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One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

Published

Author(s)

Stephen Lincoln, Tina Hambuch, Justin Zook, Sara Bristow, Kathryn Hatchell, Rebecca Truty, Michael Kennemer, Brian Shirts, Andrew Fellowes, Shimul Chowdhury, Eric Klee, Shazia Mahamdallie, Megan Cleveland, Peter Vallone, Yan Ding, Sheila Seal, Wasanthi DeSilva, Farol Tomson, Catherine Huang Huang, Russell Garlick, Nazneen Rahman, Marc L. Salit, Stephen Kingsmore, Matthew Ferber, Swaroop Aradhya, Robert Nussbaum

Abstract

Next-generation sequencing (NGS) is widely used and cost-effective. However, depending on the specific methods used, NGS can have limitations with certain technically challenging variant types. These types are poorly represented in some validation studies despite the fact that they are prevalent in patients and often medically important. Positive control specimens for such variants can be difficult to obtain, and thus we evaluated a scalable solution to this problem using synthetic constructs. Methods: 23 technically challenging variants, all previously observed in patients, were synthesized and introduced into a known genomic background. This specimen was sequenced by 7 laboratories using 9 different NGS workflows. Results: Twelve of 23 variants were detected by all 9 workflows, but just three workflows detected all 23. Many but not all of these limitations were previously known. The specimen was compatible with diverse NGS targeting methodologies and presents similar technical challenges as had corresponding patient specimens. Conclusions: Although actual patient specimens are also critical to use, multiplexed synthetic controls can help efficiently assess the analytic range of NGS based tests and can help laboratories develop and validate methods for technically challenging variants.
Citation
Genetics in Medicine

Keywords

genomics, next-generation sequencing, NGS

Citation

Lincoln, S. , Hambuch, T. , Zook, J. , Bristow, S. , Hatchell, K. , Truty, R. , Kennemer, M. , Shirts, B. , Fellowes, A. , Chowdhury, S. , Klee, E. , Mahamdallie, S. , Cleveland, M. , Vallone, P. , Ding, Y. , Seal, S. , DeSilva, W. , Tomson, F. , Huang, C. , Garlick, R. , Rahman, N. , Salit, M. , Kingsmore, S. , Ferber, M. , Aradhya, S. and Nussbaum, R. (2021), One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation, Genetics in Medicine, [online], https://doi.org/10.1038/s41436-021-01187-w, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=923823 (Accessed May 17, 2022)
Created May 18, 2021, Updated October 14, 2021