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.
Researchers at the Garvan Institute of Medical Research 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. The researchers 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, they 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.
Schematic overview illustrating the design and use of RNA sequins
(a) Schematic workflow using RNA sequins and chrIS_R. (b) 78 artificial gene loci are encoded within chrIS_R (top). Most genes encode multiple mRNA isoforms, each of which are represented as an RNA sequin standard. (c) Inset shows example of R1_82 gene, with two alternative isoforms generated through alternative splicing. By varying the concentration of the two isoforms between two staggered mixtures (A and B), we can emulate the alternative splicing of three internal exons (RNA-seq coverage indicated by gray histograms).