DETECTOR OLIGO DESIGN
TempO-Seq’s whole trancriptome probe design pipeline creates detector oligos by maximizing the number of isoforms targeted per gene, selecting for the optimal GC content/thermodynamic properties, selecting against hairpins, avoiding homopolymer stretches and repetitive elements, and avoiding detector oligos pairs that overlap SNPs. Additionally, the TempO-Seq detector oligo design pipeline designs detector oligo pairs with maximum ligation efficiency and specificity. The design pipeline takes advantage of the ligation properties of the TempO-Seq assay to design probes that can specifically detect all genes in the transcriptome with the ability to distinguish a 1 base-pair mismatch with 99% specificity. The design pipeline also minimizes background noise by controlling for nonspecific interactions even at transcriptome level multiplexing. The output of the probe design pipeline is a pair of detector oligos that produce a robust signal with little non-specific background. Additionally, our probe design pipeline, like the TempO-Seq assay, is highly modular with the ability to add and remove detector oligos pairs to/from the TempO-Seq assay without affecting comparison to previously run data. Options permit the design of detector oligos to measure different isotypes of the same gene, exon junctions, splice variants, fusions, and mutations.