1 1 RNA sequencing can measure both gene or isoform expression, and reconstruct novel and complex spliced isoforms. However, the sheer size and complexity of the transcriptome, as well as technical bias, can confound analysis with RNA-seq. To assess the impact of these variables, we have developed a set of RNA sequins that represent synthetic genes that act as internal controls during RNA sequencing. Each RNA sequin represents an individual isoform, with multiple isoforms forming artificial

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... loci that are encoded within the in silico chromosome (chrIS). By modulating the relative abundance of individual sequin isoforms we can emulate alternative splicing, whilst modulating the abundance of multiple isoforms we can emulate gene expression. Accordingly, RNA sequins are mixed at different concentration to emulate differences in gene expression and alternative splicing. By sequentially diluting sequins, we can establish a reference ladder across a range of gene expressions. We formulate multiple alternative mixtures that differ in the concentration of individual sequins. By comparing mixtures, we can emulate differential gene expression and alternative splicing between samples. By contrast, RNA sequins with invariant concentrations between mixtures provide static scaling factors that enable quantitative normalization between multiple RNAseq libraries. The RNA sequin mixture is added to a user's RNA sample at a fractional concentration prior to library preparation. The combined sample and the sequins then together undergo sequencing. The sequins can then be distinguished in the output library by their synthetic sequence, and analyzed as internal controls. For further detailed background on the design, validation and use of sequins, we refer users to 'Spliced synthetic genes as internal controls in RNA sequencing experiments' by Hardwick et al., (2016) NAture Methods