Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing
Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing
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DOI：
10.1101/gr.092924.109
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发表时间：
2009-10-01
期刊：
影响因子：
7
通讯作者：
Holt, Robert A.
中科院分区：
文献类型：
--
作者：
Freeman, J. Douglas;Warren, Rene L.;Holt, Robert A.
T细胞受体（TCR）基因组位点经历体细胞V（D）J重组，加上在重组连接处添加/减去非模板化碱基，以产生抗原识别所必需的结构多样性T细胞的库。TCR β亚基可以通过其高变CDR 3（补体决定区3）序列明确鉴定。这是V（D）J重组的位点，编码抗原接触的主要位点。T细胞库的复杂性和动态性仍然未知，因为潜在库的大小使得常规序列分析难以处理。在这里，我们使用5 '-RACE、Illumina测序和一种新的短读段组装策略来从外周血中的人T淋巴细胞中采样CDR 3（β）多样性。4050万个短读段的组装鉴定了33,664个不同的TCR β克隆型，并提供了CDR 3（β）长度多样性、非模板化碱基的使用、序列收敛性以及TRBV（T细胞受体β可变基因）和TRBJ（T细胞受体β连接基因）基因使用和配对的偏好的精确测量。TRBV和TRBJ的保守残基之间的CDR 3长度为21至81个核苷酸（nt）。TRBV基因使用率范围从TRBV 17的0.01%到TRBV 20 -1的24.6%。TRBJ基因使用率范围为TRBJ 2 -6的1.6%至TRBJ 2 -1的17.2%。我们确定了1573个收敛的例子，其中相同的氨基酸翻译由不同的CDR 3（β）核苷酸序列指定。直接序列为基础的免疫分析可能会被证明是一个有用的工具，了解库动态响应免疫挑战，没有抗原的先验知识。
T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D) J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR beta clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.