Institute of Biomedical Sciences

Title

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

Comments

Presented at Research Days 2019.

This document is currently not available here.

Share

COinS
 

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.