The CDC, the Association of Public Health Laboratories (APHL), and state and local PHLs collaborated to form the NGS Quality Initiative, which assists clinical and PHLs in developing an NGS-focused QMS. The Initiative brought together public health leaders, laboratory scientists, microbiologists, bioinformaticians, quality managers, and other subject matter experts for a consensus-based approach to identify challenges in establishing and maintaining NGS assays in clinical and PHLs.

Using CLSI’s 12 QSEs as a guide, the NGS Quality Initiative works to develop and publish customizable tools, resources, standard operating procedures (SOPs), forms, and logs, among other products, which can be used as a foundational system or integrated into a laboratory’s existing QMS (9). To promote open access to its tools, the NGS Quality Initiative developed a publicly accessible website that contains NGS-related information and all its QMS resources (https://www.cdc.gov/lab-quality/php/ngs-quality-initiative/; Supplemental materials). Since 2019, the Initiative has published 115 documents (Table 1) and several tools. From 2020 to 2025, NGS Quality Initiative products have been downloaded approximately 29,000 times, with roughly 48,000 unique visitors to the website (Fig. 2; Table S1), and information gathered through various channels (e.g., emails, surveys, and workgroups [data not shown]) indicates many laboratory personnel are interested in using the Initiative’s resources (8, 9, 14). The NGS Quality Initiative builds on previously published work, consensus guidelines, and best practices (when possible) to develop an expanded framework of resources to further laboratory implementation of quality practices for NGS workflows under research, surveillance, and diagnostic applications (14).

Outlined here by QSE, we provide brief descriptions of associated challenges and resources (i.e., NGS Quality Initiative’s published products) that help to mitigate the impact of such challenges (Table 1).

This QSE describes requirements for onboarding and retaining qualified, trained, and competent personnel who perform and manage laboratory activities (9, 11, 14). Personnel Management is vital to the laboratory; this QSE ensures that staff possess the requisite qualifications, undergo comprehensive training, demonstrate competency, exhibit the ability to perform assigned tasks proficiently, actively engage in continuous improvement, and implement and maintain the QMS effectively. The complexity of NGS workflows poses various challenges related to Personnel Management, including the need for specialized NGS training and assessments, the variety of sequencing platforms and associated equipment, specialized training to build and run bioinformatic pipelines, and the need to comply with testing regulations (e.g., CLIA regulations, or applicable regulatory and accreditation requirements) (10, 13). Proof of competency is also required, e.g., a biochemist operating an NGS assay must have demonstrated proficiency operating that assay under direct supervision (14). To overcome technical and quality-related challenges related to this QSE, the NGS Quality Initiative has published 25 products specific to laboratory personnel, including training procedures, sequencer-specific competency assessments, and training forms (Table 1; Table S1 [documents 1–25]). Since 2020, there have been over 4,000 downloads of Personnel Management-related resources (Fig. 2B; Table S3). For instance, since training bioinformatics personnel via a consistent methodology remains a challenge for NGS laboratories, the NGS QI developed the Bioinformatics Employee Training SOP, which outlines a proper sequence of training and criteria for the demonstration of competency.

Equipment Management is critical for ensuring that laboratory instruments function according to established criteria; effective management includes robust preventive maintenance and calibration programs as well as proper documentation of equipment-related problems (11, 14). Equipment used for NGS can be highly specialized, requiring detailed knowledge of proprietary engineering and associated software to troubleshoot and repair (7, 15). Additionally, laboratories performing testing under CLIA require procedures that ensure adherence to manufacturers’ recommendations for both hardware and software, or applicable regulatory and accreditation requirements (10). To reduce the impact and risks associated with NGS equipment, the NGS Quality Initiative published 36 equipment-related, customizable tools, resources, and SOPs (Table 1; Table S1 [documents 26–61]). Since 2020, there have been over 4,000 downloads of Equipment Management resources (Fig. 2B; Table S2). The three most downloaded tools for equipment are the pre-installation checklists for MiSeq, iSeq 100, and MinION Mk1B sequencers, the most popular platforms in public health settings. These tools address some of the most consistent challenges for laboratories: equipment selection, installation preparation, maintenance, and recordkeeping.

