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.

中文翻译：

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荧光激活液滴测序 (FAD-seq) 直接提供抗体发现中的筛选命中序列


液滴微流控已成为高通量筛选（包括抗体发现）中非常强大的工具。筛选通常通过物理分选具有所需表型的细胞的液滴、将其破碎、回收封装的细胞、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来进行。这一系列罕见筛选命中的多个连续操作步骤复杂且具有挑战性，导致具有所需表型的克隆的大量损失或具有不完整抗体信息的大部分细胞。在这里，我们提出了荧光激活液滴测序，其中显示所需表型的液滴被选择性地注射 RT-PCR 试剂。随后，轻链和重链基因天然配对，融合成单链片段变体形式，并在芯片外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，可以从短短五个液滴中获得不同配对的全长抗体序列，从而满足所需的表型。通过特定测序代替物理分选克服了液滴微流体筛选的一般瓶颈，并且应该与更多应用兼容。