Introduction
Advancements in Next-Generation Sequencing (NGS) have significantly reduced sequencing costs, making the technology more accessible for research and clinical applications. According to fortune business insights, global market size of NGS is projected to grow 3-fold between 2024 and 2032 (USD 9 billion to 27 billion), exhibiting a Compound Annual Growth Rate (CAGR) of 15% during the period. In this context, development of standardised protocols for NGS data flows and robust quality management systems (QMS) are crucial for reliable, high-quality results that can be trusted for clinical diagnostics and patient care. A significant progress has been made in this regard by establishing standards, guidelines and quality control metrics for NGS workflow (1-3) (Table 1). Some of the key developments in standards and guidelines for NGS include:
- Quality Management Systems (QMS): The Centers for Disease Control and Prevention (CDC), in collaboration with the Association of Public Health Laboratories (APHL), launched the Next Generation Sequencing Quality Initiative in 2019. This initiative provides laboratories with over 100 free guidance documents and standard operating procedures (SOPs) to support high-quality sequencing data and adherence to standards. The QMS framework is based on the Clinical & Laboratory Standards Institute’s (CLSI) 12 Quality Systems Essentials (QSEs), addressing challenges in developing and implementing NGS-based tests.
- Professional Standards and Guidelines: The American College of Medical Genetics and Genomics (ACMG) has developed comprehensive guidlines for clinical laboratories utilizing NGS. These guidelines cover the interpretation and reporting of variants. The ACMG’s “Technical Standards for Clinical Genetics Laboratories” were revised in 2021 to reflect technological advancements and current best practices.
- International Standards: The Global Alliance for Genomics and Health (GA4GH) is an international consortium developing standards for responsibly collecting, storing, analyzing, and sharing genomic data. Founded in 2013, GA4GH aims to enable an “internet of genomics,” a digital ecosystem or network infrastructure that integrates genomic data, computational tools, and stakeholders to facilitate the sharing, analysis, and application of genomic information globally.
| Organization | Recommendations/Guidelines | Key Focus Areas |
|---|---|---|
| European Medicines Agency (EMA) | Technical guidance on the validation and use of NGS in clinical trials and pharmaceutical development. | Clinical Trials, Pharmaceuticals |
| US Food and Drug Administration (FDA) | Recommendations for analytical validation, bioinformatics pipelines, and clinical application of NGS-based diagnostics. | Diagnostics, Bioinformatics |
| International Organization for Standardization (ISO) | ISO 20387:2018 – Standards for biobanking, including DNA and RNA sample handling for genomics. | Standardization, Biobanking |
| National Institute for Standards and Technology (NIST) | Reference materials and quality assurance standards for NGS-based genetic testing. | Quality Assurance, Reference Materials |
| The Global Alliance for Genomics and Health (GA4GH) | Frameworks for responsible data sharing, privacy, and interoperability in genomic research. | Data Sharing, Privacy, Interoperability |
| Genome Canada | Support for genomics-based research and guidelines on data-sharing for innovation in biotechnology. | Research Support, Data Sharing |
| The Human Genome Organisation (HUGO) | Guidelines on genomic variation interpretation and international data-sharing. | Variation Interpretation, Data Collaboration |
Table 1. NGS technology guidelines and recommendations
NGS workflows are complex, consisting of multiple steps from sample preparation in the wet-lab to the generation of bioinformatics output files (4). The quality of various steps in NGS workflows can be quantitatively evaluated, providing a systematic approach to assess and optimize the overall performance of these processes (5). Condition-specific, data-driven guidelines offer a robust framework to ensure the consistency and accuracy of results while promoting the harmonization of quality management in NGS workflows. For quality measures to be comparable, their calculation must be systematic and based on standardized practices. Emerging norms are beginning to serve as de facto standards in the field. At a broader level, requirements for overall procedures in NGS-based clinical diagnostics have been established by organizations such as the GA4G) and the Encyclopedia of DNA Elements (ENCODE) Consortium. These requirements, often referred to as standards, are being adopted and implemented in conjunction with established quality management frameworks, such as ISO 15189 for medical laboratories, which also address competence documentation.
