Droplet digital pcr for mutation detection in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with sanger sequencing and pyrosequencing

Document Type

Journal Article

Publication Title

Journal of Molecular Diagnostics


Elsevier BV


School of Medical and Health Sciences




McEvoy, A. C., Wood, B. A., Ardakani, N. M., Pereira, M. R., Pearce, R., Cowell, L., . . . Gray, E. S. (2018). Droplet digital PCR for mutation detection in formalin-fixed, paraffin-embedded melanoma tissues: A comparison with sanger sequencing and pyrosequencing. Journal of Molecular Diagnostics, 20(2), 240-252. Available here


The identification of somatic mutations is crucial for guiding therapeutic decisions about personalized melanoma treatment. However, genetic analysis of tumors is usually performed on limited and often low-quality DNA from tumors with low tumor cellularity and high tumor heterogeneity. Different mutation-detection platforms exist, with varying analytical sensitivities. Here we evaluated the detection of common mutations in BRAF, NRAS, and TERT promoter in 40 melanoma FFPE tissues using Droplet Digital (dd)PCR, and compared the results to the detection rates obtained by Sanger sequencing and pyrosequencing. The cellularity of tumors analyzed ranged from 5% to 50% (n = 28) and 50% to 90% (n = 12). Overall, droplet digital (dd)PCR was more sensitive, detecting mutations in 12.5% and 23% of tumors deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The increased sensitivity of ddPCR was more apparent among tumors with



Access Rights

subscription content