Molecular and Informatics methods for whole-genome mutational profiling

描述（由申请人提供）：正向遗传学有助于了解已充分研究的模式生物（如酵母、果蝇、蠕虫和斑马鱼）中的基因功能。然而，这些模型系统中使用的分子遗传图谱方法既耗时又费力。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，下一代测序的全基因组突变谱移动遗传作图系统的基础。我们将包括化学诱导的碱基对变化和插入突变。在当前的序列通量下，对于约100 Mb的基因组，基因分型点突变体将需要短读测序仪的完整机器运行。插入突变体通常需要每个菌株少得多的序列覆盖率，因此可以在单次机器运行中以多重方式对多个突变体进行测序。作为一个现实的测试生物，我们将使用弓形虫，一种致病性真核生物与65 Mb的基因组，与草案质量的基因组序列。我们将回答一些简单但基本的、尚未回答的问题：（i）在一个典型的诱变实验中引入了多少突变？(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是如何分布的？这些基本问题的答案将有助于设计突变分析实验在未来从一个更合理的基础上，例如，能够校准诱变剂剂量的基因突变事件的期望数量。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的插入突变。公共卫生相关性：我们正在开发湿台和计算机方法，以帮助科学家了解哪些基因是致病生物体中导致人类疾病所必需的。这些方法将加快基因定位过程，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.