Analytical phases of the NGS technology
The complexity of NGS technology poses challenges in defining standard requirements for clinical testing. Despite these challenges, efforts are underway by organizations like the GA4GH, the Clinical Genome Resource (ClinGen), and others to develop guidelines and best practices to standardize NGS for clinical applications. NGS technology can be divided into distinct steps:
- Nucleic acid extraction and library preparation: This includes the wet-lab, which will be different among other things for amplification-based and hybrid capture-based assay designs.
- Sequencing: Different vendors apply different detection methods and each platform requires different assay designs to optimise detection of different genomic variant types.
- Bioinformatics pipelines: This includes “dry-lab” data processing starting from the detector signal all the way to variant identification.
- Medical interpretation: Interpretation and reporting can be automated to some extent, depending on the complexity of the diagnostic procedure. It is based on the biomedical and genetic knowledge available in multiple dispersed electronic data repositories.
Technology Advancement
Technological advancements in NGS have expanded its applications in clinical diagnostics, personalized medicine, and large-scale genomic studies, driving innovation in genomics research (6). The continuous advancement of platforms, methodologies, genomic regions of interest, and bioinformatics tools presents significant challenges to standardizing the NGS process. In such a dynamic field, standardization efforts must either be highly agile to keep pace with rapid advancements or sufficiently broad and generic to remain relevant despite constant evolution. Each approach comes with its own set of challenges and complexities.
Current Guidelines, Recommendations and Regulations
Like any laboratory test, NGS is subjected to regulatory standards. Clinical laboratories are required to comply with regulations set by governmental regulatory agencies to obtain accreditation. While the regulatory oversight of clinical laboratories and NGS testing differs globally, many countries align their compliance requirements with standards established by the International Standards Organization (ISO). The presence of distinct regulatory agencies in different regions or countries has led to a lack of uniformity in the implementation of these guidelines globally. Regulatory agencies work closely with professional expert bodies to develop specific guidelines and recommendations, which are then provided to the respective agencies (Table 2). For example, in Australia and New Zealand, the National Association of Testing Authorities (NATA) serves as the regulatory agency, while the Royal College of Pathologists of Australasia (RCPA) acts as the expert body. In contrast, in North America, the College of American Pathologists (CAP) functions as both the accreditation agency and the expert body for laboratories (Figure 1). In Europe, the regulation of NGS technologies involves several key agencies and frameworks:
- European Medicines Agency (EMA):The EMA oversees the evaluation and supervision of medicinal products within the European Union (EU). It provides guidance on the use of NGS in the development and assessment of medicinal products, ensuring that NGS-based diagnostics and therapeutics meet safety and efficacy standards.
- In Vitro Diagnostic Regulation (IVDR):The IVDR (Regulation (EU) 2017/746) establishes a robust regulatory framework for in vitro diagnostic medical devices, including NGS-based tests. Effective from May 26, 2022, the IVDR aims to ensure the safety and performance of diagnostic devices across the EU. It introduces stricter requirements for clinical evidence, quality management, and post-market surveillance.
- National Competent Authorities: Individual EU member states have their own regulatory bodies responsible for implementing and enforcing EU regulations, such as the IVDR, within their jurisdictions. These authorities work in coordination with the EMA and the European Commission to ensure compliance and to facilitate the integration of NGS technologies into clinical practice-
Numerous quality control guidelines for NGS are already in place, covering a wide range of areas, from test development and bioinformatics workflows to clinical reporting and data storage (7). These guidelines also encompass external quality assessment and validation processes. Many organizations provide guidelines and recommendations for quality control (QC) in NGS (Table 2). While their focus areas may vary, certain QC parameters are universally emphasized (Table 3). Notably, Base Quality (e.g., Q30) and Library QC (Insert Size, etc.) are not explicitly covered by the National Institute of Standards and Technology (NIST)/Genome in a Bottle (GIAB). NIST/GIAB focuses primarily on providing reference materials and benchmark datasets for variant detection, such as single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants, rather than covering all aspects of the NGS workflow. Other organizations like CAP, CLIA, and RCPA fill in these gaps by providing guidelines for base quality and library QC to ensure a high-quality sequencing pipeline.
