Spliced synthetic genes as internal controls in RNA sequencing experiments

Abstract

RNA sequencing (RNA-seq) can be used to assemble spliced isoforms, quantify expressed genes and provide a global profile of the transcriptome. However, the size and diversity of the transcriptome, the wide dynamic range in gene expression and inherent technical biases confound RNA-seq analysis. We have developed a set of spike-in RNA standards, termed 'sequins' (sequencing spike-ins), that represent full-length spliced mRNA isoforms. Sequins have an entirely artificial sequence with no homology to natural reference genomes, but they align to gene loci encoded on an artificial in silico chromosome. The combination of multiple sequins across a range of concentrations emulates alternative splicing and differential gene expression, and it provides scaling factors for normalization between samples. We demonstrate the use of sequins in RNA-seq experiments to measure sample-specific biases and determine the limits of reliable transcript assembly and quantification in accompanying human RNA samples. In addition, we have designed a complementary set of sequins that represent fusion genes arising from rearrangements of the in silico chromosome to aid in cancer diagnosis. RNA sequins provide a qualitative and quantitative reference with which to navigate the complexity of the human transcriptome.

RNA-seq mixology: designing realistic control experiments to compare protocols and analysis methods

“…The inclusion of controls such as the External RNA Controls Consortium spike-in mixes (1 and 2) in different samples is another enhancement that would provide transcripts with known FCs as a further tool for assessing bias. The addition of spliced spike-in controls (52) is another alternative that would better tailor the experiment for benchmarking differential splicing methods.…”

Normalizing single-cell RNA sequencing data: challenges and opportunities

“…Ongoing efforts in this context are illustrated by a recent call from the National Institute of Standard and Technology (https://federalregister.gov/a/2015-19742), to design an improved set of controls, which should (i) mimic endogenous RNA and (ii) not interfere with the measurement of endogenous RNA. More recently, [39] introduced sequins (sequencing spike-ins) — a set of extrinsic spike-ins designed for (bulk) RNA-seq experiments.…”

Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain

“…This is due to the limited availability of high-quality, post-mortem human tissue and to technical limitations associated with standard approaches: most rely on short-read RNA-Seq and the reconstruction of fragmented sequences making the disambiguation of fulllength transcripts difficult, particularly for large and complex genes. Benchmarking studies have shown that short-read RNA-Seq methodologies are unable to accurately reconstruct and quantitate the majority of transcript and protein isoforms 22,23 .…”