Process Management refers to all laboratory activities and phases associated with the testing process (i.e., pre-analytical, analytical, and post-analytical stages) and is crucial to the quality of the laboratory’s result reporting, which dictates the impact and value of testing results for the target audience and/or customer(s) (11, 14, 16). Incorporating NGS workflows into this QSE can be complex due to the lack of experienced personnel who, e.g., may need to prepare a method validation or establish a QC plan (8, 17-21). Process Management is further complicated by frequent technical modifications, which are often required due to updates in software, sequencing platforms, and bioinformatic analysis tools. In addition, turnover in kit chemistries can create significant challenges, as changes to kit chemistries can occur frequently, sometimes in as few as 2 years. Change control plans should be built into method validation plans, especially since these changes can happen abruptly and frequently. Failure to properly manage workflow activities may result in adverse outcomes and other roadblocks. Laboratories performing testing under CLIA are required to demonstrate reproducible results and assay performance according to specifications defined by the laboratory or manufacturer, which highlights the need for uniformity in laboratory processes or applicable regulatory and accreditation requirements (10). To reduce risks associated with this QSE, the NGS Quality Initiative published 12 customizable resources (Table 1; Table S1 [documents 62–73]). Since 2020, there have been over 7,000 downloads of resources related to Process Management (Fig. 2B; Table S2). The three most downloaded products help mitigate a common challenge for NGS laboratories: method validation. These products are the NGS plan template, SOP, and summary report, all of which support the validation of NGS laboratory-developed tests.

Information Management encompasses those processes necessary for proper data management. It offers guidance on managing and accessing information, including records entered into laboratory notebooks or electronic record management systems (11). Laboratories performing NGS manage extensive amounts of data while ensuring data accuracy, validity, completeness, integrity, timeliness, and immutability (3, 22). Laboratories need policies, processes, and procedures that address data and information access. Furthermore, security plays an important role in Information Management, especially under CLIA regulations, where confidentiality and data integrity are of the utmost importance during transmission of patient data or applicable regulatory and accreditation requirements (10). To assist laboratories performing NGS-based assays, the NGS Quality Initiative has developed 14 customizable Information Management tools (Table 1; Table S1 [documents 74–87]). Since 2020, there have been over 3,000 downloads of Information Management resources (Fig. 2B; Table S2). The three most downloaded documents are Sequencing Data Storage and Retention Best Practices, Bioinformatic Pipeline Documentation Standardization—Technical Best Practices, and Bioinformatic Pipeline Documentation Management–Laboratory Management Briefing. These documents ensure consistent and accurate laboratory workflows and serve as references for laboratories, thereby standardizing procedures, reducing errors, and enhancing overall productivity. Information Management resources also provide support for bioinformatic pipeline documentation, containerization of computer resources, and practices for data sharing, storage, and retention, among other NGS-related applications.

Internal and external laboratory assessments are essential to verify that all processes meet performance and regulatory requirements (9, 11). An internal audit allows the laboratory to evaluate its processes. In contrast, external audits are conducted by entities external to the laboratory, like regulatory bodies or accrediting organizations (e.g., Centers for Medicare & Medicaid Services and the College of American Pathologists, respectively) (23). Failure to meet the requirements of any such auditing organization may result in the suspension or termination of laboratory services until deficiencies have been resolved. Due to the complexity of NGS workflows, assessments often require specialized skills and expertise in evaluating wet- and dry-bench workflows. To assist NGS laboratories, the NGS Quality Initiative has developed five customizable assessment tools (Table 1; Table S1 [documents 88–91]). These tools help evaluate laboratories’ QMS frameworks and aid in assessing bioinformatic pipelines. Because evaluating an entire QMS can be difficult, the NGS QI developed the QMS Assessment Tool, which serves as a valuable resource for users to assess their laboratory’s QMS (with a focus on NGS framework) to generate qualitative and quantitative data. The QMS Assessment Tool provides laboratories with comprehensive question sets that, once answered, produce weighted scores related to each of the 12 QSEs. This allows users to evaluate laboratory quality practices and identify gaps and areas for improvement while tracking compliance over time (e.g., by quarter, by QSE). Since 2020, there have been more than 3,500 downloads of resources related to the QSE Assessments (Fig. 2B; Table S2), approximately 2,500 of which were downloads of the QMS Assessment Tool.

