Abstract
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.
Highlights
The accurate measurement of clone size is fundamental to many immunological studies
We demonstrate the importance of choosing the appropriate combination of experimental approaches and analytical tools to measure B-cell clone size
One has to know what scale of clone sizes is of interest, which means visualizing the repertoire as a whole on a diversity or clone copy number cut-off plot
Summary
The accurate measurement of clone size is fundamental to many immunological studies. B cells that are clonally related derive from a common progenitor cell. With respect to the data generation, there are already several excellent protocols for immune repertoire profiling by NGS from DNA, RNA, and single cells [11,12,13,14,15,16,17,18] These different methods can be compared against one another on the same sample, along with procedures such as digital droplet PCR to perform experimental estimates on clone size [8]. We describe our procedures using a large number of independently amplified sequencing libraries from the spleen of one organ donor and in a newly generated data set of spleen samples from eight different organ donors (Figure 1) Using these deep and survey-level sequencing data sets, we illustrate measures of within- and between-individual clonal size and diversity analysis. Genomic DNA (normalized for the B cell fraction) was separately amplified and sequenced (two biological replicates per subject)
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