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. 2023 Sep 27;13:16200. doi: 10.1038/s41598-023-43233-4

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Sequencing workflow and bioinformatics processing. Hybridomas of interest are sequenced using a workflow consisting of RNA extraction, cDNA synthesis, and semi-nested PCR amplification with IgG-specific primers followed by the addition of unique Illumina barcodes to each sample. Illumina libraries are then generated, and adapters are ligated for sequencing on the MiSeq platform. Bioinformatics processing is shown on the right panel. Reads from the Illumina sequencing are run through HTStream for base quality trimming and other read processing. Next, they are passed through DADA2 for amplicon denoising followed by SAbPred ANARCI tool based on the IMGT numbering scheme. All ASVs, metadata, and other quality metrics are uploaded to the NeuroMabSeq database and website where further information and tools are provided to the end users. This includes but is not limited to BlastIR results, BLAT searches across the database, and recommended high quality sequences for recombinant antibody design. Annotations of internally generated scores are provided in addition to other database information. Finally, high quality sequences are used in the design of gene fragments for generation of R-mAb and scFv expression plasmids.