Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria

Abstract

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4–6 bacterial isolates per sequencing run (20–26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

“…Sequencing runs of genomic Escherichia coli DNA were downloaded from the NCBI Sequence Read Archive (SRA; ). We selected three runs each for MiniSeq (SRR20643069, SRR20643071, SRR20643072; generated by the GenomeTrakr project), iSeq (SRR14617007, SRR14617041, SRR14617075) ( Mitchell et al 2022 ), and MiSeq (SRR19575967, SRR19575968, SRR19575973; generated by the National Microbiology Laboratory).…”

Making microbial genomics work for clinical and public health microbiology

“…‘Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella , Escherichia coli and Listeria ’ [11]…”

“…an inter-laboratory study' [10] • 'Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria' [11] • 'Centre-specific bacterial pathogen typing affects infection-control decision making' [12] • 'Harmonization of whole-genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci' [13] • 'Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods' [14] • 'GenomeTrakr proficiency testing for foodborne pathogen surveillance: An exercise from 2015' [15] We encourage new submissions to the Implementation Collection that present the results of inter-laboratory evaluation of genomics methods, tools and databases; frameworks for implementation and training; articles evaluating genomics methods against established standards; and proposing standards, proficiency tests or reporting proforma for applied microbial genomics. We also welcome articles proposing and/or evaluating new genomics-based methodologies for clinical or public health microbiology (including bacteriology, virology, parasitology and mycology).…”