NGS working group provides recommendations for germline variant confirmation

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A joint report from the Association for Molecular Pathology (AMP) and National Society of Genetic Counselors published Wednesday in the Journal of Molecular Diagnostics provides evidence-based recommendations for standardizing orthogonal confirmation practices for germline variants detected by next-generation sequencing (NGS).

The goal of the new report is to provide recommendations for orthogonal confirmation practices — two essentially different methods used to measure the same value, ensuring measurement reliability. In concert with existing guidelines, these recommendations are meant to help promote standardization, transparency, and quality control among laboratories.

NGS, also called massively-parallel sequencing, enables sequencing of many DNA strands at the same time, instead of one at a time as with traditional Sanger sequencing. NGS, often used to study disease-associated genetic variation, has revolutionized the field of clinical molecular genetic testing. However, confirmation practices may vary between laboratories when investigating germline variants — gene changes in an egg or sperm cell that then become incorporated into the DNA of every cell in the offspring’s body.

The AMP Clinical Practice Committee convened the NGS Germline Variant Confirmation Working Group to assess current evidence and standardize orthogonal confirmation practices to limit the reporting of false positives. The group included organizational representation from the National Society of Genetic Counselors to identify best practices to support high-quality patient care.

Based on published literature review, empirical data, current laboratory practice surveys, public comment feedback, and professional experiences, their report offers the following eight recommendations:

  • Clinical laboratories offering germline testing using NGS should establish written policies regarding orthogonal confirmation of NGS results.
  • Laboratories’ orthogonal confirmation policies should be overseen by appropriately certified medical professionals trained and experienced in NGS.
  • Laboratories’ confirmatory methods, platforms, and associated bioinformatics should be validated and maintained under appropriate regulatory oversight.
  • Laboratories’ confirmatory methods should be orthogonal. Discrepant results between NGS and a confirmatory assay should be investigated and resolved.
  • Laboratories should perform confirmatory testing for reported germline variants with significant clinical implications, except for variant calls meeting technical criteria demonstrated to ensure high positive predictive value from NGS alone.
  • Laboratories should clearly articulate their specific policies, criteria, and methods regarding orthogonal confirmation in written materials available upon request.
  • Every clinical test report should summarize their laboratory’s orthogonal confirmation policy; any policy exceptions made should be clearly indicated.
  • Special considerations apply to certain NGS-based test types and findings.

“This new report was meant to summarize the current collective state of knowledge and guide clinical laboratory professionals regarding orthogonal confirmation of germline variants,” Dr. Susan Hsiao, Columbia University associate pathology professor and 2023 AMP Clinical Practice Committee chair, said in a statement. “AMP will continue to reassess and modify our guidelines as the methodology and bioinformatics underlying NGS-based variant detection develop, as part of our ongoing commitment to improving clinical practice and patient care.”

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