Design and testing of a custom melanoma next generation sequencing panel for analysis of circulating tumor DNA

Russell J. Diefenbach, Jenny Lee, Alexander M. Menzies, Matteo S. Carlino, Georgina V. Long, Robyn Saw, Julie Howle, Andrew J. Spillane, Richard A. Scolyer, Richard F. Kefford, Helen Rizos*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)
15 Downloads (Pure)


Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.
Original languageEnglish
Article number2228
Pages (from-to)1-16
Number of pages16
Issue number8
Publication statusPublished - 10 Aug 2020

Bibliographical note

Copyright the Author(s) 2020. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.


  • melanoma
  • circulating tumour DNA
  • targeted sequencing
  • custom panel


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