Next Generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) Working Groups
Next Generation DNA sequencing technologies are currently transitioning from research into clinical and public health settings. While the healthcare and public health benefits that can be achieved using these complex technologies are significant, adoption will require robust quality assurance and control procedures to ensure reliable test results. In the US, the Clinical Laboratory Improvement Amendments (CLIA) regulations require that specifications be established for specific performance characteristics to ensure reliable test results. These performance characteristics include accuracy, precision, analytical sensitivity, analytical specificity, reportable range, reference intervals, and other applicable metrics. At present, guidance to implement quality practices necessary to establish the required performance specifications is lacking and laboratories are challenged with translating next-generation sequencing to fit with existing regulatory and professional standards. In an effort to address these issues and to assure high quality testing, the Genetics Team within the Division of Laboratory Science and Standards (DLSS) has initiated an effort to identify principles and develop consensus guidance to help laboratories achieve reliable test results.
Nex-StoCT I Working Group
- The Genetics Team within DLSS convened a national workgroup for the Next Generation Sequencing- Standardization of Clinical Testing (Nex-StoCT) meeting on April 14-15, 2011 in Atlanta, Georgia tasked with identifying principles and developing recommendations for assuring quality laboratory practices relevant to the clinical use of NGS for the detection of germline (heritable) sequence variations.
- The discussions focused on the detection of germline sequence variations with recognition that many of the principles may also apply to cancer and infectious disease testing.
- The workgroup included 41 invited participants, including clinical and research laboratory professionals, physicians, NGS test platform and software developers, bioinformatics experts, and representatives from the National Institutes of Health, the National Institute of Standards and Technology, the Centers for Medicare and Medicaid Services (from the CLIA program), and the Food and Drug Administration.
- The workgroup developed recommendations for test validation, quality control, proficiency testing/alternate assessment, and reference materials.
- As a result of these discussions, the DLSS Genetic Testing Reference Materials Coordination Program (GeT-RM) initiated a collaborative project to coordinate the characterization of reference materials for clinical NGS applications. The project will create a Variation Call Format (VCF) file that describes differences between the consensus sequence HG19 and two deeply sequenced genomes from the 1000 Genome Project: samples NA19240 and NA12878. Pre-existing sequence data as well as new data from a variety of clinical laboratories will be combined in the VCF file. This data product will be annotated with the level of sequence certainty across one or both genomes and will be an evolving data set as technology and informatics processes evolve.
- The Nex-StoCT workgroup outcomes are available in the November 2012 issue of Nature Biotechnology in an article titled "Assuring the quality of next-generation sequencing in clinical laboratory practice". http://www.nature.com/nbt/journal/v30/n11/full/nbt.2403.html
Nex-StoCT II Working Group
- A second working group was convened on October 11-12, 2012 in Atlanta Georgia to identify principles and develop guidance for the optimization of the informatics pipeline in preparation for clinical test validation.
- The primary focus was use of NGS for the detection of germline (heritable) sequence variations. The workgroup also discussed and identified issues unique to other applications including cancer and infectious disease testing.
- The workgroup included bioinformatics experts; clinical and research laboratory professionals as well as physicians with extensive NGS experience; NGS test platform and software developers; and representatives from federal agencies, accreditation bodies and professional laboratory organizations.
- The workgroup developed recommendations for de-multiplexing, sequence alignment, variant annotation, variant/gene classification, and prioritization (ranking). The workgroup also discussed metrics and controls for evaluation of each step of the process, and considerations for choosing and optimizing software.
Outcomes and Next Steps for Nex-StoCT II:
- The Genetics Team, along with the Nex-StoCT II working group, is developing a manuscript that will describe workgroup findings and recommendations.
- At Nex-StoCT II, a breakout group discussed issues relevant to the application of NGS to infectious disease testing. There was enthusiasm for expanding Nex-StoCT to include infectious disease NGS applications. We plan to engage experts both inside and outside CDC, to explore this opportunity.
Publications and Presentations
November, 2012: Publication of Nex-StoCT workgroup outcomes:
Gargis AS, et al. Assuring the Quality of Next-Generation Sequencing in Clinical Laboratory Practice. Nature Biotechnology. 30, 1033–1036 (2012). http://www.nature.com/nbt/journal/v30/n11/full/nbt.2403.html
August 23, 2012, Washington DC: Presentation at the Next Generation Dx Summit: “Molecular Diagnostics for Infectious Disease. Principles and Recommendations for the Implementation of Next Generation Sequencing into Diagnostic Labs”. Click here [PDF 1.4MB].
May 7, 2012, Atlanta, GA: Presentation at the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) workgroup: “Next Generation Sequencing: Assuring Analytical Validity”, Click here [PDF 2.15MB].
November 18, 2011, Grapevine, TX: Poster presentation at the 2011 Association for Molecular Pathology annual meeting, “Next Generation Sequencing - Standardization of Clinical Testing: Approaches to Quality Assurance and Complying with Regulatory and Professional Standards”, Click here [PDF 1.03MB].
October 20, 2011, Montreal, Canada: Poster presentation at the 2011 American Society of Human Genetics annual meeting, “Next Generation Sequencing: Guiding the translation from research to clinical applications” were presented; click here [PDF 395KB].
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