Numerous guidelines already exist. They run the gamut from test development and bioinformatics pipelines to clinical reporting and data storage. The guidelines likewise address external quality assessment and validation along with aspects of patient and laboratory director education. Although the recommendations and guidelines are fairly similar among all expert bodies, there are still distinct differences in the quality measures employed (Table 2). For instance, reads mapped is a recommended measure in the guideline of EuroGentest, but neither CAP nor RCPA recommend its use. Similarly, CAP does not require the monitoring of GC Bias, whereas it is considered to be important by EuroGentest. Thus, although the regulatory agencies have been set up for the same purpose of ensuring good lab practices, the differences in guidelines and recommendations make standardisation difficult.
| Organization | Focus on NGS QC |
|---|---|
| CAP (College of American Pathologists) | Comprehensive QC metrics for clinical diagnostics; emphasis on pre-analytical, analytical, and post-analytical validation. |
| CLIA (Clinical Laboratory Improvement Amendments) | Standards for sample quality, test validation, and proficiency testing in U.S. clinical laboratories. |
| ESHG/EuroGentest | European guidelines for diagnostic NGS, focusing on analytical sensitivity, specificity, and bioinformatics. |
| NIST/GIAB (National Institute of Standards and Technology/Genome in a Bottle) | Provides reference genomes for benchmarking analytical accuracy of variant detection. |
| ACMG | Technical standards for clinical NGS, including variant classification and reporting. |
| AMP (Association for Molecular Pathology) | Standards and Guidelines for Validating NGS Bioinformatics Pipelines: |
| RCPA (Royal College of Pathologists of Australasia) | QA/QC in pathology, including NGS, with a focus on external proficiency testing. |
| ACGS (Association for Clinical Genomic Science) | UK-based guidelines for clinical NGS, emphasizing bioinformatics validation and reproducibility. |
| GA4GH | WGS (whole genome sequencing) quality control standards. |
Table 2: Core organizations involved in NGS quality control and their specific focus areas
| QC Parameter | CAP | CLIA | EuroGentest | NIST/GIAB | ACMG | AMP | RCPA | ACGS |
|---|---|---|---|---|---|---|---|---|
| Sample Quality | x | x | x | x | x | x | x | x |
| DNA/RNA Integrity | x | x | x | x | x | x | x | x |
| Library QC (Insert Size, etc.) | x | x | x | x | x | x | x | |
| Depth of Coverage | x | x | x | x | x | x | x | x |
| Base Quality (e.g., Q30) | x | x | x | x | x | x | x | |
| Mapping/Alignment Quality | x | x | x | x | x | x | x | x |
| GC Bias | x | x | x | x | x | x | x | x |
Table 3: QC parameters covered by different organizations
Summary
The rapid advancements and significant cost reductions in NGS technologies in recent years have enabled their clinical application. However, the inherent complexity of NGS poses challenges to standardizing usage procedures. Various regulatory agencies and organizations have been established to develop recommendations and guidelines for the clinical use of NGS. Yet, these efforts are often limited to the national level, with significant variations between professional expert bodies, hindering global harmonization.
Despite these challenges, existing guidelines are already robust, encompassing all aspects of clinical NGS from wet-lab processes and bioinformatics pipelines to subsequent consumer education. Nevertheless, international initiatives like the GA4GH play a pivotal role in accelerating and broadening the scope of standardization efforts.
For successful implementation, emerging guidelines must be practical and straightforward for individual laboratories. Harmonized guidelines will facilitate the adoption of NGS technologies, enhancing their accessibility and expanding their applications in clinical and research settings.
References
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