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. 2020 Oct 21;10:17925. doi: 10.1038/s41598-020-74567-y

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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 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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The 278 RNA-seq pipelines applied to the SEQC-benchmark dataset differ in terms of gene expression accuracy, precision, and reliability. In each heatmap, the rows are different settings for 13 aligners and the columns are combinations of three quantification and seven normalization methods. (a) Accuracy is defined as the deviation of pipeline-derived log ratios of gene expression from the corresponding qPCR-based log ratios. Median accuracy of all genes (i.e., 10,222 genes) is encoded as color, with red representing the highest accuracy, or the lowest deviation from qPCR. (b) Precision is defined as the coefficient of variation (CoV) of gene expression over replicate libraries. Median precision of all genes is encoded as color, with red indicating the highest precision, or the lowest CoV. (c) Reliability is defined as the intraclass (or intra-sample in our context) correlation that quantifies how similar replicate libraries of a sample are to one another using analysis of variance techniques. Median reliability of all genes is encoded as color, with red representing the highest reliability, or the highest intraclass correlation. Refer to the “Method” section for mathematical definitions of accuracy, precision, and reliability in the context of RNA-seq pipelines.