B cell clones expand and contract during adaptive immune responses and can persist or grow uncontrollably in lymphoproliferative disorders. 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). 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. 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. We provide a detailed, step-by-step procedure for performing these analyses using two different data sets of spleen samples from human 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. In the second data set, B cell clones are compared in eight different organ donors. We comment upon frequently encountered pitfalls and offer practical advice with alternative approaches. 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.
Bibliographical noteFunding Information:
We thank LiveOnNY and the donor families for making this study possible. We acknowledge the Penn Flow Cytometry Shared Resource and the Human Immunology Core facility. This work was supported by NIH P01 AI106697 and NIH P30 CA016520.
© 2018 Rosenfeld, Meng, Chen, Zhang, Granot, Farber, Hershberg and Luning Prak.
- B cell
- Immune repertoire
- Next generation sequencing
ASJC Scopus subject areas
- Immunology and Allergy