Institute of Biomedical Sciences
An ORF-based whole-genome gain-of-function library for Trypanosoma brucei
Document Type
Poster
Abstract Category
Immunology/Infectious Diseases
Keywords
trypanosomes, drug resistance, genetic screen
Publication Date
Spring 5-1-2019
Abstract
Despite decades of active discovery in all areas of trypanosome research, more than 50% of the Trypanosoma brucei genome is annotated as hypothetical genes of unknown function. Further progress in understanding both the pathogenesis and basic biology of Trypanosoma species requires the development of versatile approaches for genome-scale functional analysis. A whole-genome RNAi loss-of-function library has proved instrumental in the identification of new genetic functions and pathways in T. brucei. Previously, use of shotgun cloning and digested genomic DNA have been implemented in the formation of whole-genome over expression libraries with some success. To better capture the majority of T. brucei gene full open reading frames (ORFs), we have produced an ORF-based whole-genome gain-of-function library that contains >90% of the targeted genes. The library is induced in the context of a genetic screen to isolate cells resulting in a desired phenotype. Following a genetic screen, next-generation sequencing libraries are enriched using a unique oligo that targets the gain-of-function library for PCR amplification and Illumina platform sequencing. Preliminary experiments returned insufficient read depth and suggested that further optimization of sequencing libraries was required. To this end, we developed a qPCR method for assessing the amount of gain-of-function specific DNA in each next-generation library preparation. Assessment of library quality by qPCR enabled further optimization of sequencing libraries, through modifications to the enrichment PCR, and more accurate pooling of samples for Illumina sequencing. This tool was then applied to a preliminary screen for the identification of genes associated with melarsoprol resistance, in which a specific melarsoprol resistant population was isolated following induction of the gain-of-function library. The tools and optimizations made for gain-of-function library implementation will be discussed as well as early findings from a melarsoprol genetic screen.
Open Access
1
An ORF-based whole-genome gain-of-function library for Trypanosoma brucei
Despite decades of active discovery in all areas of trypanosome research, more than 50% of the Trypanosoma brucei genome is annotated as hypothetical genes of unknown function. Further progress in understanding both the pathogenesis and basic biology of Trypanosoma species requires the development of versatile approaches for genome-scale functional analysis. A whole-genome RNAi loss-of-function library has proved instrumental in the identification of new genetic functions and pathways in T. brucei. Previously, use of shotgun cloning and digested genomic DNA have been implemented in the formation of whole-genome over expression libraries with some success. To better capture the majority of T. brucei gene full open reading frames (ORFs), we have produced an ORF-based whole-genome gain-of-function library that contains >90% of the targeted genes. The library is induced in the context of a genetic screen to isolate cells resulting in a desired phenotype. Following a genetic screen, next-generation sequencing libraries are enriched using a unique oligo that targets the gain-of-function library for PCR amplification and Illumina platform sequencing. Preliminary experiments returned insufficient read depth and suggested that further optimization of sequencing libraries was required. To this end, we developed a qPCR method for assessing the amount of gain-of-function specific DNA in each next-generation library preparation. Assessment of library quality by qPCR enabled further optimization of sequencing libraries, through modifications to the enrichment PCR, and more accurate pooling of samples for Illumina sequencing. This tool was then applied to a preliminary screen for the identification of genes associated with melarsoprol resistance, in which a specific melarsoprol resistant population was isolated following induction of the gain-of-function library. The tools and optimizations made for gain-of-function library implementation will be discussed as well as early findings from a melarsoprol genetic screen.
Comments
Presented at Research Days 2019.