Menzel Ulrike, Greiff Victor, Khan Tarik A, Haessler Ulrike, Hellmann Ina, Friedensohn Simon, Cook Skylar C, Pogson Mark, Reddy Sai T
Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
PLoS One. 2014 May 8;9(5):e96727. doi: 10.1371/journal.pone.0096727. eCollection 2014.
High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.
抗体组库文库的高通量测序（HTS）已成为系统免疫学领域的强大工具。然而，HTS工作流程中存在许多偏差来源，可能会影响所获得的抗体组库数据。抗体文库制备中的关键步骤是添加短的平台特异性核苷酸接头序列。到目前为止，接头添加方法对实验文库制备以及由此产生的抗体组库HTS数据集的影响尚未得到充分研究。因此，我们通过对来自小鼠抗体分泌细胞的抗体可变重链基因进行Illumina HTS，比较了三种标准文库制备方法。克隆重叠和秩统计表明，所研究的方法产生了等效的HTS数据集。基于PCR的方法在速度、效率和实用性方面在实验上优于连接法。最后，我们使用基于两步PCR的方法建立了一种抗体组库文库生成方案，可以从低至1 ng的总RNA起始材料开始。总之，本研究朝着抗体HTS标准化实验框架迈出了重要一步，从而为基于系统的抗体组库交叉实验荟萃分析开辟了潜力。
