Using mixtures of biological samples as process controls for RNA-sequencing experiments

BMC Genomics

Methodology Article

Michele Mehaffey¹  Jennifer McDaniel²  Sarah Munro³  Jerod Parsons³  P. Scott Pine³  Marc Salit³ 

[1] Leidos Biomedical Research Inc., P.O. Box B Bldg 428, 21702, Frederick, MD, USA;Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, 20899, Gaithersburg, MD, USA;Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, 20899, Gaithersburg, MD, USA;Department of Bioengineering, Stanford University, 443 Via Ortega, 94305, Stanford, CA, USA;

关键词: RNA sequencing;    RNA-seq;    Gene expression;    Mixture deconvolution;    Expression deconvolution;    Process control;    Spike-in control;    ERCC;   

DOI  :  10.1186/s12864-015-1912-7

 received in 2015-04-20, accepted in 2015-09-09,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundGenome-scale “-omics” measurements are challenging to benchmark due to the enormous variety of unique biological molecules involved. Mixtures of previously-characterized samples can be used to benchmark repeatability and reproducibility using component proportions as truth for the measurement. We describe and evaluate experiments characterizing the performance of RNA-sequencing (RNA-Seq) measurements, and discuss cases where mixtures can serve as effective process controls.ResultsWe apply a linear model to total RNA mixture samples in RNA-seq experiments. This model provides a context for performance benchmarking. The parameters of the model fit to experimental results can be evaluated to assess bias and variability of the measurement of a mixture. A linear model describes the behavior of mixture expression measures and provides a context for performance benchmarking. Residuals from fitting the model to experimental data can be used as a metric for evaluating the effect that an individual step in an experimental process has on the linear response function and precision of the underlying measurement while identifying signals affected by interference from other sources. Effective benchmarking requires well-defined mixtures, which for RNA-Seq requires knowledge of the post-enrichment ‘target RNA’ content of the individual total RNA components. We demonstrate and evaluate an experimental method suitable for use in genome-scale process control and lay out a method utilizing spike-in controls to determine enriched RNA content of total RNA in samples.ConclusionsGenome-scale process controls can be derived from mixtures. These controls relate prior knowledge of individual components to a complex mixture, allowing assessment of measurement performance. The target RNA fraction accounts for differential selection of RNA out of variable total RNA samples. Spike-in controls can be utilized to measure this relationship between target RNA content and input total RNA. Our mixture analysis method also enables estimation of the proportions of an unknown mixture, even when component-specific markers are not previously known, whenever pure components are measured alongside the mixture.

【 授权许可】

CC BY   
© Parsons et al. 2015