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; 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）：很长一段时间以来，已经研究了免疫球蛋白库的组成，但由于当前研究重排频率，通过PCR，克隆和测序的单个重排的当前协议受到了极大的限制。因此，必要的是，得出结论的数据库非常受限制，实际上，我们对Pro-B细胞中整个基因座的VH基因的重排模式知之甚少。下一代测序（深度测序，大量平行的高通量测序）的最新出现技术提供了前所未有的能力，可以在单个实验中迅速获得数百万个序列。然后，对序列进行分析。使用这项新技术，我们现在可以对Pro-B细胞中的整个IGH曲目进行深入测序，因此可以准确地确定在初始曲目中每个单独的V，D和J基因的相对用法。我们将使用Roche 454基因组Sequencer FLX系统使用cDNA和DNA来优化使用下一代测序的条件。可以通过我们将获得的数百万个序列来阐明非随机性在主要Ig库生成的许多方面的程度。由于现在完全测序了C57BL/6基因组，因此我们知道所有V基因在基因座中的确切位置以及其所有侧翼DNA的序列，包括RSS和启动子。我们将确定哪些VH基因被过度利用，哪些在此主要曲目中未充分利用，重要的是，我们将使用此信息来阐明影响不平等的V基因使用情况以及控制重排的可访问性。我们假设我们可能会发现邻近V基因组的群体在较高或较低的频率下的区域均高于平均值。如果是这样，则这些V基因在重排期间的压实和循环期间的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基因在局部局部压实过程中产生的环的基础距离较近还是更远。因此，我们将将重排频率与CTCF/粘蛋白位点的位置进行比较。或者，此外，这种热点和冷点可能是表观遗传调节的结果。这两个假设将被探讨。 Chip-Seq开始用于转录因子，我们将在AIM 2中获得一些用于转录因子和表观遗传修饰的CHIP-SEQ数据。我们将比较有关转录因子结合和表观遗传景观的全球数据与VH基因使用的全局数据的全局数据，以确定是否经常重新重新排列VH基因的VH基因具有某些转录因子或构造蛋白的范围，或者构成了一定的epigial或构造蛋白。通过对可能影响可及性并因此影响重排频率的转录因子的结合位点的分析，我们将获得对控制VH基因座不同部分的可访问性的机制的新见解，以进行重排。 ） 公共卫生相关性：最佳方案的健康相关性开发和生物信息学工具，用于分析高吞吐量测序平台的VDJ序列，对于所有对任何对库库分析的研究者（无论是免疫球蛋白还是TCR）都有兴趣。重要的是，一旦对正常曲目进行了分析，就可以扩展该技术以检查疾病状态中曲目的潜在扰动，例如自身免疫性（例如，狼疮，类风湿关节炎，糖尿病），或遵循某些clonotypes的命运（均通过cdr3和v vertiancive vernige）（vertiancive contegence cdr3 and cdr 3）（均为vertiance conterigation）。抗原。此外，V（d）J重排的正调可能导致易位导致淋巴瘤和白血病，因此通过此下一代测序获得的数据将使我们能够更充分了解V（d）J重组过程的严格调节。 ）