The QSE Continual Improvement defines a strategy and provides information for identifying opportunities to improve laboratory processes (10, 12). Because laboratories constantly refine their workflows and methods, continual improvement is paramount for risk mitigation and risk management. Without a laboratory policy that is strongly aligned with the principles of continual improvement, organizations may struggle to implement and maintain successful risk management programs. Further care must also be taken in standardizing processes for validation of NGS assays.

NGS test systems are divided into wet- and dry-laboratory workflows (e.g., nucleic acid, library preparation, and bioinformatic analysis; Fig. 1). In addition, each step requires specific equipment, protocols, and QC metrics. Such requirements can be challenging, especially as laboratories work to implement adequate monitoring and improvement processes that ensure compliance with regulations, such as those defined by CLIA, or applicable regulatory and accreditation requirements (11). To address these challenges, the NGS Quality Initiative developed two tools, which provide a framework for setting goals and identifying, monitoring, and assessing progress (Table 1; Table S1 [documents 92 and 93]). One of the tools, an SOP entitled Identifying and Monitoring NGS Key Performance Indicators, facilitates the monitoring of test performance, laboratory processes, and potential test system drift through metric tracking and evaluation. Since 2020, there have been over 2,000 downloads of Continual Improvement resources (Fig. 2B; Table S2).

Laboratory facilities must meet physical requirements and established processes and procedures that ensure personnel safety and that provide proper environmental conditions (11). This includes providing information about the laboratory’s physical environment and implementing the facility maintenance and safety programs needed to support the laboratory and its activities (9, 24). Due to the nature of some NGS workflows, laboratory personnel face potential risks. For example, chemical exposure involving sequencing reagents (e.g., formamide and phenol) can be toxic in nature. Plans to mitigate and handle such risks and exposures are critical, including training to understand and follow safety data sheets (e.g., how to use appropriate personal protective equipment). Additional considerations include risk assessments and near-miss events; potential equipment failures in high-containment laboratories present an added layer of complexity due to access being restricted to specialized personnel. To assist laboratories with such challenges, the NGS Quality Initiative has created five customizable waste disposal documents for Illumina and Oxford Nanopore instruments (Table 1; Table S1 [documents 94–98]), including the Oxford Nanopore Flow Cell Reuse or Return SOP, which clarifies reuse, return, and disposal methods for flow cells, processes that are critical but sometimes convoluted or confusing. Since 2020, there have been over 700 downloads of QSE Facilities and Safety Management resources (Fig. 2B; Table S1).

Efficient and cost-effective laboratory operations require the constant availability of quality reagents, supplies, and services. Laboratories should describe agreements regarding the acquisition of equipment, materials, products, and services while also ensuring that all specified criteria for critical supplies and services are consistently met (10, 12). Inventory management, with an adherence to CLIA regulations or applicable regulatory requirements, is paramount in a QMS, whether related to reagents, lots, or storage conditions. However, due to the complexities of NGS (e.g., frequent reagent changes, large numbers of reagent lots to be cataloged and stored, and planning and risk assessments related to changing inventory), satisfying all regulatory criteria can be taxing on the laboratory (11). To aid NGS laboratories in managing inventory, the NGS Quality Initiative has developed three tools related to QSE Supplier and Inventory Management (Table 1; Table S1 [documents 99–101]). The inventory manager is a stand-alone solution that facilitates inventory creation and management, as well as the forecasting of reagent needs. Since 2022, more than 600 downloads have been documented for the QSE Supplier and Inventory Management (Fig. 2B; Table S2).

This QSE describes the creation, management, maintenance, control, archival, and retention of documents for the laboratory’s QMS and workflow (12). Laboratory documents and records, whether paper or electronic, must be completed, dated, and populated with correct information to ensure accurate results and patient safety. However, documents and records management can be difficult due to NGS complexity, the large amount of data generated by each test, the format and number of files generated during a sequencing run, and applicable regulatory requirements (Table 1). Record retention times vary based on the regulatory body and accrediting organization. For example, CLIA requires that records be retained for at least 2 years, even in the event of discontinued procedures or methods; this includes emails, method validation plans, and laboratory results, such as sequencing data or QC metrics (11). To help laboratories that perform NGS-based assays, the NGS Quality Initiative has created a Documents and Records Management job aid (Table S1 [document 102]). This tool can help to create documents, establish policies, and determine retention periods according to laboratory needs. Since 2023, there have been nearly 150 downloads of the QSE Documents and Records Management resources (Fig. 2B; Table S2).

