Longitudinal studies of B cell responses, such as those tracking influenza-binding B cell clones ( 4, 5) require methods for measuring clone sizes and comparing them at different time points. B cell clones can be viewed as the unit of selection in an immune response ( 1) the successful recruitment of clones results in diversification and expansion of cells with the appropriate antigen specificity and effector function ( 2, 3). B cells that are clonally related derive from a common progenitor cell. The accurate measurement of clone size is fundamental to many immunological studies. ![]() Overall, we provide a series of pragmatic analytical approaches and show how different clone size measures can be used to study the clonal landscape in bulk B cell immune repertoire profiling data. We comment upon frequently encountered pitfalls and offer practical advice with alternative approaches. In the second data set, B cell clones are compared in eight different organ donors. In the first data set, 19 independently generated biological replicates from a single individual are analyzed for B cell clone size, diversity and sampling sufficiency for clonal overlap analysis. We provide a detailed, step-by-step procedure for performing these analyses using two different data sets of spleen samples from human organ donors. We define three different measures of B cell clone size-copy numbers, instances, and unique sequences-and show how these measures can be used to rank clones, analyze their diversity, and study their distribution within and between individuals. ![]() Here, we describe a series of computational approaches for estimating B cell clone size in NGS immune repertoire profiling data of antibody heavy chain gene rearrangements. One way to monitor and track B cell clones is to perform large-scale sampling of bulk cell populations, amplifying, and sequencing antibody gene rearrangements by next-generation sequencing (NGS). 4Department of Microbiology and Immunology, Drexel College of Medicine, Drexel University, Philadelphia, PA, United Statesī cell clones expand and contract during adaptive immune responses and can persist or grow uncontrollably in lymphoproliferative disorders.3Columbia Center for Translational Immunology, Columbia University, New York, NY, United States.2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, United States. ![]() Chen 2 Bochao Zhang 1 Tomer Granot 3 Donna L.
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