OligoY: a pipeline for the design of repetitive oligopaint probes for the Y Chromosome
Authors: Isabela Almeida 1; Henry Bonilla Bruno 1; Mara Pinheiro 1; Amanda Luvisotto 1; Antonio Carvalho 2; Maria Vibranovski 1
Affiliations: 1) Universidade de São Paulo, USP, São Paulo/SP, Brazil; 2) Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro/RJ, Brazil
Keywords: q. other (Pipeline); t. bioinformatic and genome tools
Studies with the Y chromosome offer a series of obstacles, one of them being the heterochromatic and highly repetitive state of this structure, leading to difficulties in assembling scaffolds and contigs and, thus, in a lack of final assembled sequences for it. The Y chromosome of Drosophila melanogaster, the model organism used in this research, has an estimated size of 41Mb of repeat-rich sequences, but only 10% of them are assembled in the most recent genome release. In contrast, the protocol for designing probes used in full chromosome fluorescent labeling experiments (FISH Oligopaint) does not include repetitive sequences to avoid off-target hybridization. For this reason, there are currently less than 1500 oligopaint probes for this Y chromosome model, which is a value at least ten times smaller when compared to the one observed for other chromosomes of the same species. Furthermore, this amount is insufficient to carry out FISH Oligopaint assays efficiently. The main objective of this research is to develop a pipeline that allows the design of oligopaint probes for the Y chromosome off any species of interest. The final pipeline includes the use of open-source and existing tools in Bioinformatics, identification of sequences unique to the chromosome of interest, guarantees the user the autonomy to choose parameters and effectively uses repetitive sequences unique to the target chromosome to design probes, therefore maximizing overall efficiency of cytogenetic experiments. After extensive tests and validations in silico and in situ, it was verified that the application of the developed pipeline, OligoY, allows staining the Y chromosome without generating off-target signal, despite the use of repetitive sequences for oligopaint probe design. In addition, some of the OligoY pipeline strategies' can be used in other chromosomes to address gaps in repeat-rich regions that otherwise would not be stained.