Abstract:Data that defines IGHV (immunoglobulin heavy chain variable) germline gene inference using sequences of IgM-encoding transcriptomes obtained by Illumina MiSeq sequencing technology are described. Such inference is used to establish personalized germline gene sets for in-depth antibody repertoire studies and to detect new antibody germline genes from widely available immunoglobulin-encoding transcriptome data sets. Specifically, the data has been used to validate (Parallel antibody germline gene and haplotype a… Show more
“…(iv) Two pairs of genes, V3-9 and V1-8 , and V5-10-1 and V3-64D , are deleted in a mutually exclusive manner. This pattern has previously been observed for single haplotypes 18,31,32 . Here we show the prevalence of this pattern among a large cohort.Fig.…”
Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding immunoglobulin genes is incomplete, resulting in conflicting VDJ gene assignments and biased genotype and haplotype inference. Haplotypes can be inferred using IGHJ6 heterozygosity, observed in one third of the people. Here, we propose a robust novel method for determining VDJ haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, enabling inference of deletions and copy number variations in the entire population. To test this method, we generated a multi-individual data set of naive B-cell repertoires, and found allele usage bias, as well as a mosaic, tiled pattern of deleted IGHD and IGHV genes. The inferred haplotypes may have clinical implications for genetic disease predispositions. Our findings expand the knowledge that can be extracted from antibody repertoire sequencing data.
“…(iv) Two pairs of genes, V3-9 and V1-8 , and V5-10-1 and V3-64D , are deleted in a mutually exclusive manner. This pattern has previously been observed for single haplotypes 18,31,32 . Here we show the prevalence of this pattern among a large cohort.Fig.…”
Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding immunoglobulin genes is incomplete, resulting in conflicting VDJ gene assignments and biased genotype and haplotype inference. Haplotypes can be inferred using IGHJ6 heterozygosity, observed in one third of the people. Here, we propose a robust novel method for determining VDJ haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, enabling inference of deletions and copy number variations in the entire population. To test this method, we generated a multi-individual data set of naive B-cell repertoires, and found allele usage bias, as well as a mosaic, tiled pattern of deleted IGHD and IGHV genes. The inferred haplotypes may have clinical implications for genetic disease predispositions. Our findings expand the knowledge that can be extracted from antibody repertoire sequencing data.
“…Such analysis ( 19 ) was performed using IgDiscover ( 15 ) to define the lymphocyte donors’ IGHV germline gene and allele makeup. We furthermore used a haplotype quality-control approach to define the validity of many of the allele calls ( 18 , 19 ). This approach also allowed us to validate novel alleles not present in the IMGT reference directory used for gene assignments ( 19 ).…”
Section: Resultsmentioning
confidence: 99%
“…The germline gene repertoire of the donors have been inferred using IgDiscover ( 15 ) using the IgM-encoding transcriptomes of the donors’ BM, and has, when possible, been quality controlled by haplotype-based analysis ( 18 , 19 ). Eleven commonly expressed germline genes (IGHV1-8, IGHV1-18, IGHV2-5, IGHV3-7, IGHV3-11, IGHV3-21, IGHV3-23, IGHV4-39, IGHV4-59, IGHV5-51, and IGHV6-1) (Table 1 in Supplementary Material) mostly encoded by a single or a few highly related alleles, representing six germline gene subgroups, were selected for further analysis (Table 1 ).…”
B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.
“…Although many studies analyzed patterns of V-D-J pairing (23, 24), there is still a shortage of studies of unusual recombination events such as V(DD)J recombination incorporating two D genes into a single unusually long CDR3 with tandem fused IGHD genes (or tandem CDR3 ). Meek et al (25) were the first to reveal a few tandem CDR3s, thus confirming the V(DD)J recombination conjecture put forward by Kurosawa and Tonegawa (26).…”
The V(D)J recombination forms the immunoglobulin genes by joining the variable (V), diversity (D), and joining (J) germline genes. Since variations in germline genes have been linked to various diseases, personalized immunogenomics aims at finding alleles of germline genes across various patients. Although recent studies described algorithms for
de novo
inference of V and J genes from immunosequencing data, they stopped short of solving a more difficult problem of reconstructing D genes that form the highly divergent CDR3 regions and provide the most important contribution to the antigen binding. We present the IgScout algorithm for
de novo
D gene reconstruction and apply it to reveal new alleles of human D genes and previously unknown D genes in camel, an important model organism in immunology. We further analyze non-canonical V(DD)J recombination that results in unusually long CDR3s with tandem fused IGHD genes and thus expands the diversity of the antibody repertoires. We demonstrate that tandem CDR3s represent a consistent and functional feature of all analyzed immunosequencing datasets, reveal ultra-long CDR3s, and shed light on the mechanism responsible for their formation.
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