T细胞受体（TCR）基因的深度测序在分析免疫谱方面功能强大。为了制备TCR测序文库，多重聚合酶链反应（mPCR）被广泛应用并且非常高效。也就是说，大多数mPCR产物都包含对于抗原识别至关重要的区域，这也表明V（D）J重组正常。但是，多重PCR可能会受到引物偏倚的困扰。5'-RACE是一个很有前途的替代方案，它仅应用一对引物即可避免引物偏倚。然而，在5'-RACE数据中，已观察到非规则的V（D）J重组（例如，不带V基因片段的TCR序列），并且两次研究之间的频率发生变化（30-80％）。这表明科学界尚未知道引起非常规TCR序列的原因或如何减少其表达。尽管可以通过比较5'-RACE方案推测原因，但仍需要仔细的实验确认，这样的系统研究仍不可用。在这里，我们检查了商业试剂盒的5'-RACE方案，并证明了修饰如何将常规TCR-β序列的比例提高至> 85％。我们还发现短DNA片段的比例与非规则TCR-β序列的百分比之间存在很强的线性相关性，这表明文库中短DNA片段的存在是非规则TCR-β序列的主要原因。因此，从5'-RACE库中彻底去除短DNA片段是提高数据效率的关键。我们强烈建议您在测序之前进行片段长度分析，短DNA片段的比例可用于估算非规则TCR序列的百分比。由于TCR基因的深度测序仍然相对昂贵，因此良好的质量控制应该很有价值。
Deep sequencing of T-cell receptor (TCR) genes is powerful at profiling immune repertoire. To prepare a TCR sequencing library, multiplex polymerase chain reaction (mPCR) is widely applied and is highly efficient. That is, most mPCR products contain the region critical for antigen recognition, which also indicates regular V(D)J recombination. Multiplex PCR, however, may suffer from primer bias. A promising alternative is 5'-RACE, which avoids primer bias by applying only one primer pair. In 5'-RACE data, however, non-regular V(D)J recombination (e.g., TCR sequences without a V gene segment) has been observed and the frequency varies (30-80%) between studies. This suggests that the cause of or how to reduce non-regular TCR sequences is not yet well known by the science community. Although it is possible to speculate the cause by comparing the 5'-RACE protocols, careful experimental confirmation is needed and such a systematic study is still not available. Here, we examined the 5'-RACE protocol of a commercial kit and demonstrated how a modification increased the fraction of regular TCR-β sequences to >85%. We also found a strong linear correlation between the fraction of short DNA fragments and the percentage of non-regular TCR-β sequences, indicating that the presence of short DNA fragments in the library was the main cause of non-regular TCR-β sequences. Therefore, thorough removal of short DNA fragments from a 5'-RACE library is the key to high data efficiency. We highly recommend conducting a fragment length analysis before sequencing, and the fraction of short DNA fragments can be used to estimate the percentage of non-regular TCR sequences. As deep sequencing of TCR genes is still relatively expensive, good quality control should be valuable.
