Application of Quality Assurance in Future Advanced Sequencing Diagnostics

Background

Over the past decade, Next-Generation Sequencing (NGS) has made revolutionary progress in the diagnosis of Rare Neurological Diseases (RNDs). However, the lack of technical, interpretative, and reporting standards has posed challenges in ensuring consistent and high-quality diagnostics globally. To address this issue, the European Reference Network for Rare Neurological Diseases (ERN-RND) collaborated with the European Molecular Genetics Quality Network (EMQN) to establish an NGS-based external quality assessment scheme for RND diagnostics.

In the 21st century, NGS methods have gradually become the foundation for diagnosing rare neurological diseases. Due to the clinical complexity and overlapping presentations of neurological disorders, as well as their extensive genetic heterogeneity, NGS methods provide a rapid and cost-effective approach to molecular diagnosis. Comprehensive genomic methods, including exome and whole-genome sequencing, are increasingly considered the first-line genetic tests for various RNDs.

Although the advantages of NGS in diagnosing RNDs are undeniable, NGS is a complex diagnostic method involving multiple stages that affect the final diagnostic results. Laboratories providing NGS diagnostic tests use various methods for sequencing library preparation, bioinformatics analysis, variant interpretation, and reporting. This diversity also leads to variability in test results, with many studies showing medically significant variability among laboratories in test characteristics, variant interpretation, and clinical reporting. Therefore, there is a need to promote harmonization of the NGS testing process and establish mechanisms to support high-quality NGS diagnostic testing.

Research Source

This paper was led by Aleš Maver, Katja Lohmann, Fran Borovečki, Nicola Wolstenholme, Rachel L. Taylor, Malte Spielmann, Tobias B. Haack, Matthias Gerberding, Borut Peterlin, and Holm Graessner, with authors from various research institutions across multiple countries. The paper was published in the European Journal of Human Genetics in 2024. As part of ERN-RND, the research team is dedicated to improving the quality of genetic testing in RND diagnostic laboratories across Europe.

Research Objective

ERN-RND aims to improve the level of care for RND patients within the European Union, particularly in ensuring comparable NGS test results for RNDs. To achieve this goal, ERN-RND established an external quality assessment scheme. The first pilot test of this scheme was conducted in 2021, followed by a second round of evaluation in 2022. This paper summarizes the experiences of these two rounds of external quality assessment and outlines the current state of NGS diagnostics provided in ERN-RND centers.

Research Process

Scheme Establishment

The ERN-RND external quality assessment scheme was established in 2021 in collaboration with EMQN. External quality assessment was conducted according to standard operating procedures. Participants performed molecular testing, interpretation, and reporting using provided case DNA samples and clinical information, and provided feedback on test methods and bioinformatics algorithms used.

Evaluation Procedure

The assessment focused on three main areas: molecular detection, interpretation, and documentation accuracy, with each area having a maximum score of 2.0 points. Points were deducted if laboratories failed to meet minimum standards or made errors. The laboratory’s score depended on the severity of the deviation.

Participation

In the first year, 29 laboratories applied to participate, and 25 submitted final reports; in the second year, 42 laboratories applied, and 37 submitted reports. Participating laboratories in the first year came from 17 different European countries, and from 18 countries in the second year.

Methods and Strategies

Comprehensive genomic methods were widely adopted by participating laboratories for RND diagnostics. In the pilot run, 56% of laboratories used exome sequencing, and 24% used gene panels. In the second year, 89.2% of laboratories reported using exome, clinical exome, or whole-genome sequencing. Most laboratories reported using internally developed data analysis pipelines and orthogonal validation of sequence variants.

Laboratory Performance

In the 2021 pilot run, 88% of laboratories reported correct molecular results for all three cases. In 2022, 94.6% of laboratories reported correct molecular results for all three cases. The detection included assessment of complex variant detection, such as CNV detection and interpretation of VCP-related dementia.

Results Analysis

The pilot program and subsequent runs demonstrated the overall accuracy and reproducibility of NGS in RND diagnostics. However, there was still widespread inter-laboratory variation in variant interpretation and data analysis reporting. This study revealed inconsistencies among laboratories in providing library preparation and sequencing methods, analysis quality parameters, gene content, and interpretation standards.

At the same time, widespread inter-laboratory variation was observed, particularly in reporting arrangements, adoption of interpretation standards, and clinical interpretation and reporting. For specific cases, such as VCP variant-related cases, there were significant differences in clinical interpretation among laboratories.

Reporting Format

There was also considerable variation in the reporting formats and content provided. Despite the complexity of NGS testing, it is recommended to concisely state the main results and conclusions of the test on the first page of the report, avoiding unnecessary length and complexity.

Suggestions for Future Improvement

To improve the quality and application value of diagnostic tests, it is recommended to promote consistent adherence to diagnostic test standards, variant interpretation, and reporting standards through continuous monitoring of external quality assessment and development of expert opinions or best practice guidelines applicable to RND-specific genetic backgrounds. The following measures are further recommended:

• Provision of sequencing platform and capture reagent information: Laboratories should provide detailed information on sequencing platforms, capture reagents, and their versions in the report.
• Quality parameter reporting: Laboratories should report important quality parameters such as sequencing depth and coverage.
• Variant interpretation standards: Laboratories need to provide information on variant interpretation standards, such as reporting evidence codes and their assigned strength of evidence when using ACMG guidelines.
• Clinical interpretation and guidance: Laboratories should concisely describe the relevance of test results to clinical referral and recommend genetic counseling and further genetic testing.

Conclusion

NGS technology shows significant potential in the diagnosis of RNDs, but continuous improvement is still needed in terms of consistency in variant interpretation and reporting. This study emphasizes the need to standardize the application of NGS testing in RND diagnostics through the creation of clear guidelines and ongoing external quality assessment, ensuring high-quality and consistent genetic diagnostic results, thereby improving patient care.