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

Mok, Calvin; Au, Vinci; Thompson, Owen; Edgley, Mark L.; Gevirtzman, Louis; Yochem, John; Lowry, Josh; Memar, Nadin; Wallenfang, Matthew R.; Rasoloson, Dominique; Bowerman, Bruce; Schnabel, Ralf; Seydoux, Géraldine; Moerman, Donald G.; Waterston, Robert H.

doi:10.1101/150862

Cited by 7 publications

(24 citation statements)

References 60 publications

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“…Having observed the high error rate and inefficiency of WGS, we asked if a targeted sequencing approach could improve copy number estimation while at the same time requiring fewer overall sequence reads. Molecular inversion probes (MIPs) offer a means to target specific loci for sequencing at high depth (Hiatt et al 2013;Mok et al 2017). Previously, single molecule MIPs (smMIPs) have been used to capture and sequence short tandem repeats, another class of repetitive DNA elements (Carlson et al 2015).…”

Section: Molecular Inversion Probe-based Copy Number Estimation Yieldmentioning

confidence: 99%

Challenges and Approaches to Genotyping Repetitive DNA

Morton¹,

Hall

Kwan³

et al. 2020

G3 Genes|Genomes|Genetics

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Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

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Section: Molecular Inversion Probe-based Copy Number Estimation Yieldmentioning

confidence: 99%

Challenges and Approaches to Genotyping Repetitive DNA

Morton¹,

Hall

Kwan³

et al. 2020

G3 Genes|Genomes|Genetics

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“…Of the remaining wild isolate SNV sites, a 350 bp selection window was used to identify potential MIP sites. The list of candidate MIP sites was used to design and score MIPs based on previously published criteria (Mok et al 2017). The list of designed MIPs was subdivided into each individual strain where the highest-scoring MIP for each linkage group was identified.…”

Section: Mip Site Selection and Designmentioning

confidence: 99%

“…MIPs were pooled based on worm pools being tested and generated as previously published (Mok et al, 2017). Individual MIPs were normalized to a concentration of 100 uM and pooled to a maximum volume of 85 ul.…”

Section: Mip Library Pooling Preparation and Sequencingmentioning

confidence: 99%

“…In this manner, a specific phenotype can be screened across hundreds of thousands of mutated genomes, thereby querying a very large search space (Brenner 1974;De Stasio and Dorman 2001;Kevin et al 2006). The identification of causal variants across this space can be a laborious process although a variety of methods now exist to aid in the sequencing and mapping of mutant genomes (Doitsidou et al 2010;Minevich et al 2012;Jaramillo-Lambert et al 2015;Mok et al 2017). In contrast, a reverse genetics screen by RNAi, generates a smaller potential search space by querying a collection of specific gene knock-down targets for a detectable phenotype in a limited number of genetic backgrounds (Fraser et al 2000;Kamath et al 2003;Lehner et al 2006a).…”

Section: Introductionmentioning

confidence: 99%

“…To assay the mutations and thus the representation of each strain in these pools, rather than use costprohibitive whole genome sequencing, we adapted molecular inversion probes (MIPs) to identify strainspecific variants (Hiatt et al 2013). We previously used MIPs for the genetic mapping of temperature-sensitive alleles in a collection of C. elegans mutant strains (Mok et al 2017); here we analyse population growth in a multi-generational competitive fitness assay to phenotype via MIPs (PhenoMIP) by quantifying the proportion of each strain in a pool. As a proof of principle, we utilized the Million Mutation Project as a source for our strains.…”

Section: Introductionmentioning

confidence: 99%

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PhenoMIP: High-Throughput Phenotyping of Diverse Caenorhabditis elegans Populations via Molecular Inversion Probes

Mok

Belmarez

Edgley

et al. 2020

G3 Genes|Genomes|Genetics

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Whether generated within a lab setting or isolated from the wild, variant alleles continue to be an important resource for decoding gene function in model organisms such as Caenorhabditis elegans. With advances in massively parallel sequencing, multiple whole-genome sequenced (WGS) strain collections are now available to the research community. The Million Mutation Project (MMP) for instance, analysed 2007 N2-derived, mutagenized strains. Individually, each strain averages ~400 single nucleotide variants amounting to ~80 protein-coding variants. The effects of these variants, however, remain largely uncharacterized and querying the breadth of these strains for phenotypic changes requires a method amenable to rapid and sensitive high-throughput analysis. Here we present a pooled competitive fitness approach to quantitatively phenotype subpopulations of sequenced collections via molecular inversion probes (PhenoMIP). We phenotyped the relative fitness of 217 mutant strains on multiple food sources and classified these into five categories. We also demonstrate on a subset of these strains, that their fitness defects can be genetically mapped. Overall, our results suggest that approximately 80% of MMP mutant strains may have a decreased fitness relative to the lab reference, N2. The costs of generating this form of analysis through WGS methods would be prohibitive while PhenoMIP analysis in this manner is accomplished at less than one-tenth of projected WGS costs. We propose methods for applying PhenoMIP to a broad range of population selection experiments in a cost-efficient manner that would be useful to the community at large.

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A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia

Acker

Smith

Devine

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

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MIP-MAP: High Throughput Mapping ofCaenorhabditis elegansTemperature Sensitive Mutants via Molecular Inversion Probes

Abstract: 34Temperature sensitive (TS)

Cited by 7 publications

References 60 publications

Challenges and Approaches to Genotyping Repetitive DNA

Challenges and Approaches to Genotyping Repetitive DNA

PhenoMIP: High-Throughput Phenotyping of Diverse Caenorhabditis elegans Populations via Molecular Inversion Probes

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia

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