Molecular and Informatics methods for whole-genome mutational profiling

描述（由申请人提供）：正向遗传学对于理解酵母、果蝇、蠕虫和斑马鱼等经过充分研究的模式生物的基因功能很有帮助。然而，在这些模型系统中使用的分子遗传作图方法是费时费力的。此外，这些方法不能用于遗传上不可接近的生物体，其中许多（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，下一代测序全基因组突变谱移动遗传作图到一个系统的立足点。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100 Mb左右的基因组，基因分型点突变将需要一台完整的短读测序仪。插入突变通常需要每个菌株较少的序列覆盖，因此可以在一台机器运行中以多路复用方式对多个突变进行测序。作为一种现实的试验生物，我们将使用弓形虫，一种具有65 Mb基因组的致病性真核生物，具有草稿质量的基因组序列。我们将回答一些简单但基本但尚未得到解答的问题：(i)在一次典型的诱变实验中引入多少突变？（ii）诱变剂剂量与突变事件数量之间的关系是什么？（iii）对于我们正在测试的两种诱变剂，这种关系是否相同？（iv）相对于基因的功能亚基、跨染色体以及区域核苷酸组成，突变事件在基因组中的分布情况如何？这些基本问题的答案将有助于在未来从更合理的基础上设计突变分析实验，例如，能够根据基因中所需的突变事件数量校准诱变剂剂量。最后，我们应用我们开发的分析方法来绘制弓形虫发病机制所必需的表型下的插入突变。公共卫生相关性：我们正在开发湿台式和计算机方法，以帮助科学家了解致病生物体中哪些基因是引起人类疾病所必需的。这些方法将加快这一基因定位过程，并广泛适用于大量的生物体。

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.