Genomics of Acute myelogenous leukemia (AML): Whole Genome Resequencing

这个项目的长期目标是识别唯一的体细胞、非同义突变 通过对至少10个人类AML基因组进行全基因组、全面的重新测序 来自精心挑选的AML患者的肿瘤样本数量；这些变化的频率将是 在另外187例急性髓系白血病中确定。尽管目前全基因组的高昂成本 重新测序很高，我们预计在拟议的研究期内，成本将继续下降。 因此，我们将使用这项研究创建的数据和信息学平台来分析尽可能多的AML 基因组越多越好。我们选择对AML最常见的亚型之一FAB进行初步研究 M1没有相关的突变，已经被证明是导致这种情况的原因 疾病。选定进行分析的样品具有以下特点： 1.有足够数量的骨髓和皮肤DNA可以进行10倍全基因组 测序覆盖率，如有必要(即每个样品5微克，非扩增)。 2.骨髓标本中70%的成髓细胞，以确保AML细胞的浓缩。 3.两个或两个以下克隆性细胞遗传学异常，以及肿瘤和生殖系DMA的完整分析 在500K Affy SNP阵列和2.1M Long Ono CGH阵列平台上用于拷贝数变体的样本。 4.基因表达谱分析后的M1 AML样本的“典型”表达特征。 我们当地银行的AML样本中有10个FAB M1样本符合所有标准，而且大多数已经达到 在最初的资助期，有几个基因被重新测序。重要的是，对这些基因突变的检测 通过定向聚合酶链式反应和重测序，保证了样品全基因组重测序的质量。vbl.使用 根据这10个案例的材料，我们提出了以下目标： 具体目标1：我们将使用Solexa大规模并行测序平台执行30 对至少10个FAB M1 AML样本进行测序(10倍基因组覆盖，每次1 GB)，以及 与前两个急性髓细胞白血病基因组的皮肤DNA样本匹配。其他AML样本将分析为 成本许可。 具体目标2：我们将使用基于Solexa的生物信息学分析管道和我们自己的阅读 将Solexa短片段与人类参考基因组序列反复比对的映射策略。 具体目标3：我们将开发识别、验证(在核心D中)和注释所有非同义词的方法 对AML基因组中的体细胞突变进行了测序，经过READ作图和 识别高质量差异。这些突变的频率将在187年度进行评估 核心D中的其他AML病例及其与发病机制和预后的相关性将在#年进行评估 其他PPG项目。

The long-term goal of this project is to identify all of the somatic, non-synonymous mutations in the unique portion of at least 10 human AML genomes by performing genome-wide, comprehensive resequencing of a number of tumor samples from carefully selected AML patients; the frequency of these changes will be determined in an additional 187 cases of AML. Although the current high cost of whole genome resequencing is high, we anticipate that costs will continue to fall through the proposed research period. Hence, we will use the data and informatics platforms created by this study to analyze as many AML genomes as possible. We have chosen to initially study one of the most common subtypes of AML, FAB M1, which has no associated mutations that have been shown to be responsible for the initiation of this disease. Samples chosen for analysis have the following characteristics: 1. Adequate amounts of bone marrow and skin DMA are available to perform 10X whole genome sequencing coverage, if necessary (i.e. 5 ug of each sample, non-amplified). 2. > 70% myeloblasts in the bone marrow sample to assure enrichment of AML cells. 3. Two or fewer clonal cytogenetic abnormalities, and complete analysis of tumor and germline DMA samples on the 500K Affy SNP array and the 2.1 M long oligo CGH array platforms for copy number variants. 4. "Typical" expression signature of M1 AML samples after gene expression profiling analysis. 10 FAB M1 samples from our locally banked AML samples meet all criteria, and most have already had several genes resequenced in the initial funding period. Importantly, the detection of mutations in these samples by directed PCR and resequencing assures their quality for whole genome resequencing. Using material from these 10 cases, we propose the following Aims: Specific Aim 1: We will use the Solexa massively parallel sequencing platform to perform 30 sequencing runs (10X genomic coverage at 1Gb per run) on at least 10 FAB M1 AML samples, and matched skin DNA samples for the first two AML genomes. Additional AML samples will be analyzed as cost permits. Specific Aim 2: We will use the Solexa-based bioinformatics analysis pipeline and our own read mapping strategy to iteratively align the short Solexa reads onto the human reference genome sequence. Specific Aim 3: We will develop approaches to identify, validate (in Core D), and annotate all nonsynonymous somatic mutations in the AML genomes sequenced, following read mapping and identification of high quality discrepancies. The frequency of these mutations will be evaluated in 187 additional AML cases in Core D, and their relevance for pathogenesis and outcomes will be evaluated in other PPG projects.