由于免疫库 (IR) 的多样性，使用传统的短读长测序重建全长 IR 已被证明具有挑战性。通过线性滚环扩增和纳米孔测序开发了全长 IR 测序 (FLIRseq) 工作流程。其准确性和定量能力通过质粒混合物和基于短读长的商业 B 细胞受体/T 细胞受体测序 (BCR/TCR-seq) 进行了验证。使用FLIRseq对8例急性淋巴细胞白血病患者、3例过敏性疾病患者、4例银屑病患者和5例前列腺癌患者的组织和外周血中的IR进行分析。FLIRseq读数的错配率和间隙率较低，识别率较高速率高于纳米孔读数（所有 P < 2.2 × -16）。 FLIRseq对成分的相对定量与实际定量一致（P > 0.05）。 FLIRseq 优于 BCR/TCR-seq，提供长互补决定区 3、B 细胞同种型和很少使用的 V 基因序列。 FLIRseq 观察到缓解期白血病患者克隆型多样性增加（P < 0.05），异常 BCR/TCR 百分比下降。对于患有过敏性疾病或牛皮癣的患者，FLIRseq 提供了有关 V(D)J 重组和特定免疫球蛋白类别的直接见解。与前列腺癌组织相比，银屑病组织淋巴细胞中偏向 T 细胞受体 β 链的全长 V 段表现出更一致的 AlphaFold2 预测的蛋白质结构 (P < 0.05)。 FLIRseq 能够对直接 V(D)J 重组和免疫球蛋白类别，从而有助于表征致病机制、监测微小残留疾病和定制过继细胞疗法。© 诊断协会

Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging.A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq.FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × -16). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor β chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05).FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.© Association for Diagnostics & Laboratory Medicine 2024.