Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories

Abstract

Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials.

FIG 1

General NGS workflow. NGS workflows contain both wet-lab (and sample processing steps) and dry-lab (bioinformatics pipeline) steps. Sequence generation (primary analysis) occurs on the instrument and is the process of taking images or signals from the instrument and converting them into base calls that are assigned quality scores. During secondary analysis, primary sequence data are further processed and assessed for quality before either alignment to a reference sequence or de novo assembly is performed. During tertiary analysis, results are interpreted, clinically significant findings are identified, and a final report is generated. These workflow steps will vary depending on platform and application-specific requirements. Asterisk indicates metagenomics, or unbiased sequencing applications, do not require culture or isolation steps.

FIG 2

Assay validation framework. Proposed frameworks for clinical NGS implementation divide the analytical validation process into three phases: test development, assay validation, and quality management (18, 26). This figure was adapted in part from frameworks that were previously described (18, 26). SOP, standard operating procedure; QC, quality control; PT, proficiency testing; AA, alternate assessment.