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Only alignments that were in-frame and could produce functional protein were further considered. Additionally, data filtering steps were implemented to remove potential PCR contaminants and improperly amplified PCR products that were inappropriately found in multiple samples (see Experimental Procedures). We also chose to exclude sequences lacking both VD and DJ junctional bases, since these are more likely to occur independently in different individuals. Sequences with unassigned D regions were retained in our analysis. After filtering, the read count per sample varied between 450 and 9,000, with a median count of 2,000 ( Table S1). Clonal expansions of immune cells have been observed in many pathological states such as viral and bacterial infections and hematological malignancies (Thors��lius Crenolanib et?al., 2006; Wang and Palese, 2009; Zhou et?al., 2002). VH sequencing has facilitated the identification of prominent clonal B cell populations in lymphocytic malignancies, without prior selection for specific classes of B cells (Cleary et?al., 1984; Warnke and Levy, 1978). Less-prominent B cell clones are also evident in RecBCD sequence data sets from healthy individuals and can be detected by identification of identically rearranged sequences in PCR pools?generated from independent cellular DNA aliquots from the same sample (Boyd et?al., 2009). Such VH ��coincident sequences�� were found to be extremely rare between samples from different individuals, due to the substantial combinatorial and junctional diversity of immunoglobulin gene rearrangements (Boyd et?al., 2009; Glanville et?al., 2011). The presence of clonal populations in a sample may be estimated using various diversity indices (for example, Hill, 1973; Jost, 2006) to calculate clonality scores from a single DNA pool per sample. In our study, we apply a replicate library sequencing approach to robustly test for the presence of clonal B cell populations in an individual by sequencing from multiple distinct DNA aliquots isolated from the same lymphocyte sample (Boyd et?al., 2009). We use the probability of identifying clonally related sequences in arbitrary sequence pairs from different aliquots of a single sample (P[collision]) (Schnabel, 1938) as a metric to track clonality. P(collision) captures the probability that any two randomly chosen Antidiabetic Compound Library B cells in an individual share a clonal origin, with mean values of P(collision) independent of sequencing depth (Figures S1A and S1B). Consistent with an active immune response, P(collision) was higher in acute-phase samples compared to convalescent or postconvalescent samples from the same individual (p?= 0.0004 and p?
