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Cited by 266Gene expression and cancer classificationGenomics and Phylogenetic StudiesSingle-cell and spatial transcriptomics

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Power analysis and sample size estimation for RNA-Seq differential expression

Open access
Ching, Travers, Huang, Sijia, Garmire, Lana X.·2014·RNA
DOI 10.1261/rna.046011.114PMC PMC4201821
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Abstract

It is crucial for researchers to optimize RNA-seq experimental designs for differential expression detection. Currently, the field lacks general methods to estimate power and sample size for RNA-Seq in complex experimental designs, under the assumption of the negative binomial distribution. We simulate RNA-Seq count data based on parameters estimated from six widely different public data sets (including cell line comparison, tissue comparison, and cancer data sets) and calculate the statistical power in paired and unpaired sample experiments. We comprehensively compare five differential expression analysis packages (DESeq, edgeR, DESeq2, sSeq, and EBSeq) and evaluate their performance by power, receiver operator characteristic (ROC) curves, and other metrics including areas under the curve (AUC), Matthews correlation coefficient (MCC), and F -measures. DESeq2 and edgeR tend to give the best performance in general. Increasing sample size or sequencing depth increases power; however, increasing sample size is more potent than sequencing depth to increase power, especially when the sequencing depth reaches 20 million reads. Long intergenic noncoding RNAs (lincRNA) yields lower power relative to the protein coding mRNAs, given their lower expression level in the same RNA-Seq experiment. On the other hand, paired-sample RNA-Seq significantly enhances the statistical power, confirming the importance of considering the multifactor experimental design. Finally, a local optimal power is achievable for a given budget constraint, and the dominant contributing factor is sample size rather than the sequencing depth. In conclusion, we provide a power analysis tool ( http://www2.hawaii.edu/~lgarmire/RNASeqPowerCalculator.htm ) that captures the dispersion in the data and can serve as a practical reference under the budget constraint of RNA-Seq experiments.

