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Using next-generation sequencing to isolate mutant genes from forward genetic screens

Key Points

  • Next-generation sequencing enables simultaneous mapping and identification of a causal mutation though sequencing bulk populations of mutant recombinant organisms in an approach known as mapping-by-sequencing.

  • The initial mapping interval can be found in several ways. The ratio of homozygous and heterozygous markers, as well as the difference in the allele counts between pools of mutant and wild-type organisms, allows the analysis of linkage without prior knowledge of any genetic marker. Allele frequency analyses are more accurate but require a genome-wide list of genetic markers.

  • The analysis of large genomes can be simplified using various methods. High-throughput RNA sequencing (RNA-seq), targeted enrichment sequencing or restriction-site-associated DNA sequencing (RAD-seq) are powerful alternatives to whole-genome sequencing that can also allow the mapping of causal regions.

  • Mutation identification by direct sequencing of mutant genomes is challenged by the large amounts of background mutations in a mutant genome. Additional information, such as prior mapping intervals, can be used narrow down the list of possible candidate mutations.

  • Mutation identification can be as simple as comparing the genomes of independently generated allelic mutant lines and searching for genes that are affected in all genomes.

  • Mapping-by-sequencing methods can be used to decipher the genetic architecture of complex traits.

Abstract

The long-lasting success of forward genetic screens relies on the simple molecular basis of the characterized phenotypes, which are typically caused by mutations in single genes. Mapping the location of causal mutations using genetic crosses has traditionally been a complex, multistep procedure, but next-generation sequencing now allows the rapid identification of causal mutations at single-nucleotide resolution even in complex genetic backgrounds. Recent advances of this mapping-by-sequencing approach include methods that are independent of reference genome sequences, genetic crosses and any kind of linkage information, which make forward genetics amenable for species that have not been considered for forward genetic screens so far.

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Figure 1: Mapping-by-sequencing reveals the genetic basis of an inner ear mutant of zebrafish.
Figure 2: Analysis strategies for mapping-by-sequencing.
Figure 3: Pros and cons of different sequence data types for mapping-by-sequencing.
Figure 4: Mutation identification without linkage information, reference sequences and recombination.

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Acknowledgements

The author thanks J. M. Jiménez-Gómez, E.-M. Willing and H. Sun for comments on the manuscript, and N. Obholzer for comments on figure 1. The development of methods for mapping-by-sequencing in K.S.'s group has been generously supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG SPP-1530).

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Glossary

Forward genetic screens

Genetic screens in which mutants are isolated on the basis of their phenotypes. The mutations responsible are identified by positional cloning or by a candidate gene approach.

Genetic markers

Genetic differences that are used to distinguish between different alleles of the respective DNA loci.

Next-generation sequencing

(NGS). In the context of this Review, sequencing methods that have emerged since 2005 and that produce millions of typically short sequence reads (50–400 bases) from amplified DNA clones.

Dominance

A genetic interaction between the two alleles at a locus, such that the phenotype of heterozygotes deviates from the average of the two homozygotes.

Penetrance

The proportion of individuals with a specific genotype who manifest the genotype at the phenotypic level. If the penetrance of an allele is 100%, then all individuals carrying that allele will express the associated phenotype, and the genotype is said to be completely penetrant.

Fixed

Pertaining to the point at which an allele (such as a mutation) has completely displaced other alleles; that is, the allele is present in a homozygous state in every individual in the population.

Non-synonymous nucleotide substitution

A change in nucleotide sequence that alters the encoded amino acid.

Second-site mutations

Mutations introduced in organisms that are already genetically compromised for a given pathway or process in order to isolate mutations that either suppress or enhance the effect of the first mutation.

RAD sequencing

(RAD-seq). A DNA sequencing method in which DNA is cut with restriction enzymes prior to sequencing, which exclusively targets the DNA associated with restriction sites.

Syntenic

Pertaining to the presence of collinear homologous DNA sequences in related chromosomal regions.

Quantitative trait loci

(QTLs). Genetic loci that control quantitative traits. They are identified on the basis of statistical association between genetic markers and phenotypes that can be measured.

Structural sequence variation

A genomic alteration that changes the number of copies or the arrangement of regions of the genome.

Epistatic interactions

Non-additive interactions between two or more mutations at different loci, such that their combined effect on a phenotype deviates from the sum of their individual effects.

Saturated screens

Genetic screens that have reached the point at which no new gene mutations can be found.

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Schneeberger, K. Using next-generation sequencing to isolate mutant genes from forward genetic screens. Nat Rev Genet 15, 662–676 (2014). https://doi.org/10.1038/nrg3745

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