Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne 1015, Switzerland.
European Molecular Biology Laboratory, Heidelberg 69117, Germany.
Proc Natl Acad Sci U S A. 2024 Sep 10;121(37):e2405342121. doi: 10.1073/pnas.2405342121. Epub 2024 Sep 6.
Droplet microfluidics has become a very powerful tool in high-throughput screening, including antibody discovery. Screens are usually carried out by physically sorting droplets hosting cells of the desired phenotype, breaking them, recovering the encapsulated cells, and sequencing the paired antibody light and heavy chain genes at the single-cell level. This series of multiple consecutive manipulation steps of rare screening hits is complex and challenging, resulting in a significant loss of clones with the desired phenotype or large fractions of cells with incomplete antibody information. Here, we present fluorescence-activated droplet sequencing, in which droplets showing the desired phenotype are selectively picoinjected with reagents for RT-PCR. Subsequently, light and heavy chain genes are natively paired, fused into a single-chain fragment variant format, and amplified before off-chip transfer and downstream nanopore sequencing. This workflow is sufficiently sensitive for obtaining different paired full-length antibody sequences from as little as five droplets, fulfilling the desired phenotype. Replacing physical sorting by specific sequencing overcomes a general bottleneck in droplet microfluidic screening and should be compatible with many more applications.
液滴微流控技术已成为高通量筛选的一种非常强大的工具，包括抗体发现。筛选通常通过物理方式对具有所需表型的细胞的液滴进行分类，打破液滴，回收包裹的细胞，并在单细胞水平上对配对的抗体轻链和重链基因进行测序。对稀有筛选命中的这一系列多个连续操作步骤非常复杂和具有挑战性，导致具有所需表型的克隆或具有不完全抗体信息的大量细胞的显著损失。在这里，我们提出了荧光激活的液滴测序，其中显示所需表型的液滴被选择性地用 RT-PCR 试剂进行皮内注射。随后，轻链和重链基因以原生形式配对，融合成单链片段变体格式，在离片转移和下游纳米孔测序之前进行扩增。该工作流程足够灵敏，可从少至五个液滴中获得不同的配对全长抗体序列，满足所需的表型。通过特定的测序取代物理分类克服了液滴微流控筛选的一般瓶颈，并且应该与更多的应用兼容。