Nonconforming Event (NCE) Management consists of the requirements for documenting and investigating events that do not conform to the laboratory’s established policies, processes, procedures, or other specified requirements (9, 11). The QSE NCE is critical to mitigate risk and correct deviations from normal practices before larger issues occur. This QSE describes processes to detect and record nonconformances, manage products and services that fail to meet specified requirements, classify the nonconformances for analysis, and correct the problems presented by nonconformances. Due to NGS complexity, laboratories can encounter more frequent NCEs related to personnel, equipment, and process management. The consequences of NCEs can be significant, ranging from minor to critical (e.g., using a sequencer with outdated calibration can lead to incorrect sequencing or misinterpretation that can result in incorrect patient diagnoses and treatments). Adding to the intricacy of this QSE, CLIA requires establishing procedures for monitoring out-of-compliance events (11). To reduce the difficulties associated with this QSE, the NGS Quality Initiative has developed six NCE tools, including procedures for identifying and documenting NCEs, analyzing root causes, and undertaking corrective actions (Table 1; Table S1 [documents 103–108]). Since 2022, there have been over 1,000 downloads of NCE Management resources (Fig. 2B; Table S2).

The QSE Organization and Leadership outlines the criteria for structured roles and responsibilities while also highlighting key leadership functions essential for a laboratory’s success in meeting regulatory and customer requirements (10). Laboratories should have a documented organizational plan and structure that ensures the delivery of services and that details a system-wide approach to quality (12). It is crucial to clearly document the scope of laboratory services, including a description of all testing services provided and the customers served. CLIA requires that all personnel roles, responsibilities, and reporting relationships be documented and communicated effectively (11). This ensures that all staff members are aware of their respective roles within the organization. Poor organization and communication can negatively impact results, final reports, customer satisfaction, and the laboratory’s reputation. To reduce the challenges associated with this QSE, the NGS Quality Initiative has developed three documents with examples of organizational structures and communication plans (Table 1; Table S1 [documents 109–111]). For instance, the NGS Laboratory Communication Plan Guide provides considerations and best practices for conveying essential information to and from leadership, laboratory professionals, internal customers, and external customers. Since 2023, there have been nearly 200 downloads of Organization and Leadership resources (Fig. 2B; Table S2).

The QSE Customer Focus describes the laboratory’s need to identify customers and their expectations, formalize contractual agreements for laboratory services, and manage customer feedback (10, 12). Customers rely on laboratories for critical and highly specialized work, expertise, accurate results, and timely reports, all of which are essential in building and maintaining customer trust and satisfaction. For a laboratory performing NGS, customers can include patients, physicians, genetic counselors, epidemiologists, and others. Failure to meet customers’ expectations may result in delayed decisions and errors, thereby impacting clinical outcomes and/or public health efforts. CLIA requires establishing a system to communicate results and customer feedback during the total testing process (11). Nevertheless, it can be challenging to report NGS results that meet all customer needs. To help laboratories communicate results effectively, the NGS Quality Initiative has created three resources: NGS Result Report Interpretation Guide, NGS Result Report Template, and NGS Training Resource Repository (Table 1; Table S1 [documents 112–114]). Since 2023, there have been over 300 downloads of QSE Customer Focus resources (Fig. 2B; Table S2).

Establishing a QMS for laboratories performing NGS is a complex process. Many challenges persist due to the variation in techniques and applications, complexity of the NGS assays, lack of guidance for the development of reference materials, lack of consensus or gold-standard materials for infectious disease testing, and the need for additional-application and agent-specific prescriptive guidance (10, 11, 14, 20, 25). In response, the NGS Quality Initiative has moved beyond producing guidance documents and progressed toward creating practical, implementable tools that address the recurring operational barriers laboratories face when integrating NGS. By mapping resources to the CLSI’s 12 QSEs and providing customizable SOPs, checklists, training materials, validation templates, and bioinformatics toolkits, the Initiative translates high-level quality concepts into concrete workflows and decision aids. These products directly support laboratories in tackling common obstacles (e.g., workforce training, equipment installation and maintenance, data and pipeline documentation, method validation, inventory forecasting, and nonconforming event management), thereby enabling more consistent, reproducible, and auditable NGS practice across diverse settings. The Initiative’s emphasis on usability (e.g., platform-specific pre-installation and maintenance checklists), assessment (e.g., the QMS Assessment Tool), and shared technical solutions helps operationalize quality practices and shorten the time to effective implementation (8). Continued collaboration with federal and nonfederal partners will be essential to refine these tools, expand agent- and application-specific guidance, and advance external quality assurance mechanisms. The NGS Quality Initiative’s practical, QSE-aligned toolkit therefore represents a substantive contribution toward making robust NGS quality management actionable and sustainable in clinical and public health laboratories.

