Samples were collected at Hemacares Southern California donor center. focused on circulating B cells.3Here, we examine the circulating B cell populations of ten human subjects and present the largest single collection of adaptive immune receptor sequences described to date, comprising almost 3 billion antibody heavy chain sequences. This dataset allows genetic study of the baseline human antibody repertoire at unprecedented depth and granularity, revealing largely TAK-715 unique repertoires for each individual studied, a subpopulation of universally shared antibody clonotypes, and exceptional overall repertoire diversity. Eighteen sequencing libraries were generated for each of ten subjects (Physique ED1). These libraries yielded 2.90109raw reads. Following annotation,4which included duplicate removal using unique TAK-715 molecular identifiers,5we obtained 3.64108productive antibody sequences (Table ED1). Amplification was reproducible, with comparable gene usage between replicates (Figures1A,ED2). The frequencies of IgM-encoding (0.620.94) and IgG-encoding (0.060.38) sequences were consistent with the expected frequency of circulating B cells expressing these isotypes (Physique 1B).6Although V-gene, J-gene and CDRH3 length (VJ-CDR3len) distributions were comparable between subjects (Figures 1C, E-F), differences were large enough that individual repertoires could conceivably be distinguished using only these features. We reduced sequence subsamples to VJ-CDR3len frequency distributions and quantified similarity using the Morisita-Horn similarity index.7,8Subject repertoires were clearly distinguishable using as few as 104sequences (Figures1D,ED4) and did not cluster by age, gender or ethnicity (Physique 1G). The IgG+repertoires were least similar, suggesting that subjects unique immunological histories are a significant contributor to repertoire individuality (Physique 1H). A one-versus-rest support vector machine (SVM) classifier trained on VJ-CDR3len data from 5 of the 6 biological replicates from each subject accurately assigned the remaining replicate using test/train datasets of as few as 500 sequences from each replicate (Physique 1I). == Physique 1. Uniqueness of the repertoires of individual subjects. == a) Frequency comparison of V/J combinations in biological replicates from subject 326650. V/J combinations are colored according to the V-gene used. b) Sequence frequency by antibody isotype. Subjects are colored as in (c). Each point represents a single biological replicate. Mean of all samples is usually indicated for each isotype. c) CDRH3 length distribution for each subject. CDRH3 lengths were decided using the IMGT numbering scheme. d) Morisita-Horn similarity of pairwise comparisons between subject 316188 and each of the other subjects. Lines indicate mean similarity of 20 bootstrap samplings and shaded areas indicate 95% confidence intervals. Data from subject 316188 is usually TAK-715 representative; plots for all other subjects can be found inFigure ED4. V-gene (e) and J-gene (f) use by subject. Increased color intensity indicates higher frequency. Subjects are colored as in (c). g) Clustered distance matrix of subjects, using pairwise VJ-CDR3len Morisita-Horn similarity as the distance measure. Distance matrix was computed using single-linkage clustering (Euclidean distance metric). Subject colors are as in TAK-715 (c). A dendrogram representation of the distance matrix is also shown around the left side of the distance matrix. h) Comparison of intra- and inter-subject VJ-CDR3len similarity, using either all sequences, IgM sequences with fewer than two nucleotide mutations, IgM sequences with two or more mutations, or IgG sequences. Points represent individual intra- or inter-subject comparisons. Boxplots show the median line and span the 25th-75th percentile, with whiskers indicating the 95% confidence interval. i) Mean receiver operating characteristic (ROC) area under the curve (AUC) for a one-versus-rest SVM classifier. The ROC AUC does not drop below 1.0 for any subject when Mouse monoclonal to CD3.4AT3 reacts with CD3, a 20-26 kDa molecule, which is expressed on all mature T lymphocytes (approximately 60-80% of normal human peripheral blood lymphocytes), NK-T cells and some thymocytes. CD3 associated with the T-cell receptor a/b or g/d dimer also plays a role in T-cell activation and signal transduction during antigen recognition the test/training datasets include 500 sequences each, and that threshold is indicated with a dashed vertical line. To estimate repertoire diversity while minimizing the effects of sequencing and amplification error, we first considered clonotype diversity. An antibody clonotype is usually a collection of sequences using the same V/J-genes and encoding an identical CDRH3 amino acid sequence.9For each subject, all sequences from each biological replicate were collapsed into a set of unique clonotypes. Any clonotypes repeatedly observed after pooling deduplicated biological replicates must be derived from different cells, providing a straightforward means of quantifying multiple occurrence. For clarity,.