Accurate profiling of full-length Fv in highly homologous antibody libraries using UMI tagged short reads

Itay Levin, Marek Štrajbl, Yair Fastman, Dror Baran, Shir Twito, Jessica Mioduser, Adi Keren, Sharon Fischman, Michael Zhenin, Guy Nimrod, Natalie Levitin, May Ben Mayor, Meital Gadrich, Yanay Ofran

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Deep parallel sequencing (NGS) is a viable tool for monitoring scFv and Fab library dynamics in many antibody engineering high-throughput screening efforts. Although very useful, the commonly used Illumina NGS platform cannot handle the entire sequence of scFv or Fab in a single read, usually focusing on specific CDRs or resorting to sequencing VH and VL variable domains separately, thus limiting its utility in comprehensive monitoring of selection dynamics. Here we present a simple and robust method for deep sequencing repertoires of full length scFv, Fab and Fv antibody sequences. This process utilizes standard molecular procedures and unique molecular identifiers (UMI) to pair separately sequenced VH and VL. We show that UMI assisted VH-VL matching allows for a comprehensive and highly accurate mapping of full length Fv clonal dynamics in large highly homologous antibody libraries, as well as identification of rare variants. In addition to its utility in synthetic antibody discovery processes, our method can be instrumental in generating large datasets for machine learning (ML) applications, which in the field of antibody engineering has been hampered by conspicuous paucity of large scale full length Fv data.

Original languageEnglish
Pages (from-to)E61
JournalNucleic Acids Research
Issue number11
StatePublished - 23 Jun 2023

Bibliographical note

Publisher Copyright:
© 2023 The Author(s).


The research was funded by Biolojic Design, Ltd. Funding for open access charge: Biolojic Design, Ltd.

FundersFunder number
Biolojic Design, Ltd.


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