MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes

Abstract

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans. Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2. We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures.

Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

“…Despite all the advantages, large scale studies in C. elegans involving essential genes are still not well developed. Temperature-sensitive alleles allow the temporal suppression of gene function, however very few essential genes count with a ts allele [5,6]. Another possibility is the use of chromosome balancers, which cover already 85% of the genome and a big effort is being carried out for having balancers for all C. elegans essential genes by CRISPR [12] and will all be fluorescently labelled (Caenorhabditis Genetics Center (CGC), Personal Communication).…”

“…Among them, temperature-sensitive (ts) alleles are extensively used, where protein function can be disturbed specifically upon temperature shift at any desired time. Although the number of identified ts alleles is rapidly increasing [6][7][8][9], not even 10% of the ~7,000 essential genes in C. elegans have yet a ts allele. An alternative to maintain and propagate lethal mutations is the use of balancer chromosomes.…”

A portable, low-cost device for precise control of specimen temperature under stereomicroscopes

“…However, recent efforts, including improved methods for mapping mutations, have contributed many new temperature sensitive alleles and a resurged interest in their utility [18][19][20]. Of particular interest is the discovery that many temperature sensitive alleles are fast acting, inducing the mutant phenotype only a minute or minutes after the switch from permissive to restrictive temperature [10,18].…”

“…We thank the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of their shared equipment, services and expertise, in particular the valued assistance of graphic designer Tim Whelan for his contributions to the technical drawing and Dr. Aaron Lifland in the Optical Microscopy Core for his help with imaging. 20…”

Employing the one-cell C. elegans embryo to study cell division processes

“…In addition, RNAi reveals the first severe defect that develops following inhibition of gene function, limiting functional analysis at different temporal stages. Temperature-sensitive ( ts ) alleles provide a powerful method for the temporal control and extent of gene inhibition (Ward and Miwa 1978; O’Rourke et al 2011; Mok et al 2017). Ts mutants typically have singleamino acid changes that weaken protein function.…”