Phenotype to genotype: A new and rapid approach using whole-genome sequencing

TL;DR

This paper introduces WheresWalker, a new method that uses whole-genome sequencing to quickly identify genes responsible for specific traits in organisms like zebrafish.

Contribution

WheresWalker is a novel algorithm that improves the speed and accuracy of mutation identification in forward genetic screens.

Findings

WheresWalker successfully identified mutations in zebrafish genes mttp, apobb.1, and mia2.

The method also discovered a new locus, slc3a2a, involved in ApoB-lipoprotein metabolism.

WheresWalker works across species and supports rapid positional cloning.

Abstract

Forward genetic screening is a powerful approach to assign functions to genes and can be used to elucidate the many genes whose functions remain unknown. A key step in forward genetic screening is mapping: identification of the gene causing the phenotype. Existing mapping methods use a bioinformatic mapping-by-sequencing approach based on allelic frequency calculations that often identify large genomic regions which contain an intractable number of candidate genes for testing. Here, we describe WheresWalker, a modern mapping-by-sequencing algorithm that identifies a mutation-containing interval and then supports positional cloning to shrink the interval, which drastically reduces the number of potential candidates, allowing for extremely rapid mutation identification. We validated this method using mutants from a forward genetic mutagenesis screen in zebrafish for modifiers of ApoB-lipoprotein metabolism. WheresWalker correctly mapped and identified novel zebrafish mutations in mttp, apobb.1, and mia2 genes, as well as a previously published mutation in maize. Further, we used WheresWalker to identify a previously unappreciated ApoB-lipoprotein metabolism-modifying locus, slc3a2a.

Forward genetic mutagenesis screening is an unbiased approach for the identification of mutations linked to a phenotype of interest. While this approach can be a powerful tool for uncovering novel gene functions, identification of the specific phenotype-causing mutation can be time-consuming, and is a limiting factor. Here, we introduce WheresWalker, a mapping tool that uses whole genome sequencing data to identify a region of the genome linked to the phenotype. WheresWalker retrieves genetic markers from the linked interval that are used to narrow the region, until a small number of candidate genes remain; these candidates are tested using high-efficiency CRISPR/Cas9 editing. WheresWalker works in multiple species, and was applied to identify mutations from a recent forward genetic screen in zebrafish for genes that modulate ApoB-lipoprotein metabolism, a key factor in the progression of metabolic disease. Using WheresWalker, we have identified a novel locus, slc3a2a, that is involved in ApoB-lipoprotein biogenesis.

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