OligoY Pipeline for designing Y chromosome oligopaint probes including repetitive sequences

Author(s)

Isabela Pimentel de Almeida

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Matemática e Estatística (IME/SBI)
• Defense date: 2022-01-26

Examining board members

Maria Dulcetti Vibranovski; Leonardo Barbosa Koerich; Ariane Machado Lima; Mainá Bitar Lourenço

• Advisor: Maria Dulcetti Vibranovski; Antonio Bernardo de Carvalho

Abstract

One of the main obstacles in studies with the Y chromosome is related to 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 41 Mb 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 of 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, thus 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. (AU)

• FAPESP's process: 19/14878-4 - A technique for constructing complete oligopaints from heterochromatic sequences using Drosophila melanogaster Y chromosome as a model
• Grantee: Isabela Pimentel de Almeida
• Support Opportunities: Scholarships in Brazil - Master