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

Jerod Parsons, Sarah Munro, P. Scott Pine, Jennifer McDaniel, Michele Mehaffey, Marc Salit

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


Background: Genome-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. Results: We 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. Conclusions: Genome-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.

Original languageEnglish (US)
Article number708
JournalBMC Genomics
Issue number1
StatePublished - Sep 17 2015
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2015 Parsons et al.


  • ERCC
  • Expression deconvolution
  • Gene expression
  • Mixture deconvolution
  • Process control
  • RNA sequencing
  • RNA-seq
  • Spike-in control


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