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FIGURE 1. Power curves based on the number of samples per condition for the six public data sets and five RNA-Seq differential expression analysis packages. Library sizes were estimated from the gene counts of the real data sets. Per-gene dispersion was estimated through the Cox–Reid adjusted profile likelihood. (A) Power curves relative to sample size and differential expression methods in six public data sets. The four unpaired-sample data sets (Bottomly, Bullard, Huang, M–P) were analyzed with edgeR, DESeq, DESeq2, EBSeq, and sSeq. The paired-sample data sets (Tuch and Qian) were analyzed with edgeR, DESeq, DESeq2, and sSeq. Note that EBSeq is not included as it is currently not adapted to analyzing paired-sample data. (B) Heatmap of averaged power over the differential expression methods in six public data sets.
FIGURE 1. Power curves based on the number of samples per condition for the six public data sets and five RNA-Seq differential expression analysis packages. Library sizes were estimated from the gene counts of the real data sets. Per-gene dispersion was estimated through the Cox–Reid adjusted profile likelihood. (A) Power curves relative to sample size and differential expression methods in six public data sets. The four unpaired-sample data sets (Bottomly, Bullard, Huang, M–P) were analyzed with edgeR, DESeq, DESeq2, EBSeq, and sSeq. The paired-sample data sets (Tuch and Qian) were analyzed with edgeR, DESeq, DESeq2, and sSeq. Note that EBSeq is not included as it is currently not adapted to analyzing paired-sample data. (B) Heatmap of averaged power over the differential expression methods in six public data sets.CC BY-NC · PubMed Central
FIGURE 2. Performance comparison with receiver operator characteristics (ROC) curves and other metrics for the six public data sets and five RNA-Seq differential expression analysis packages. Sensitivity and 1 − specificity were estimated in each simulation for n = 4 samples per condition. The simulations were conducted as in Figure 1. (A) ROC curve comparison. True positive rate (TPR) versus false positive rate (FPR) was plotted. (B) Other performance metrics. Area under the curve (AUC) was measured up to FPR = 0.5 of the ROC curves in A. Matthew correlation coefficient (MCC) and F-measure were measured at the threshold of α = 0.05.
FIGURE 2. Performance comparison with receiver operator characteristics (ROC) curves and other metrics for the six public data sets and five RNA-Seq differential expression analysis packages. Sensitivity and 1 − specificity were estimated in each simulation for n = 4 samples per condition. The simulations were conducted as in Figure 1. (A) ROC curve comparison. True positive rate (TPR) versus false positive rate (FPR) was plotted. (B) Other performance metrics. Area under the curve (AUC) was measured up to FPR = 0.5 of the ROC curves in A. Matthew correlation coefficient (MCC) and F-measure were measured at the threshold of α = 0.05.CC BY-NC · PubMed Central
FIGURE 3. Paired versus single-factor power analysis of paired-sample data sets (Qian and Tuch). The data sets were evaluated with pairing information (paired analysis, solid line) or without pairing information (single-factor analysis, dashed line), using the standard analysis pipelines for the respective packages as in Figure 1. Note that EBSeq is not included as it is currently not adapted to analyzing paired-sample data.
FIGURE 3. Paired versus single-factor power analysis of paired-sample data sets (Qian and Tuch). The data sets were evaluated with pairing information (paired analysis, solid line) or without pairing information (single-factor analysis, dashed line), using the standard analysis pipelines for the respective packages as in Figure 1. Note that EBSeq is not included as it is currently not adapted to analyzing paired-sample data.CC BY-NC · PubMed Central
FIGURE 4. Power of protein coding genes versus long noncoding RNA (lincRNA) transcripts. The comparison was made using the Huang data set, which used ribosomal RNA removal for RNA library construction. The transcriptome was separated into protein coding genes (solid line) or lincRNA (dashed line) categories. Power was estimated in each simulation for these two categories, using the standard analysis pipelines for the respective packages as in Figure 1.
FIGURE 4. Power of protein coding genes versus long noncoding RNA (lincRNA) transcripts. The comparison was made using the Huang data set, which used ribosomal RNA removal for RNA library construction. The transcriptome was separated into protein coding genes (solid line) or lincRNA (dashed line) categories. Power was estimated in each simulation for these two categories, using the standard analysis pipelines for the respective packages as in Figure 1.CC BY-NC · PubMed Central
FIGURE 5. Optimization of power given a budget constraint. The cost of RNA-Seq per sample is dependent on the cost of constructing the RNA-Seq library and the cost of single-end sequencing under the multiplex arrangement, where multiple samples could be barcoded to share one lane of the HiSeq flow cell. Both sequencing depth and sample size are variables under the budget constraint. (A) Power curves relative to samples, exemplified by increasing budgets of $3000, $5000, and $10,000 among five RNA-Seq differential expression analysis packages. (B) Optimal powers achieved for given budget constraints. (C) Biological replicates required to obtain optimal powers for given budget constraints. (D) Sequencing depths required to obtain optimal powers for given budget constraints.
FIGURE 5. Optimization of power given a budget constraint. The cost of RNA-Seq per sample is dependent on the cost of constructing the RNA-Seq library and the cost of single-end sequencing under the multiplex arrangement, where multiple samples could be barcoded to share one lane of the HiSeq flow cell. Both sequencing depth and sample size are variables under the budget constraint. (A) Power curves relative to samples, exemplified by increasing budgets of $3000, $5000, and $10,000 among five RNA-Seq differential expression analysis packages. (B) Optimal powers achieved for given budget constraints. (C) Biological replicates required to obtain optimal powers for given budget constraints. (D) Sequencing depths required to obtain optimal powers for given budget constraints.CC BY-NC · PubMed Central

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Metadata

Authors
Ching, T., Huang, S., & Garmire, L. X.
Affiliations
University of Hawaiʻi at Mānoa (US)
Corresponding author
Garmire, Lana X.
ORCID
Venue
RNA
Publisher
Cold Spring Harbor Laboratory
Published
2014-09-22
Volume
20
Issue
11
Pages
1684-1696
ISSN
  • 1355-8382 (ISSN-L)
  • 1469-9001
DOI
10.1261/rna.046011.114
PMC
PMC4201821
OpenAlex
W2157710407
Cited by
263
References
45
Citations by year
2026: 17 · 2025: 24 · 2024: 17 · 2023: 23 · 2022: 19 · 2021: 24 · 2020: 28 · 2019: 31 · 2018: 27 · 2017: 20 · 2016: 23 · 2015: 6 · 2014: 3
FWCI
7.10
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • RNA (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • HighWire Press Open Archive (submittedVersion)
Funders
National Institute of General Medical Sciences (P20 COBRE GM103457)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
RNA-seq, statistical power, sample size determination, differential gene expression, negative binomial distribution, DESeq2, edgeR, sequencing depth, long intergenic non-coding RNA
MeSH terms
Animals, Computer Simulation, Humans, Regression Analysis, Research Design, RNA, Messenger, Software, Reproducibility of Results, Sequence Analysis, RNA, Sample Size, Gene Expression Profiling, Databases, Genetic, Mice, High-Throughput Nucleotide Sequencing

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