Kathleen Zeglinski from the Walter and Eliza Hall Institute of Medical Research in Australia presented the ten minute recorded session entitled “NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells” at the Nanopore Community Meeting 2022. Zeglinski explained that hybridomas and B cells are common ways to generate antibodies in order to produce large quantities of antibodies. However, they mentioned that hybridomas are unstable and sometimes lose their antibody genes, and that a large percentage of hybridomas are actually producing a mix of antibodies. Interestingly, Zeglinski shared an article that mentioned that reproducibility the reproducibility crisis can be blamed (partially) on antibodies! The current approach is to use Sanger sequencing to verify antibodies. This process is costly and labor intensive. Using Nanopore sequencing with NAb-seq reduces time, is cheaper (as low as $30 per antibody!), and sequences the transcriptome and therefore is not dependent on primers. The NAb-seq process, Zeglinski explained, is whole transcriptome sequencing in which FACS sorted B cells are used for RNA extraction, cDNA synthesis, PCR barcoding kit, and Nanopore sequencing. The team, Zeglinski explained, worked on a pipeline to simplify analysis. The pipeline is available from Github as a nextflow pipeline and generates an HTML report. Zeglinski explained that NAb-seq detects the presence of multiple productive heavy and light chains in a sample. Zeglinski concluded that NAb-seq is cheap, fast, and effective for determining the sequence of antibodies from hybridomas and B cells. The process “can generate 100% accurate sequences” and facilitate verification of antibodies in research. I had never considered this application and learned about a different approach that combines transcriptomics and antibody verification. I also appreciate the development of a simple tool for analyses that can be used along with simple barcoding kits.
