Forward genetics has been instrumental to understand gene function in well-studied model organisms such as yeast, fruitfly, the worm, and zebrafish. However, molecular genetic mapping methods used in these model systems are time-consuming and laborious. Moreover, these methods cannot be used for organisms that are genetically inaccessible, many of which (e.g. eukaryotic parasites) have direct medical significance. We propose to use high-throughput, next generation sequencing for whole-genome mutational profiling to move genetic mapping to a systematic footing. We will include both chemically induced base-pair changes and insertional mutations. At current sequence throughput, for genomes around 100 Mb, genotyping point mutants will require a full machine run of a short-read sequencer. Insertional mutants typically require much less sequence coverage per strain and thus multiple mutants may be sequenced in a single machine run, in a multiplexed fashion. As a realistic test organism, we will use Toxoplasma gondii, a pathogenic eukaryote with a 65 Mb genome, with draft-quality genome sequence. We will answer simple but fundamental, as yet unanswered questions: (i) How many mutations are introduced in a typical mutagenesis experiment? (ii) What is the relationship between mutagen dosage and the number of mutation events? (iii) Is this relationship the same for the two mutagenic agents we are testing? (iv) How are the mutation events distributed in the genome relative to the functional subunits of genes, across chromosomes, and in terms of regional nucleotide composition? The answer to these basic questions will be instrumental in designing mutational profiling experiments in the future from a more rational footing, e.g. by being able to calibrate mutagen dosage for the desired number of mutation events in genes. Finally, we apply the profiling methods we develop to map insertional mutations underlying a phenotype essential for Toxoplasma pathogenesis.
We are developing wet-bench and computer methods to help scientist understand which genes are essential in pathogenic organisms for causing human diseases. These methods will speed up this gene-mapping process, and are widely applicable across a large number of organisms.
