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  4. Fluorescence-activated droplet sequencing (FAD-seq) directly provides sequences of screening hits in antibody discovery
 
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research article

Fluorescence-activated droplet sequencing (FAD-seq) directly provides sequences of screening hits in antibody discovery

Autour, Alexis  
•
Merten, Christoph A.  
September 10, 2024
Proceedings Of The National Academy Of Sciences Of The United States Of America (PNAS)

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.

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Type
research article
DOI
10.1073/pnas.2405342121
Scopus ID

2-s2.0-85203420077

PubMed ID

39240970

Author(s)
Autour, Alexis  
•
Merten, Christoph A.  
Date Issued

2024-09-10

Publisher

National Academy of Sciences

Published in
Proceedings Of The National Academy Of Sciences Of The United States Of America (PNAS)
Volume

121

Issue

37

Article Number

e2405342121

Subjects

antibodies

•

droplet microfluidics

•

heavy

•

light chain pairing

•

single-cell and droplet sorting

Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LBMM  
FunderFunding(s)Grant NumberGrant URL

JPIAMR

01KI822

Available on Infoscience
January 24, 2025
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/243759
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