Chorus2: design of genome‐scale oligonucleotide‐based probes for fluorescence in situ hybridization

Abstract

Oligonucleotide (oligo)-fluorescence in situ hybridization (FISH) has rapidly becoming the new generation of FISH technique in plant molecular cytogenetics research. Genome-scale identification of single-copy oligos is the foundation of successful oligo-FISH experiments. Here, we introduce Chorus2, a software that is developed specifically for oligo selection. We demonstrate that Chorus2 is highly effective to remove all repetitive elements in selection of single-copy oligos, which is critical for the development of successful FISH probes. Chorus2 is more effective than Chorus, the original version of the pipeline, and OligoMiner for repeat removal. Chorus2 allows to select oligos that are conserved among related species, which extends the usage of oligo-FISH probes among phylogenetically related plant species. We also implemented a new function in Chorus2 that allows development of FISH probes from plant species without an assembled genome. We anticipate that Chorus2 can be used in plants as well as in mammalian and other non-plant species. Chorus2 will broadly facilitate the design of FISH probes for various types of application in molecular cytogenetics research.

Single Copy Oligonucleotide Fluorescence In Situ Hybridization Probe Design Platforms: Development, Application and Evaluation

“…For FISH experiments in plant species, whose genomes comprise large amounts of repetitive sequences, improving the specificity of oligos in the genome is the principal consideration. Traditional strategies based on alignment may not eliminate repeats well [51]. Combination of alignment-based and k-mer-based methods shows improved specificity of oligos in genome.…”

“…For those plants with large genomes, such as maize and wheat, potential repetitive sequences might not be well documented. When designing oligo probes in these genomes, de novo repeats detection and filtering based on shotgun sequences is another approach that can be used to improve the specificity of oligos [49][50][51]. Moreover, it should be noted that oligos might bind to unintended sites with minor mismatches (Figure 1d).…”

“…Probes designed by this pipeline showed high specificity and resolution across the cucumber chromosome 3 [8]. In 2021, the developer of Chorus upgraded their pipeline and named it Chorus2 [51]. Chorus2 has a faster probe design process because of the replacement of BLAT by the NGS aligner BWA [48].…”

“…Chorus software [8] was initially implemented to design oligo-FISH probe for plants and then updated (named Chorus2 [51]) to design robust oligo probes in plants and other species using k-mer scoring and NGS filtering methods.…”

LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci

“…The development of fluorescent in situ hybridization (FISH) facilitated their identification because each chromosome in a spread could be identified by cytological markers or by chromosome painting (He et al 2018). Detection by FISH analysis requires chromosome-specific probes (Cremer et al 1988), which can be readily developed using an oligonucleotide-based methodology (Zhang et al 2021).…”