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. 2019 Mar 5;9:3542. doi: 10.1038/s41598-019-40026-6

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© The Author(s) 2019

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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Optimization of the Sequencing-by-ligation on-chip. (a) Performance is assessed by averaging the RCPs’ quality within their corresponding sequencing probe groups (A, C, G and T), downscaling each one of them by a factor of four, stacking them and normalizing the result to the off-chip control. RCP quality is computed as the ratio of the assigned sequencing probe’s fluorescence intensity (i.e. strongest signal) to the sum of all sequencing probes’ fluorescence (all signals excluding anchor primer). Essentially, RCPs’ mean qualities stack being equal or higher than 1 denotes a base-calling as robust or better than control. The red squares highlight the first occurrence of a parameter’s final value, which was retained in the optimized, device-enabled, protocol. (b) Comparison of detection probes stripping efficiency between off-chip and on-chip. (c) RCPs’ mean qualities for the four bases of the barcode, normalized to off-chip. The steady value through different cycles indicates good stability of the RCPs. Error bars correspond to within-sample variation (n = 1).