Next generation sequence analysis of the IgH repertoire

描述（由申请人提供）：免疫球蛋白库的组成已经研究了很长时间，但受到目前通过PCR、单个重排的克隆和测序来研究重排频率的协议的极大限制。因此，得出结论的数据库是非常有限的，事实上，我们对前b细胞中Vh基因的重排模式所知甚少。最近出现的下一代测序（深度测序，大规模并行高通量测序）技术提供了前所未有的能力，可以在单个实验中快速获得数百万个序列。然后对序列进行生物信息学分析。利用这项新技术，我们现在能够对前b细胞中的整个IgH库进行深度测序，从而首次准确确定初始库中每个单独的V， D和J基因的相对使用情况。我们将利用罗氏454基因组测序器FLX系统优化利用cDNA和DNA进行下一代测序的条件。非随机性的程度，在许多方面的产生主要的Ig曲目可以阐明数以百万计的序列，我们将获得。由于C57BL/6基因组现在已经完全测序，我们知道所有V基因在位点内的精确位置，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不平等V基因使用的因素，并控制重排的可及性。我们假设我们可能会发现相邻V基因组重排的频率高于或低于平均水平的区域。如果是这样的话，在重排过程中，这些V基因在基因座三维结构中的相对位置会增强或抑制重排，这取决于V基因离基因座压实过程中产生的环的基部是近还是远。因此，我们将比较重排频率与CTCF/内聚蛋白位点的位置。另外，这些热点和冷点也可能是表观遗传调控的结果。这两种假设都将被探讨。ChIP-seq开始用于转录因子，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。我们将把这些转录因子结合和表观遗传景观的全球数据与Vh基因使用的全球数据进行比较，以确定频繁重排的Vh基因是否有某些转录因子或建筑蛋白结合在附近，或某些表观遗传谱。通过对可能影响可达性并因此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位点不同部分的可达性以进行重排的机制获得新的见解。

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. )