(c) Cumulative distribution of normalized VDJ triple frequencies used for simulation: HIP1 (= 4,373 unique VDJ triples), HIP2 (= 4,351 unique VDJ triples) and HIP3 (= 4,372 unique VDJ triples)

(c) Cumulative distribution of normalized VDJ triple frequencies used for simulation: HIP1 (= 4,373 unique VDJ triples), HIP2 (= 4,351 unique VDJ triples) and HIP3 (= 4,372 unique VDJ triples). shared by all three). Some of the B cell clonotypes had thousands of clones (somatic variants) within the clonotype lineage. While some of these shared lineages might be driven by exposure to common antigens, prior foreign antigen exposure was not the only pressure shaping the shared repertoires, as we also identified shared clonotypes present in both human cord blood samples and in all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease. Determination of the complete set of expressed recombined human immune receptor genes is usually of general interest to understand fundamental aspects of the development and maintenance of the immune system (such as comparing na?ve and memory or neonatal and adult repertoires)2,3. We sought to estimate the size and diversity of human B cell receptor (BCR) repertoires of healthy adults or neonates by sequencing samples to remarkable depth. We designated B cell recombined variable region sequences as members of a single if the sequences were encoded by the same BCR VH/JH, V/J or V/J gene segments and possessed identical amino acids in the third complementarity determining region (CDR3). The V3J clonotype provides a minimal representation for a BCR sequence that can applied across different immune repertoire sequencing methods. We isolated large numbers of peripheral blood mononuclear cells (PBMCs) by leukapheresis from three healthy adults, designated HIP1 (female, age 47 y), HIP2 (male age 22 y) or HIP3 (male age 29 y), obtaining 13, 21, or 30 billion PBMCs, respectively (Extended Data Table 1). To increase sequencing depth, we used diverse methods and primer sets (Extended Data Tables 2, ?,3,3, and ?and44). The sequencing reactions yielded 1.4, 1.5 or 1.3 109 Meloxicam (Mobic) natural sequencing reads for subjects HIP1, 2 or 3 3. We processed the sequences to remove low-quality reads (see Supplementary Methods), obtaining about 5.8, 6.3, or 5.1 108 sequences after quality control filtering for subject HIP1, 2 or 3 3, respectively. After filtering, sequences were designated Experimental sequencing yielded about 17.1 million Ig heavy V3J clonotypes for subject HIP2. The species richness endpoint estimate was 17,110,333. Extrapolation gave a species richness estimate of 20,210,426. (Experimental sequencing yielded about 9.0 million V3J clonotypes for Meloxicam (Mobic) HIP3. The endpoint species richness estimate was 8,989,812. Extrapolation Meloxicam (Mobic) gave a species richness estimate of 11,984,340. (HIP1+2+3) was 0.3% (n = 29,062 unique V3J clonotypes). We found a similar extent of sharing in our subjects V3J clonotypes (0.3 to 0.6% shared) with each of three BCR repertoires in an independently derived data set8, even though very different methodologies were used for sequencing. The median HCDR3 length of HIP1+2+3 (n = 22,408 unique CDR3s) was 13 amino acids, which was shorter than the median length of 16 amino acids for HIP1+2+3 (n = 30,156,947 unique CDR3s) (Extended Data Fig. 2a). Open in a separate window Physique 2. Shared clonotypes between three healthy adult subjects (HIP1, 2 and 3).(a) Shared V3J clonotypes from sequenced Ig heavy chains. (b) (= 3,641 common clonotypes) ranked highest in the 1,000 comparisons giving a = 1.0 10?4 (see Extended Data Fig. 2e for normalized histogram of Rabbit Polyclonal to RAD51L1 common clonotypes between synthetic sets). (c) Fold change in VH+JH usage between SHIP1+2+3 (= 29,062 unique clonotypes) and all HIP subjects (designated:.