COD-FISH: Contrastive Oligonucleotide Design for Fluorescence In-Situ Hybridization to Detect Single RNA Molecules with High Specificity

Single molecule fluorescence in situ hybridization or smFISH with oligonucleotide probes is a prominent technique in the field of RNA detection; it is used in a variety of ways, from mapping organisms’ development to identifying the presence of malignant genes with single molecule sensitivity. Optimal smFISH probe design is an active area of research, with probe specificity being an important consideration. In this paper we introduce COD-FISH, a probe design program that targets probe specificity and optimizes probes set selection for RNA smFISH. Probes generated by COD-FISH are compared in-silico with those created by the programs from the Arjun Raj Lab, Oligominer, and Stellaris. Our results indicate that COD-FISH produces probe sets with significantly higher specificity as compared to other approaches, with a 13% relative improvement overall and over 80% relative improvement in short sequence RNA targets.