Facilitating application of NGS to analysis of livestock T-cell receptor repertoires

许多LMIC的畜牧业部门受到病原体的负面影响，目前尚无可行的疫苗可供使用。已知T细胞有助于对许多病原体(如结核病、东海岸热)的保护，目前的努力旨在更好地了解这些细胞在赋予免疫力方面所起的作用，并确定可用于使未来疫苗开发合理化的“保护相关性”。T细胞反应的综合分析中的一个参数是表达的TCR谱，它可用于研究病原体特异性T细胞的克隆型结构、动力学和回忆。TCR是由α(TRA)和β(TRB)链组成的异二聚体、克隆型分布的受体，决定了单个T细胞的抗原特异性。体细胞V(D)J重组和V(D)J连接的非胚系编辑产生了一个非常多样化的TCR谱系，在人类中估计为~2×10‘7。在疫苗接种或病原体暴露期间，当遇到同源抗原时，T细胞经历克隆型扩增，这可以通过克隆型TRA和/或TRB链的表达增加来评估。然而，在开发NGS平台之前，获得足够的TCR谱系的测序深度以可靠地检测高度复杂的T细胞群体(例如，体内PBMC或原代T细胞培养)中的这种克隆性扩张是困难的。在过去的十年中，一些生物信息管道(如MiTCR和IMGT-HighV-Quest)的发展使得使用NGS数据的TCR分析能够应用于人类和小鼠T细胞群体的研究。这些分析的先决条件是：i)产生无偏TCR链扩增片段的TCR链特异性PCR方法，ii)V和J基因片段序列的全面数据库，以及iii)复杂的算法，可以将TCR序列与相关的V和J基因片段数据库比对(从而定义V和J的使用以及单个TCR链的高变量CDR3序列)，还可以补偿和纠正不可避免的PCR/测序错误，这些错误可能会显著影响序列数据的解释。用于人类和小鼠TCR分析的生物信息学管道所使用的原理是通用的，在最近的研究中，我们已经证明，给定适当的V和J基因数据库，管道可以被修改并应用于来自其他物种(在这种情况下是欧洲牛)的NGS序列数据。在这个项目中，我们建议：1-设计聚合酶链式反应程序，以实现从相关家畜群体中无偏扩增TRA和TRB链。猪、山羊、绵羊和水牛TRA和TRB链的优化的基于SMART的PCR扩增方案将得到验证。2-测序足够的全长TRA/TRB扩增片段，以实现对表达的TRA和TRB谱带的全面覆盖。将来自不同LMIC和欧洲品种的5个代表性个体的扩增产物进行PacBio测序，获得全长扩增子序列。3-生物信息学解构体细胞重组TRA和TRB链，构建胚系V和J基因片段序列数据库。将开发解构重组TRA/TRB链的管道，以建立组成V和J基因片段的胚系序列，并填充一个全面的数据库。4-修改现有算法，使这些数据库能够用于TCR谱系的NGS分析(并向可公开访问的TCR分析算法的管理者提供TRBV、TRBJ、TRIV和TRAJ数据库)。CTLGH生物信息库将建立和托管MiTCR算法的修订版，该算法将能够利用牲畜物种数据库。此外，这些数据库将由可公开使用的Milboratory网站托管。

The livestock sectors of many LMIC are negatively impacted by pathogens for which no viable vaccines are currently available. T-cells are known to contribute to protection against a number of these pathogens (e.g. tuberculosis, East Coast Fever) and current efforts are directed to better understanding the role these cells plays in conferring immunity and defining the 'correlates of protection' that can be used to rationalise future vaccine development. One parameter that has been advocated for inclusion in comprehensive analysis of T-cell responses is the repertoire of the expressed TCR, which can be exploited to study the clonotypic structure, kinetics and recall of pathogen-specific T-cells. TCRs are heterodimeric, clonotypically distributed receptors composed of alpha (TRA) and beta (TRB) chains that determine the antigenic-specificity of individual T-cells. Somatic V(D)J recombination and non-germline editing of the V(D)J junction generates a vastly diverse TCR repertoire, that in humans has been estimated to be ~2x10'7. Upon encountering their cognate antigen, either during vaccination or pathogen exposure, T-cells undergo clonotypic expansion which can be evaluated by the increased expression of the clonotypic TRA and/or TRB chains. However, achieving sufficient sequencing depth of the TCR repertoire to reliably detect such clonal expansions in highly complex T-cell populations (e.g. in vivo PBMC or primary T-cell cultures) was difficult before development of NGS platforms. In the last decade, development of a number of bioinformatic pipelines (e.g. MiTCR and IMGT-HighV-Quest) has enabled TCR analysis using NGS data to be applied to the study of human and murine T-cell populations. The pre-requisites for these analyses are i) TCR chain-specific PCR protocols that generate unbiased TCR chain amplicons, ii) a comprehensive database of V and J gene segment sequences and iii) sophisticated algorithms that can align TCR sequences to the relevant V and J gene segment databases (and so define V and J usage and the hypervariable CDR3 sequence of individual TCR chains) and also compensate and correct for the inevitable PCR/sequencing errors that can significantly compromise sequence data interpretation. The principles used in bioinformatic pipelines developed for human and murine TCR analysis are generic and in recent studies we have demonstrated that given the appropriate V and J gene databases the pipelines can be modified and applied to NGS sequence data from other species (in this case European Bos taurus cattle). In this project we propose to:1 - Design PCR protocols to enable unbiased PCR amplification of TRA and TRB chains from the relevant livestock populations. Optimised SMART-based PCR amplification protocols for pig, goat, sheep and water buffalo TRA and TRB chains will be validated.2 - Sequence sufficient full length TRA/TRB amplicons to enable comprehensive coverage of the expressed TRA and TRB repertoires. Amplicons generated from 5 representative individuals from different LMIC and European breeds will be subjected to PacBio sequencing to obtain full length amplicon sequence.3 - Bio-informatically de-construct somatically recombined TRA and TRB chains to construct database of germline V and J gene segments sequences. Pipelines to de-construct recombined TRA/TRB chains will be developed to enable germline sequences of the constituent V and J gene segments to be established and a comprehensive database populated.4 - Modify existing algorithms to enable these databases to be employed in NGS analysis of TCR repertoires (and provide TRBV, TRBJ, TRAV and TRAJ databases to managers of publicly accessible TCR analysis algorithms). A modified version of the MiTCR algorithm which will enable the utilisation of the livestock species databases will be established and hosted by the CTLGH bioinformatics repository. In addition, the databases will be hosted by the publicly available Milaboratory website.