Minimal requirements for ISO15189 validation and accreditation of three next generation sequencing procedures for SARS-CoV-2 surveillance in clinical setting

Abstract

Rapid and recurrent breakthroughs of new SARS-CoV-2 strains (variants) have prompted public health authorities worldwide to set up surveillance networks to monitor the circulation of variants of concern. The use of next-generation sequencing technologies has raised the need for quality control assessment as required in clinical laboratories. The present study is the first to propose a validation guide for SARS-CoV-2 typing using three different NGS methods fulfilling ISO15189 standards. These include the assessment of the risk, specificity, accuracy, reproducibility, and repeatability of the methods. Among the three methods used, two are amplicon-based involving reverse transcription polymerase chain reaction (Artic v3 and Midnight v1) on Oxford Nanopore Technologies while the third one is amplicon-based using reverse complement polymerase chain reaction (Nimagen) on Illumina technology. We found that all methods met the quality requirement (e.g., 100% concordant typing results for accuracy, reproducibility, and repeatability) for SARS-CoV-2 typing in clinical setting. Additionally, the typing results emerging from each of the three sequencing methods were compared using three widely known nomenclatures (WHO, Pangolineage, and Nextclade). They were also compared regarding single nucleotide variations. The outcomes showed that Artic v3 and Nimagen should be privileged for outbreak investigation as they provide higher quality results for samples that do not meet inclusion criteria for analysis in a clinical setting. This study is a first step towards validation of laboratory developed NGS tests in the context of the new European regulation for medical devices and in vitro diagnostics.

Enhancing Clinical Utility: Utilization of International Standards and Guidelines for Metagenomic Sequencing in Infectious Disease Diagnosis

“…The data processing specific to mNGS should follow the general requirement under the ISO24420 standards. To be used in clinical laboratories, assays should follow ISO15189 [ 46 , 47 ], a global quality standard for use in clinical laboratories, and standardized standard operating procedures (SOP). Changing any component of an assay should be validated and meet acceptable performance criteria before testing on patients, including reagents, equipment, sequencing instruments, and bioinformatics tools.…”

“…The in-house bioinformatics pipelines were compared with popular metagenomics pipelines using simulated in silico datasets and published raw patient datasets [ 211 ]. On the other hand, wet lab validation ensures the accuracy and reliability of mNGS results as it involves experimentally verifying the sequencing results with various laboratory techniques and reagents based on ISO15189 and ISO20397 [ 46 , 211 ]. In both dry and wet labs, mNGS validation should be based on the 5M1E methodology to enhance problem-solving capabilities, optimize procedures, and maintain high-quality assurance standards.…”