Sample size calculation based on generalized linear models for differential expression analysis in RNA-seq data

2016 ◽  
Vol 15 (6) ◽  
Author(s):  
Chung-I Li ◽  
Yu Shyr
Keyword(s):  
Sample Size ◽  
Linear Models ◽  
Negative Binomial ◽  
Differential Expression Analysis ◽  
Sample Size Calculation ◽  
Rna Seq ◽  
Optimal Sample Size ◽  
Optimal Sample ◽  
Gene Count ◽  
Downstream Analysis

AbstractAs RNA-seq rapidly develops and costs continually decrease, the quantity and frequency of samples being sequenced will grow exponentially. With proteomic investigations becoming more multivariate and quantitative, determining a study’s optimal sample size is now a vital step in experimental design. Current methods for calculating a study’s required sample size are mostly based on the hypothesis testing framework, which assumes each gene count can be modeled through Poisson or negative binomial distributions; however, these methods are limited when it comes to accommodating covariates. To address this limitation, we propose an estimating procedure based on the generalized linear model. This easy-to-use method constructs a representative exemplary dataset and estimates the conditional power, all without requiring complicated mathematical approximations or formulas. Even more attractive, the downstream analysis can be performed with current R/Bioconductor packages. To demonstrate the practicability and efficiency of this method, we apply it to three real-world studies, and introduce our on-line calculator developed to determine the optimal sample size for a RNA-seq study.

scholarly journals Adjusted Sample Size Calculation for RNA-seq Data in the Presence of Confounding Covariates

2021 ◽  
Vol 1 (2) ◽  
pp. 47-63
Author(s):  
Xiaohong Li ◽  
Shesh N. Rai ◽  
Eric C. Rouchka ◽  
Timothy E. O’Toole ◽  
Nigel G. F. Cooper
Keyword(s):  
Sample Size ◽  
Differential Expression ◽  
Expression Analysis ◽  
Negative Binomial ◽  
Differential Expression Analysis ◽  
Sample Size Calculation ◽  
Size Estimation ◽  
Rna Seq ◽  
Simulation Based ◽  
The Relationship

Sample size calculation for adequate power analysis is critical in optimizing RNA-seq experimental design. However, the complexity increases for directly estimating sample size when taking into consideration confounding covariates. Although a number of approaches for sample size calculation have been proposed for RNA-seq data, most ignore any potential heterogeneity. In this study, we implemented a simulation-based and confounder-adjusted method to provide sample size recommendations for RNA-seq differential expression analysis. The data was generated using Monte Carlo simulation, given an underlined distribution of confounding covariates and parameters for a negative binomial distribution. The relationship between the sample size with the power and parameters, such as dispersion, fold change and mean read counts, can be visualized. We demonstrate that the adjusted sample size for a desired power and type one error rate of α is usually larger when taking confounding covariates into account. More importantly, our simulation study reveals that sample size may be underestimated by existing methods if a confounding covariate exists in RNA-seq data. Consequently, this underestimate could affect the detection power for the differential expression analysis. Therefore, we introduce confounding covariates for sample size estimation for heterogeneous RNA-seq data.

scholarly journals Comparison and evaluation of statistical error models for scRNA-seq

2021 ◽  
Author(s):  
Saket Choudhary ◽  
Rahul Satija
Keyword(s):  
Linear Models ◽  
Negative Binomial ◽  
Statistical Error ◽  
Rna Seq ◽  
Multiple Sources ◽  
Error Models ◽  
Wide Range ◽  
Data Driven Approach ◽  
Downstream Analysis ◽  
Experimental Processing

