Molecular and Informatics methods for whole-genome mutational profiling

描述(申请人提供)：正向遗传学已被用来理解研究得很好的模式生物中的基因功能，如酵母、果蝇、蠕虫和斑马鱼。然而，在这些模型系统中使用的分子遗传作图方法既耗时又费力。此外，这些方法不能用于遗传上无法接近的生物体，其中许多生物体(例如真核寄生虫)具有直接的医学意义。我们建议使用高通量的下一代测序进行全基因组突变图谱分析，以将遗传图谱转移到系统化的基础上。我们将包括化学诱导的碱基对变化和插入突变。在目前的序列吞吐量下，对于大约100Mb的基因组，对点突变进行基因分型将需要一台完整的机器运行短读测序仪。插入突变体通常需要每个菌株少得多的序列覆盖率，因此可以在一次机器运行中以多路复用的方式对多个突变体进行测序。作为一个现实的测试有机体，我们将使用弓形虫，一种具有65Mb基因组的致病真核生物，具有草稿质量的基因组序列。我们将回答简单但基本但尚未回答的问题：(I)在一个典型的突变实验中引入了多少突变？(2)诱变剂的剂量与诱变事件的数量之间有什么关系？(Iii)对于我们正在测试的两种诱变剂，这种关系是否相同？(4)突变事件在基因组中相对于基因功能亚基、在染色体上以及在区域核苷酸组成方面是如何分布的？这些基本问题的答案将有助于在未来更合理的基础上设计突变图谱实验，例如，通过能够校准所需数量的基因突变事件的诱变剂剂量。最后，我们应用我们开发的图谱分析方法来定位弓形虫致病所必需的表型下的插入突变。与公共卫生相关：我们正在开发湿法工作台和计算机方法，以帮助科学家了解哪些基因是导致人类疾病的病原体的关键基因。这些方法将加快这一基因图谱的进程，并广泛适用于大量生物体。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.