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

许多低收入国家的畜牧业受到病原体的不利影响，目前没有可行的疫苗。已知T细胞有助于保护免受许多这些病原体（例如结核病，东海岸热）的侵害，目前的努力旨在更好地理解这些细胞在赋予免疫力和定义可用于合理化未来疫苗开发的“保护相关因素”中所起的作用。已经提倡包括在T细胞应答的综合分析中的一个参数是所表达的TCR的库，其可以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是异二聚体的、克隆型分布的受体，其由决定单个T细胞的抗原特异性的α（TRA）和β（TRB）链组成。体细胞V（D）J重组和V（D）J接头的非种系编辑产生了非常多样化的TCR库，其在人中估计为~ 2x 10 - 7。在接种疫苗或病原体暴露期间遇到其同源抗原时，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，实现TCR库的足够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细胞培养物）中的此类克隆扩增是困难的。在过去的十年中，许多生物信息学管道（例如MiTCR和IMGT-HighV-Quest）的开发使得使用NGS数据的TCR分析能够应用于人类和小鼠T细胞群的研究。这些分析的先决条件是i）产生无偏TCR链扩增子的TCR链特异性PCR方案，ii）V和J基因片段序列的综合数据库，和iii）可以将TCR序列与相关的V和J基因片段数据库比对的复杂算法（并因此定义V和J的使用以及单个TCR链的高变CDR 3序列），并且还补偿和校正不可避免的PCR/PCR扩增。测序错误可能会严重损害序列数据解释。在为人类和小鼠TCR分析开发的生物信息学管道中使用的原理是通用的，并且在最近的研究中，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的NGS序列数据（在这种情况下，欧洲普通牛）。在这个项目中，我们建议：1 -设计PCR方案，使公正的PCR扩增TRA和TRB链从相关的牲畜种群。将验证猪、山羊、绵羊和水布法罗TRA和TRB链的优化的基于SMART的PCR扩增方案。2-测序足够的全长TRA/TRB扩增子，以能够全面覆盖表达的TRA和TRB库。从来自不同LMIC和欧洲品种的5个代表性个体产生的扩增子将进行PacBio测序以获得全长扩增子序列。3-生物信息学解构建体细胞重组的TRA和TRB链以构建种系V和J基因片段序列的数据库。将开发去构建重组TRA/TRB链的管道，以使能够建立组成V和J基因区段的种系序列并填充综合数据库。4-修改现有算法，以使这些数据库能够用于TCR库的NGS分析（并向公共可访问TCR分析算法的管理者提供TRBV、TRBJ、TRAV和TRAJ数据库）。将由CTLGH生物信息学储存库建立和托管MiTCR算法的修改版本，该算法将使牲畜物种数据库的利用成为可能。此外，数据库将由公开的Milaboratory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.