VH and JH gene usage and clonality of Ab repertoires in different immune compartments. (A) Tissue sampling from D20. IgG HC libraries from blood, BM, draining iLN, spleen, and gut (ileum) were constructed and deep sequenced. (B and C) After processing, the sequences were assigned to the VH (B) and JH (C) germline alleles identified in the D20 animal. The genes are shown familywise (VH1–VH7), with the most frequently used VH (B) and JH (C) allele in each family, based on the IgM library, at the top. (D) Top: Sequencing metadata. Total reads, number of raw sequences generated using the MiSeq 2× 300-bp sequencing platform in libraries made from different tissue compartments; merged reads, number of paired sequences; barcode clusters, number of sequences after collapsing sequences with identical barcodes and HCDR3 into a single consensus sequence (including singletons); unique VDJ sequences, total number of uniquely barcoded in-frame Ab sequences; clonotypes, number of unique clonotypes defined as sequences with the same VH and JH gene assignments, identical HCDR3 length, and 80%* HCDR3 sequence identity, also depicted in the lower panel (data from one independent experiment). (E) HCDR3 length distribution shown as the percentage of sequences with a given HCDR3 length of the total number of clonotypes. The Next Generation Sequencing experiment was performed once, and the analyses, at least twice.