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. 2021 Mar 3;11:5098. doi: 10.1038/s41598-021-84513-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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ddPCR analysis of CSF ctDNA across platforms, with and without pre-amplification. The effect of DNA pre-amplification on BioRad platform test sensitivity was evaluated using ctDNA extracted from H3.3K27M mutant CSF (DMG-1-C), non-tumor CSF (H-C) and non-template controls (blank). (A) ddPCR with Assay A and (B) Assay B, after PCR pre-amplification of input DNA using the same respective primer set resulted in greater droplet counts compared to ddPCR without prior DNA PCR pre-amplification. A further increase in droplet detection was achieved following DNA pre-amplification using a 300 bp H3F3A primer (Assay C), with no observed change in detected MAF with either assay after pre-amplification. (C) RainDance platform results. The effect of DNA pre-amplification on RainDance platform test sensitivity was evaluated using ctDNA extracted from H3.3K27M mutant CSF (DMG-70-C). PCR pre-amplification of input DNA using Assay A or Assay B increased detected droplet counts, relative to ddPCR analysis alone by Assay A, with no effect on observed MAF. % values = Mutation Allelic Frequency (MAF); * = Statistically significant difference in MAF (t-test, p < 0.05).