9.4.1 flow cell. B) Phylogenetic tree including Illumina, hybrid and Nanopore isolates sequenced with R10.4.1 flow cell. Blue boxes highlight the STs for which Nanopore-sequenced isolates (NPF and NPU) did not cluster with their hybrid or Illumina counterparts (HYB and ILL) due to the high number of allelic differences

Fig. 3

Phylogenetic trees based on cgMLST (scaling factor 10). A-D: isolates B11-34 A) Illumina assembly; B) Hybrid assembly; C) Nanopore (R9.4.1 flowcell) with Flye assembly; D) Nanopore (R9.4.1 flowcell) with Unicycler assembly. Red and blue boxes highlight the distinction between PCR ribotype RT066 (B18 and B23) from RT078 in the ST11 branch. E-H: isolates B39-51. E) Illumina assembly; F) Hybrid assembly; G) Nanopore (R10.4.1 flowcell) with Flye assembly; H) Nanopore (R10.4.1 flowcell) with Unicycler assembly