There remains a need to develop and improve methods that continuously promote NGS testing quality, standardization, and implementation while also increasing understanding of test performance (9, 26, 27). Many organizations, both federal and nonfederal, have attempted to satisfy these needs, in part, through the application of prescriptive guidance, regulatory guidelines, and guardrails. Though success has been demonstrated, the need for additional uptake across diverse partners remains. Although the NGS Quality Initiative does not focus on specific international requirements, CLSI’s QSEs can overlap with ISO 15189, which provides users with the potential to customize tools for specific international quality requirements (10).

The NGS Quality Initiative continues to develop tools and resources that close these gaps and address quality-related challenges for NGS laboratories.

Given the increased reliance on sequencing techniques, high-throughput systems will likely become more ubiquitous within public health and clinical settings. At the same time, machine learning and artificial intelligence (AI) are already applied in many laboratories; these tools will continue to improve and grow, and such growth has the potential to greatly benefit those laboratories equipped to harness it. Utilization of machine learning algorithms is already advantageous in clinical practice and can provide enhanced detection and/or identification of disease states, with additional insights into the mechanisms driving disease (28, 29). Other changes could pertain to the evaluation of current applications and the effectiveness of AI in bioinformatics and NGS-related tasks, including research, coding (e.g., pipeline development), and visualization capabilities (28-30). In line with advances in AI, the Initiative is currently exploring natural language processing models to assess method validation plans and increase the efficiency of this process (data not shown).

Advances specific to NGS itself are also expected in the coming years. Along with the rapid growth of NGS-based methods for surveillance and diagnostics comes the requirement for additional standards and further guidance. In silico data set standards may be generated according to sequencing-machine parameters and then engineered to include complex genetic traits that might not otherwise be captured in sample cohorts traditionally used to test methods; however, without prescriptive guidance as to the development of gold standards, each laboratory is left to govern the development, quality, and use of data sets. In addition, automated platforms may save time for personnel (e.g., sample-to-result NGS platforms require minimal hands-on tasks), but such approaches often require new training and increased regulatory considerations (31-34).

Going forward, the establishment of NGS assay performance consensus and interlaboratory comparison methods will be critical. These needs could be addressed, in part, by external quality assurance tools like challenge panels, proficiency testing (PT), or alternative assessment programs, which would allow laboratories to evaluate accuracy and reliability among peers using standardized materials and data files. For example, a Swiss PT study identified differences between bioinformatic workflows and lessons learned to develop future benchmarks for quality control (35). To help address this challenge, the NGS Quality Initiative worked with APHL on a peer network program, in which laboratories can compare workflows on a use-case basis, share sequencing data, develop/deploy new NGS-based QMS practices, and establish collaborations (36).

Additional agent-specific guidelines, gold-standard reference materials, widely available bioinformatic resources, and regulatory guidelines will be needed to standardize the application of NGS. To support this gap, the NGS Quality Initiative collaborated with CDC laboratories and external partners to support use cases for the development and optimization of bioinformatic pipelines, namely, AMR Metagenomics Detection, Simulation, Taxonomic Classification, and Read Optimization and Simulation, Phylogeny Estimation, Read Optimization, Resistance Mutation Identification and Evaluation, and Sequence Annotation. These tools were developed to support laboratories in creating and using in silico reference materials by expanding adoption of mutagenized files, evaluating their impact on NGS test development and validation, and providing accessible, reproducible workflows for laboratory generation and use (37, 38).

As the field continues to evolve, the NGS Quality Initiative will continue to connect partners, develop resources, and create new tools to meet current and future public health and clinical laboratory quality needs.

Supplemental materials (JCM00213-26-s0001.docx). Supplemental materials and Tables S1 to S3.