biostudies-literatureMoura-Sampaio JFundação para a Ciência e a TecnologiaMinistério da Ciência, Tecnologia e Ensino Superior2723-2727https://www.ebi.ac.uk/biostudies/studies/S-EPMC9168528biostudies-literatureUnknown20Next-generation sequencing (NGS) is an indispensable tool in antibody discovery projects. However, the limits on NGS read length make it difficult to reconstruct full antibody sequences from the sequencing runs, especially if the six CDRs are randomized. To overcome that, we took advantage of Illumina's cluster mapping capabilities to pair non-overlapping reads and reconstruct full Fab sequences with accurate V_L:V_H pairings. The method relies on <i>in silico</i> cluster coordinate information, and not on extensive <i>in vitro</i> manipulation, making the protocol easily deployable and less prone to PCR-derived errors. This work maintains the throughput necessary for antibody discovery campaigns, and a high degree of fidelity, which potentiates not only phage-display aComputational and structural biotechnology journalReconstruction of full antibody sequences in NGS datasets and accurate V_L:V_H coupling by cluster coordinate matching of non-overlapping reads.PMC9168528UIDB/04462/2020PD/BD/128321/2017UIDP/04462/2020Boeuf RAntunes MAMoura-Sampaio JBatista APEwert SFaustino AFfalseReconstruction of full antibody sequences in NGS datasets and accurate V_L:V_H coupling by cluster coordinate matching of non-overlapping reads.Next-generation sequencing (NGS) is an indispensable tool in antibody discovery projects. However, the limits on NGS read length make it difficult to reconstruct full antibody sequences from the sequencing runs, especially if the six CDRs are randomized. To overcome that, we took advantage of Illumina's cluster mapping capabilities to pair non-overlapping reads and reconstruct full Fab sequences with accurate V_L:V_H pairings. The method relies on <i>in silico</i> cluster coordinate information, and not on extensive <i>in vitro</i> manipulation, making the protocol easily deployable and less prone to PCR-derived errors. This work maintains the throughput necessary for antibody discovery campaigns, and a high degree of fidelity, which potentiates not only phage-display a2022-01-01T00:00:00Z20222026-07-14T14:11:14.357Z2022-07-20T01:36:06.327ZS-EPMC91685283583262310.1016/j.csbj.2022.05.054