Heterogeneity in single-cell RNA-seq (scRNA-seq) data is driven by multiple sources, including biological variation in cellular state as well as technical variation introduced during experimental processing. Deconvolving these effects is a key challenge for preprocessing workflows. Recent work has demonstrated the importance and utility of count models for scRNA-seq analysis, but there is a lack of consensus on which statistical distributions and parameter settings are appropriate. Here, we analyze 58 scRNA-seq datasets that span a wide range of technologies, systems, and sequencing depths in order to evaluate the performance of different error models. We find that while a Poisson error model appears appropriate for sparse datasets, we observe clear evidence of overdispersion for genes with sufficient sequencing depth in all biological systems, necessitating the use of a negative binomial model. Moreover, we find that the degree of overdispersion varies widely across datasets, systems, and gene abundances, and argues for a data-driven approach for parameter estimation. Based on these analyses, we provide a set of recommendations for modeling variation in scRNA-seq data, particularly when using generalized linear models or likelihood-based approaches for preprocessing and downstream analysis.

scholarly journals Sample size calculation and re-estimation based on the prevalence in a single-arm confirmatory diagnostic accuracy study

2020 ◽  
Vol 29 (10) ◽  
pp. 2958-2971 ◽  
Author(s):  
Maria Stark ◽  
Antonia Zapf
Keyword(s):  
Diagnostic Accuracy ◽  
Sample Size ◽  
Sensitivity And Specificity ◽  
Sample Size Calculation ◽  
Type I ◽  
Diagnostic Accuracy Study ◽  
Optimal Sample Size ◽  
Optimal Sample ◽  
Accuracy Study ◽  
The One

Introduction In a confirmatory diagnostic accuracy study, sensitivity and specificity are considered as co-primary endpoints. For the sample size calculation, the prevalence of the target population must be taken into account to obtain a representative sample. In this context, a general problem arises. With a low or high prevalence, the study may be overpowered in one subpopulation. One further issue is the correct pre-specification of the true prevalence. With an incorrect assumption about the prevalence, an over- or underestimated sample size will result. Methods To obtain the desired power independent of the prevalence, a method for an optimal sample size calculation for the comparison of a diagnostic experimental test with a prespecified minimum sensitivity and specificity is proposed. To face the problem of an incorrectly pre-specified prevalence, a blinded one-time re-estimation design of the sample size based on the prevalence and a blinded repeated re-estimation design of the sample size based on the prevalence are evaluated by a simulation study. Both designs are compared to a fixed design and additionally among each other. Results The type I error rates of both blinded re-estimation designs are not inflated. Their empirical overall power equals the desired theoretical power and both designs offer unbiased estimates of the prevalence. The repeated re-estimation design reveals no advantages concerning the mean squared error of the re-estimated prevalence or sample size compared to the one-time re-estimation design. The appropriate size of the internal pilot study in the one-time re-estimation design is 50% of the initially calculated sample size. Conclusions A one-time re-estimation design of the prevalence based on the optimal sample size calculation is recommended in single-arm diagnostic accuracy studies.

scholarly journals Best practices on the differential expression analysis of multi-species RNA-seq

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Matthew Chung ◽  
Vincent M. Bruno ◽  
David A. Rasko ◽  
Christina A. Cuomo ◽  
José F. Muñoz ◽  
...  
Keyword(s):  
Best Practices ◽  
Differential Expression ◽  
Expression Analysis ◽  
Differential Expression Analysis ◽  
Single Species ◽  
Rna Seq ◽  
Species Analysis ◽  
Differential Gene ◽  
Multiple Species ◽  
Downstream Analysis

AbstractAdvances in transcriptome sequencing allow for simultaneous interrogation of differentially expressed genes from multiple species originating from a single RNA sample, termed dual or multi-species transcriptomics. Compared to single-species differential expression analysis, the design of multi-species differential expression experiments must account for the relative abundances of each organism of interest within the sample, often requiring enrichment methods and yielding differences in total read counts across samples. The analysis of multi-species transcriptomics datasets requires modifications to the alignment, quantification, and downstream analysis steps compared to the single-species analysis pipelines. We describe best practices for multi-species transcriptomics and differential gene expression.

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