Next generation sequence analysis of the IgH repertoire

IgH 库的下一代序列分析

• 批准号: 8054158
• 负责人: ANN J FEENEY
• 金额: $ 33.16万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054158
• 关键词: Adult; Affect; Antigens; Area; Autoimmunity; B cell repertoire; B-Lymphocytes; Binding; Binding Sites; Bioinformatics; Biological; C57BL/6 Mouse; Cloning; Complementary DNA; Coupled; Coupling; DNA; DNA Sequence Rearrangement; Data; Data Analyses; Databases; Development; Diabetes Mellitus; Disease; Distal; Epigenetic Process; Event; Factor Analysis; Family; Frequencies; Gene Rearrangement; Generations; Genes; Genetic; Genetic Recombination; Genome; Genomics; Health; Histones; Hot Spot; IGH@ gene cluster; Immunization; Immunoglobulins; Individual; Infection; Knowledge; Location; Lupus; Modification; Mus; Pattern; Platelet Factor 4; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Binding; Protocols documentation; Regulation; Relative (related person); Research Personnel; Rheumatoid Arthritis; Sequence Analysis; Site; Spottings; System; Technology; Time; V(D)J Recombination; Variant; Work; base; cohesin; insight; interest; leukemia/lymphoma; member; new technology; next generation; novel; pathogen; promoter; research study; three dimensional structure; tool; transcription factor

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. )

描述（由申请人提供）：免疫球蛋白库的组成已经被研究了很长时间，但是受到当前通过PCR、克隆和个体重排测序来研究重排频率的方案的极大限制。因此，得出结论的数据库非常有限，而且事实上，我们对 pro-B 细胞中 Vh 基因整个基因座的重排模式知之甚少。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，可以在单次实验中快速获得数百万个序列。然后通过生物信息学方法对序列进行分析。利用这项新技术，我们现在能够对 pro-B 细胞中的整个 IgH 库进行深度测序，从而首次准确确定每个单独的 V、D 和 J 基因在 B 细胞中的相对使用情况。初始曲目。我们将使用罗氏 454 基因组测序仪 FLX 系统优化使用 cDNA 和 DNA 进行下一代测序的条件。初级 Ig 库生成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 基因组现已完全测序，我们知道基因座内所有 V 基因的精确位置，以及其所有侧翼 DNA 的序列，包括 RSS 和启动子。我们将确定在这个主要库中哪些 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 基因使用不平等的因素，并控制重排的可及性。我们假设我们可能会发现相邻 V 基因组的重排频率均高于或低于平均值的区域。如果是这样，那么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 3 维结构中的相对位置可能会增强或抑制重排，具体取决于 V 基因距离环的碱基更近还是更远它们是在轨迹压缩过程中创建的。因此，我们将比较重排频率与 CTCF/粘连蛋白位点的位置。或者或此外，这样的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讨。 ChIP-seq 开始针对转录因子进行，我们将在目标 2 中获得一些转录因子和表观遗传修饰的 ChIP-seq 数据。我们将把转录因子结合和表观遗传景观的全局数据与 Vh 的全局数据进行比较基因用途以确定频繁重排的 Vh 基因是否具有附近结合的某些转录因子或结构蛋白，或某些表观遗传特征。通过对可能影响可及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 基因座不同部分进行重排的可及性的机制获得新的见解。 ） 公共卫生相关性：健康相关性 开发最佳方案和生物信息学工具来分析来自高通量测序平台的 VDJ 序列将对所有对库分析（无论是免疫球蛋白还是 TCR）领域感兴趣的研究人员普遍使用。重要的是，一旦对正常库进行了分析，这项技术就可以扩展到检查自身免疫等疾病状态下库的潜在扰动（例如狼疮、类风湿性关节炎、糖尿病），或者追踪某些克隆型（已鉴定的克隆型）的命运。免疫或感染多种病原体/致病抗原后（通过 CDR3 和 V 基因使用）。此外，V(D)J 重排的错误调节可能导致易位，从而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全面地了解 V(D)J 重组过程的严格调节。 ）