99 B-cell repopulation after rituximab in rheumatoid arthritis B-cell receptor repertoire analysis B-cell receptor (BCR) clonotypes were defined as unique IGHV to IGHJ BCR sequences. Abundance was defined as the number of different UMIs associated with each clonotype, expressed as the percentage of total number of UMIs in the sample. Clonal expansion was calculated as the Gini index on the distribution of the number of unique UMIs per BCR clonotype in each sample, and Clonal diversity as the Shannon index on the distribution of the number of unique BCR clonotypes per clonal lineage in each sample [13]. These indices were calculated using the renyi function in the vegan R package (version 2.5-6) [14]. Analysis of somatic hypermutation was performed using the SHazaM R package (version 0.2.1) [12]. ADA testing Serum was tested for presence of ADA against rituximab performing a chemioluminescence drug-tolerant capture ELISA assay using a Meso Scale Diagnostic platform at the clinical immunology laboratory of GlaxoSmithKline Research and Development, Upper Merion, PA, USA. Dealing with missing data Three patients were excluded from the final analysis because the baseline PAXGene sample was not collected. For the remaining 28 patients (baseline characteristics in online supplementary table S1), 5 follow-up samples (out of total 112) were not collected and 14 failed BCR amplification or post-sequencing quality control (online supplementary table S2). For analysis of response prediction all patients were included based on the assumption that sample failure represented complete B cell depletion. These samples will be later referred to as imputed data. This assumption is supported by the fact that available samples taken at the earlier or later timepoint indeed did show extensive depletion (see figure 2B). No change in results was observed if these patients were, or were not included (figure 3 and figure 4 and online supplementary figure S3). In case of missing DAS28-score (5% of the data), the timepoints concerned were excluded from the analysis. For the analysis of clonality, somatic hypermutation, depletion and re-population after rituximab, patients were included if they had no (n = 18) or one (n = 5) missing follow-up timepoint (total n = 23). Statistics Data are presented as mean and standard deviation (SD) or median and interquartile range (IQR) after D’Agostino and Pearson omnibus test for normality. Differences between groups were evaluated using unpaired t-test and one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons post-test for 5
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