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

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.

Active Lysogeny in Listeria Monocytogenes Is a Bacteria-Phage Adaptive Response in the Mammalian Environment.

Some Listeria monocytogenes (Lm) strains harbor a prophage within the comK gene, which renders it inactive. During Lm infection of macrophage cells, the prophage turns into a molecular switch, promoting comK gene expression and therefore Lm intracellular growth. During this process, the prophage does not produce infective phages or cause bacterial lysis, suggesting it has acquired an adaptive behavior suited to the pathogenic lifestyle of its host. In this study, we demonstrate that this non-classical phage behavior, named active lysogeny, relies on a transcriptional response that is specific to the intracellular niche. While the prophage undergoes lytic induction, the process is arrested midway, preventing the transcription of the late genes. Further, we demonstrate key phage factors, such as LlgA transcription regulator and a DNA replicase, that support the phage adaptive behavior. This study provides molecular insights into the adaptation of phages to their pathogenic hosts, uncovering unusual cooperative interactions.