Next generation sequence analysis of the IgH repertoire

说明(申请人提供)：免疫球蛋白谱系的组成已经研究了很长时间，但受到目前通过聚合酶链式反应研究重排频率、克隆和个体重排测序的方案的极大限制。因此，得出结论的数据库非常有限，事实上，我们对前B细胞中VH基因在整个基因座上的重排模式知之甚少。新一代测序技术(深度测序、大规模并行高通量测序)的出现提供了前所未有的能力，可以在一次实验中快速获得数百万条序列。然后对序列进行生物信息学分析。使用这项新技术，我们现在能够对前B细胞的整个IgH谱系进行深度测序，从而第一次准确地确定每个单独的V、D和J基因在初始谱系中的相对用途。我们将优化使用罗氏454基因组测序仪FLX系统的下一代测序的条件。初级免疫球蛋白谱系产生的许多方面的非随机性程度可以通过我们将获得的数百万个序列来阐明。由于C57BL/6基因组现在已经完全测序，我们知道所有V基因在该基因座内的准确位置，以及它们所有侧翼DNA的序列，包括RSS和启动子。我们将确定哪些VH基因被过度利用，哪些未被充分利用，重要的是，我们将利用这些信息来阐明影响V基因不平等使用的因素，并控制重排的可及性。我们假设，我们可能会发现这样的区域，在这些区域中，相邻的V基因组都以高于或低于平均频率的频率重排。如果是这样的话，在重排过程中发生的压缩和循环过程中，这些V基因在基因座三维结构中的相对位置可能会增强或抑制重排，这取决于V基因是更接近还是更远离在基因座压缩过程中产生的环的底部。因此，我们将比较重排频率与CTCF/粘附素位点的位置。另外，这些热点和冷点可能是表观遗传调控的结果。这两个假设都将被探究。ChIP-SEQ已经开始对转录因子进行研究，我们将在AIM 2中获得一些转录因子和表观遗传修饰的CHIP-SEQ数据。我们将把这些关于转录因子结合和表观遗传格局的全球数据与VH基因使用的全球数据进行比较，以确定频繁重排的VH基因是否有特定的转录因子或结构蛋白结合在附近，或者是否有特定的表观遗传学图谱。通过对可能影响可及性从而影响重排频率的转录因子结合位点的分析，我们将对控制VH基因不同部分的可及性进行重排的机制有新的见解。) 公共卫生相关性：与健康相关从高通量测序平台分析VDJ序列的最佳方案和生物信息学工具的开发将对所有对免疫球蛋白或TCR谱系分析领域感兴趣的研究人员普遍使用。重要的是，一旦对正常谱系进行了分析，这项技术就可以扩展到检查疾病状态(如狼疮、类风湿性关节炎、糖尿病)中谱系的潜在扰动，或者跟踪某些克隆型(通过CDR3和V基因的使用来识别)在免疫或感染各种病原体/致病抗原后的命运。此外，V(D)J重排的错误调控可能导致易位导致淋巴瘤和白血病，因此通过下一代测序获得的数据将使我们能够更充分地了解V(D)J重组过程的严格调控。)

DESCRIPTION (provided by applicant): The composition of the immunoglobulin repertoire has been studied for a long time, but has been greatly limited by the current protocol of studying rearrangement frequency by PCR, cloning and sequencing of individual rearrangements. Of necessity, therefore, the database from which conclusions were made was very restricted, and in fact, we know very little about the rearrangement pattern of Vh genes throughout the locus in pro-B cells. The recent advent of next generation sequencing (deep sequencing, massively parallel high throughput sequencing) technology provides the unprecedented ability to rapidly obtain millions of sequences in a single experiment. Analysis of the sequences is then done bioinformatically. Using this new technology, we are now in a position to perform deep sequencing of the entire IgH repertoire in pro-B cells, and thus for the first time, to accurately determine the relative usage of each individual V, D and J gene in the initial repertoire. We will optimize the conditions for utilization of next generation sequencing using cDNA and DNA using the Roche 454 Genome Sequencer FLX system. The extent of non-randomness in many aspects of the generation of the primary Ig repertoire can be elucidated with the millions of sequences that we will obtain. Since the C57BL/6 genome is now completely sequenced, we know the precise location of all V genes within the loci, as well as the sequences of all of their flanking DNA including RSSs and promoters. We will determine which Vh genes are overutilized and which are underutilized in this primary repertoire, and importantly, we will use this information to elucidate factors influencing unequal V gene usage, and controlling accessibility for rearrangement. We hypothesize that we may find regions in which groups of neighboring V genes all rearrange at higher or all at lower frequencies than average. If so, the relative location of those V genes in the 3-dimensional structure of the locus during the compaction and looping that takes place during rearrangement could enhance or inhibit rearrangement depending whether the V genes are closer or further away from the base of the loops that are created during locus compaction. Therefore, we will compare rearrangement frequencies to the locations of CTCF/cohesin sites. Alternatively, or in addition, such hot spots and cold spots could be the result of epigenetic regulation. Both of these hypotheses will be explored. ChIP-seq is beginning to be done for transcription factors, and we will obtain some ChIP-seq data for transcription factor and epigenetic modifications in Aim 2. We will compare this global data on transcription factor binding and epigenetic landscape to the global data on Vh gene usage to determine if frequently rearranging Vh genes have certain transcription factors or architectural proteins bound nearby, or certain epigenetic profiles. Through this analysis of binding sites for transcription factors that may influence accessibility and therefore influence rearrangement frequency, we will gain novel insights into the mechanisms controlling accessibility of different portions of the Vh locus to undergo rearrangement. ) PUBLIC HEALTH RELEVANCE: Health relatedness Development of the optimal protocols and the bioinformatic tools to analyze VDJ sequences from high throughput sequencing platforms will be of general use to all investigators interested in any area of repertoire analyses, whether of immunoglobulin or TCR. Importantly, once analyses have been made of the normal repertoire, this technology can be expanded to examine potential perturbations of the repertoire in disease states such autoimmunity (e.g., lupus, rheumatoid arthritis, diabetes), or to follow the fate of certain clonotypes (identified by CDR3 and V gene usage) following immunization or infection with a variety of pathogens/pathogenic antigens. Furthermore, misregulation of V(D)J rearrangement can result in translocations leading to lymphomas and leukemias, and so the data obtained through this next generation sequencing will permit us to more fully understand the tight regulation of the V(D)J recombination process. )