Whole genome sequencing facilitates intragenic variant interpretation following modifier screening in C. elegans

Abstract

Background Intragenic modifiers (in-phase, second-site variants) are known to have dramatic effects on clinical outcomes, affecting disease attributes such as severity or age of onset. However, despite their clinical importance, the focus of many genetic screens in model systems is on the discovery of extragenic variants, with many labs still relying upon more traditional methods to identify modifiers. However, traditional methods such as PCR and Sanger sequencing can be time-intensive and do not permit a thorough understanding of the intragenic modifier effects in the context of non-isogenic genomic backgrounds. Results Here, we apply high throughput approaches to identify and understand intragenic modifiers using Caenorhabditis elegans. Specifically, we applied whole genome sequencing (WGS) to a mutagen-induced forward genetic screen to identify intragenic suppressors of a temperature-sensitive zyg-1(it25) allele in C. elegans. ZYG-1 is a polo kinase that is important for centriole function and cell divisions, and mutations that truncate its human orthologue, PLK4, have been associated with microcephaly. Combining WGS and CRISPR/Cas9, we rapidly identify intragenic modifiers, show that these variants are distributed non-randomly throughout zyg-1 and that genomic context plays an important role on phenotypic outcomes. Conclusions Ultimately, our work shows that WGS facilitates high-throughput identification of intragenic modifiers in clinically relevant genes by reducing hands-on research time and overall costs and by allowing thorough understanding of the intragenic phenotypic effects in the context of different genetic backgrounds.

Model Organism Modifier (MOM): a user-friendly Galaxy workflow to detect modifiers from genome sequencing data using Caenorhabditis elegans

“…MOM Galaxy workflow has been implemented to run the pipeline developed to detect genetic modifiers in genetic screening in C. elegans ( Jean et al . 2021 ) in the Galaxy environment ( The Galaxy Community 2022 ), combining various tools alongside preconfigured parameters.…”

“…2014 ) (listed in Table 2 ). Tools and parameters have been implemented as previously published by Jean et al . (2021) for an effective genetic modifier variant detection.…”

“…To facilitate the identification of candidate modifiers, we have implemented the automated filtration and annotation processes previously used by Jean et al . (2021) .…”

“…2012 ; Joseph et al . 2018 ; Jean et al . 2021 ), increases the ability at exploring more mutated genomes with potential modifier candidates and allows accurate detection of intragenic and extragenic modifiers.…”

“…To help overcome this barrier and facilitate the development of high-throughput genetic screens based on srWGS in model organisms, we have developed a pipeline that was previously used by Jean et al . (2021) .…”