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

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. Researchers at the NIST describe and evaluate experiments characterizing the performance of RNA-sequencing (RNA-Seq) measurements, and discuss cases where mixtures can serve as effective process controls.

The researchers 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. The researchers 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.

This study uses four mixtures of the same general form:

A mixture M (two per dataset in this study) is composed of a number of named components C (“B”,”L”, and”M” in the Brain/Liver/Muscle mixture or “A” and “B” in the SEQC dataset), with each component comprising a proportion of the mixture.