+1 Recommend
0 collections
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      NormExpression: An R Package to Normalize Gene Expression Data Using Evaluated Methods


      Read this article at

          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.


          Data normalization is a crucial step in the gene expression analysis as it ensures the validity of its downstream analyses. Although many metrics have been designed to evaluate the existing normalization methods, different metrics or different datasets by the same metric yield inconsistent results, particularly for the single-cell RNA sequencing (scRNA-seq) data. The worst situations could be that one method evaluated as the best by one metric is evaluated as the poorest by another metric, or one method evaluated as the best using one dataset is evaluated as the poorest using another dataset. Here raises an open question: principles need to be established to guide the evaluation of normalization methods. In this study, we propose a principle that one normalization method evaluated as the best by one metric should also be evaluated as the best by another metric (the consistency of metrics) and one method evaluated as the best using scRNA-seq data should also be evaluated as the best using bulk RNA-seq data or microarray data (the consistency of datasets). Then, we designed a new metric named Area Under normalized CV threshold Curve (AUCVC) and applied it with another metric mSCC to evaluate 14 commonly used normalization methods using both scRNA-seq data and bulk RNA-seq data, satisfying the consistency of metrics and the consistency of datasets. Our findings paved the way to guide future studies in the normalization of gene expression data with its evaluation. The raw gene expression data, normalization methods, and evaluation metrics used in this study have been included in an R package named NormExpression. NormExpression provides a framework and a fast and simple way for researchers to select the best method for the normalization of their gene expression data based on the evaluation of different methods (particularly some data-driven methods or their own methods) in the principle of the consistency of metrics and the consistency of datasets.

          Related collections

          Most cited references 11

          • Record: found
          • Abstract: found
          • Article: not found

          Synthetic spike-in standards for RNA-seq experiments.

          High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2(20) concentration range as spike-in controls to measure sensitivity, accuracy, and biases in RNA-seq experiments as well as to derive standard curves for quantifying the abundance of transcripts. We observed linearity between read density and RNA input over the entire detection range and excellent agreement between replicates, but we observed significantly larger imprecision than expected under pure Poisson sampling errors. We use the control RNAs to directly measure reproducible protocol-dependent biases due to GC content and transcript length as well as stereotypic heterogeneity in coverage across transcripts correlated with position relative to RNA termini and priming sequence bias. These effects lead to biased quantification for short transcripts and individual exons, which is a serious problem for measurements of isoform abundances, but that can partially be corrected using appropriate models of bias. By using the control RNAs, we derive limits for the discovery and detection of rare transcripts in RNA-seq experiments. By using data collected as part of the model organism and human Encyclopedia of DNA Elements projects (ENCODE and modENCODE), we demonstrate that external RNA controls are a useful resource for evaluating sensitivity and accuracy of RNA-seq experiments for transcriptome discovery and quantification. These quality metrics facilitate comparable analysis across different samples, protocols, and platforms.
            • Record: found
            • Abstract: found
            • Article: not found

            Revisiting global gene expression analysis.

            Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms. Copyright © 2012 Elsevier Inc. All rights reserved.
              • Record: found
              • Abstract: found
              • Article: not found

              SCnorm: robust normalization of single-cell RNA-seq data

              Summary Normalization of RNA-sequencing data is essential for accurate downstream inference, but the assumptions upon which most methods are based do not hold in the single-cell setting. Consequently, applying existing normalization methods to single-cell RNA-seq data introduces artifacts that bias downstream analyses. To address this, we introduce SCnorm for accurate and efficient normalization of scRNA-seq data.

                Author and article information

                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                30 April 2019
                : 10
                [1] 1School of Mathematical Sciences, Nankai University , Tianjin, China
                [2] 2College of Life Sciences, Nankai University , Tianjin, China
                [3] 3School of Biomedical Engineering, Health Science Center, Shenzhen University , Shenzhen, China
                [4] 4Institute for Systems Biology , Washington, DC, United States
                Author notes

                Edited by: Tuo Zhang, Cornell University, United States

                Reviewed by: Yudong Cai, Shanghai University, China; Naibin Duan, Shandong Academy of Agricultural Sciences, China

                These authors have contributed equally to this work

                This article was submitted to Bioinformatics and Computational Biology, a section of the journal Frontiers in Genetics

                Copyright © 2019 Wu, Liu, Jin, Ji, Wang, Glusman, Robinson, Liu, Ruan and Gao.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 3, Tables: 0, Equations: 0, References: 17, Pages: 8, Words: 0


                gene expression, normalization, evaluation, r package, scrna-seq


                Comment on this article