He Linling, Sok Devin, Azadnia Parisa, Hsueh Jessica, Landais Elise, Simek Melissa, Koff Wayne C, Poignard Pascal, Burton Dennis R, Zhu Jiang
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
1] Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA [2] IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, California 92037, USA [3] International AIDS Vaccine Initiative (IAVI), New York, NY 10004, USA [4] Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California 92037, USA.
Sci Rep. 2014 Oct 27;4:6778. doi: 10.1038/srep06778.
B-cell repertoire analysis using next-generation sequencing has become a valuable tool for interrogating the genetic record of humoral response to infection. However, key obstacles such as low throughput, short read length, high error rate, and undetermined bias of multiplex PCR method have hindered broader application of this technology. In this study, we report several technical advances in antibody repertoire sequencing. We first demonstrated the ability to sequence antibody variable domains using the Ion Torrent PGM platform. As a test case, we analyzed the PGT121 class of antibodies from IAVI donor 17, an HIV-1-infected individual. We then obtained "unbiased" antibody repertoires by sequencing the 5'-RACE PCR products of B-cell transcripts from IAVI donor 17 and two HIV-1-uninfected individuals. We also quantified the bias of previously published gene-specific primers by comparing the repertoires generated by 5'-RACE PCR and multiplex PCR. We further developed a single-molecule barcoding strategy to reduce PCR-based amplification noise. Lastly, we evaluated several new PGM technologies in the context of antibody sequencing. We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families.
使用下一代测序技术进行B细胞受体库分析已成为探究针对感染的体液免疫反应遗传记录的一项重要工具。然而，诸如低通量、短读长、高错误率以及多重PCR方法的未知偏差等关键障碍阻碍了该技术的更广泛应用。在本研究中，我们报告了抗体受体库测序方面的多项技术进展。我们首先展示了使用Ion Torrent PGM平台对抗体可变区进行测序的能力。作为一个测试案例，我们分析了来自IAVI供体17（一名感染HIV-1的个体）的PGT121类抗体。然后，我们通过对来自IAVI供体17以及两名未感染HIV-1个体的B细胞转录本的5'-RACE PCR产物进行测序，获得了“无偏差”的抗体受体库。我们还通过比较5'-RACE PCR和多重PCR产生的受体库，对先前发表的基因特异性引物的偏差进行了量化。我们进一步开发了一种单分子条形码策略以减少基于PCR的扩增噪声。最后，我们在抗体测序的背景下评估了几种新的PGM技术。我们期望，基于长读长和高保真的下一代测序技术，无偏差分析将提供更准确的整体抗体受体库视图，而条形码策略将有助于对单个抗体家族进行高分辨率分析。
