Next-generation forward genetic screens: using simulated data to improve the design of mapping-by-sequencing experiments in Arabidopsis

Abstract

Forward genetic screens have successfully identified many genes and continue to be powerful tools for dissecting biological processes in Arabidopsis and other model species. Next-generation sequencing technologies have revolutionized the time-consuming process of identifying the mutations that cause a phenotype of interest. However, due to the cost of such mapping-by-sequencing experiments, special attention should be paid to experimental design and technical decisions so that the read data allows to map the desired mutation. Here, we simulated different mapping-by-sequencing scenarios. We first evaluated which short-read technology was best suited for analyzing gene-rich genomic regions in Arabidopsis and determined the minimum sequencing depth required to confidently call single nucleotide variants. We also designed ways to discriminate mutagenesis-induced mutations from background Single Nucleotide Polymorphisms in mutants isolated in Arabidopsis non-reference lines. In addition, we simulated bulked segregant mapping populations for identifying point mutations and monitored how the size of the mapping population and the sequencing depth affect mapping precision. Finally, we provide the computational basis of a protocol that we already used to map T-DNA insertions with paired-end Illumina-like reads, using very low sequencing depths and pooling several mutants together; this approach can also be used with single-end reads as well as to map any other insertional mutagen. All these simulations proved useful for designing experiments that allowed us to map several mutations in Arabidopsis.

Easymap: A User-Friendly Software Package for Rapid Mapping-by-Sequencing of Point Mutations and Large Insertions

“…The user has to obtain paired-end (e.g., Illumina-like) or single-end (e.g., Ion Proton-like) NGS reads from a mutant carrying an insertion of partially or completely known sequence. In previous works, we used simulations to assess that a 5× read depth can be sufficient to map most insertions in a sample using the methods that we implemented in Easymap ( Wilson-Sánchez et al, 2019 ); however, as higher read depths directly translate to more reliable results, a minimum read depth of 10× is advisable. Easymap can also use reads from multiple mutants pooled into a single DNA sample, in which case the minimum read depth recommended is 10× per pooled mutant.…”

Easymap: a user-friendly software package for rapid mapping by sequencing of point mutations and large insertions

“…NGS experiment simulations can be helpful for optimizing the design of effective mapping experiments (James et al, 2013; Wilson-Sánchez et al, 2019). Therefore, Easymap includes a built-in experiment simulator that allows the user to simulate NGS data in order to test different mapping designs and parameters.…”

ATP-Dependent Clp Protease Subunit C1, HvClpC1, Is a Strong Candidate Gene for Barley Variegation Mutant luteostrians as Revealed by Genetic Mapping and Genomic Re-sequencing

“…For instance, the Mutmap (Abe et al, 2012) and Mutmap + (Fekih et al, 2013) approaches, initially applied to rice, require access to a high-quality reference genome for the genotype used for mutagenesis; the homozygosity mapping approach requires availability of genotypes with a known pedigree (Singh et al, 2013). Special attention needs to be paid to experimental design and technical decisions in order to enforce that sequencing data will allow to map a mutation of interest (Wilson-Sanchez et al, 2019). In barley, MANY-NODED DWARF 4 (MND4) was cloned by a technique similar to SHOREmap (Schneeberger et al, 2009); LAXATUM-a (LAX-a) was isolated by exome-capture sequencing (Mascher et al, 2013) of several highly informative recombinant pools (Jost et al, 2016).…”