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. 2020 Jan 22;10:916. doi: 10.1038/s41598-020-57473-1

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© The Author(s) 2020

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Variation between replicates and amplification frequency at low copy number. (A) 10 copies of linearised plasmid per reaction shows low variation between 72 replicates, (B) melt curves centred on 87 degrees C and (C) Time frequency distribution. (D) Increased variation between replicates observed at 1 copy with possible false positives (in black) corresponding to higher melt temperatures (E) corresponding to positive results after 80 minutes. The frequency distribution (F) shows the increased variation of positive results at low copy number. The amplification frequency from the 1 to 10 copies per partition data are compared to the predicted amplification frequency (dotted line) based on digital PCR calculations. (G) Amplification frequencies for positive results less than 90 minutes (green), 40 minutes (orange) and 30 minutes (purple) with (H) average Time values at each copy number for results less than 90, 40 and 30 minutes (error bars removed for simplicity) and (I) variance for assay results less than 90, 40 and 30 minutes.