Biology subjects

Rodriguez, O. L.

Publications and source records attributed to Rodriguez, O. L..

18 recordsLinked to original sources

bioRxiv2025

Germline polymorphism in the immunoglobulin kappa and lambda loci explain variation in the expressed light chain antibody repertoire

Variation in antibody (Ab) responses contributes to variable disease outcomes and therapeutic responsiveness, the determinants of which are incompletely understood. This study demonstrates that polymorphisms in immunoglobulin (IG) light chain loci dictate the composition of the Ab repertoire, establishing fundamental baseline differences that preclude functional Ab-mediated responses. Using long-read genomic sequencing of the IG kappa (IGK) and IG lambda (IGL) loci, we comprehensively resolved genetic variation, including novel structural variants, single nucleotide variants, and gene alleles. By integrating these genetic data with Ab repertoire profiling, we found that all forms of IG germline variation contributed to inter-individual gene usage differences for >70% of light chain genes in the repertoire, directly impacting the amino acids of expressed light chain transcripts, including complementarity determining region domains. The genomic locations of usage - associated variants in both intergenic and coding regions indicated that IG polymorphisms modulate gene usage via diverse mechanisms, likely including the modulation of V(D)J recombination, heavy and light chain pairing biases, and transcription/translation. Finally, relative to IGL, IGK was characterized by more extensive linkage disequilibrium and genetic co-regulation of gene usage, illuminating differential regulatory and evolutionary features between the two light chain loci. These results firmly establish the critical contribution of IG light chain polymorphism in Ab repertoire diversity, with important implications for investigating Ab responses in health and disease.

immunology↗

bioRxiv2025

Human genetic variation shapes the antibody repertoire across B cell development

The peripheral antibody repertoire is shaped by inherited genetic variation and selection during B cell development. However, how the repertoire changes across developmental stages--and the relative impact of genetics and selection in establishing the antibody repertoire--remain largely unknown. To dissect the individual and combined effects of these factors, we integrated antibody repertoire transcriptomics across pro-B, pre-B, immature, and naive B cells with single-molecule real-time long-read DNA sequencing of the immunoglobulin heavy (IGH), kappa (IGK), and lambda (IGL) chain loci. We find that IGH genetic variants establish gene usage biases at the pro-B stage that persist throughout development. In contrast, IGK and IGL repertoires are extensively remodeled during maturation, consistent with receptor editing. Surprisingly, IGH variants also exert trans effects on light chain gene usage, suggesting coordinated constraints on heavy-light chain pairing. Principal component and allele-specific analyses reveal that genetics primarily shapes the heavy chain repertoire, while selection more strongly influences light chain diversity. These findings define distinct dynamics of antibody repertoire development and underscore the importance of personal immunogenomics in understanding individual immune responses.

genomics↗

bioRxiv2025

The human immunoglobulin heavy chain constant gene locus is enriched for large complex structural variants and coding polymorphisms that vary in frequency among human populations

The human immunoglobulin heavy chain constant (IGHC) domain of antibodies (Ab) is responsible for effector functions critical to immunity. This domain is encoded by genes in the IGHC locus, where descriptions of genomic diversity remain incomplete. We utilized long-read sequencing to build an IGHC haplotype/variant catalog from 105 individuals of diverse ancestry. We discovered uncharacterized single nucleotide variants (SNV) and large structural variants (SVs, n=7), representing new genes and alleles enriched for non-synonymous substitutions, highlighting potential functional effects. Of the 221 identified IGHC alleles, 192 were novel. SNV, SV, and gene allele/genotype frequencies revealed population differentiation, including (i) hundreds of SNVs in African and East Asian populations exceeding a fixation index (FST) of 0.3, and (ii) an IGHG4 haplotype carrying coding variants uniquely enriched in Asian populations. Our results illuminate missing signatures of IGHC diversity and establish a new foundation for investigating IGHC germline variation in Ab function and disease.

genomics↗

bioRxiv2025

A Broad Survey and Functional Analysis of Immunoglobulin Loci Variation in Rhesus Macaques

Rhesus macaques (RMs) are a vital model for studying human disease and invaluable to pre-clinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed novel experimental and computational methodologies to generate the largest collection to date of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian origin RMs. We created a comprehensive resource of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs), including the curation of 1474 novel alleles, unveiling tremendous diversity, and expanding existing IG allele sets by 60%. This publicly available, continually updated resource (https://vdjbase.org/reference_book/Rhesus_Macaque) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

immunology↗

bioRxiv2024

Diverse priming outcomes under conditions of very rare precursor B cells

Rare B cells can have special pathogen-recognition features giving them the potential to make outsized contributions to protective immunity. However, rare naive B cells infrequently participate in immune responses. We investigated how germline-targeting vaccine antigen delivery and adjuvant selection affect priming of exceptionally rare BG18-like HIV broadly neutralizing antibody-precursor B cells (~1 in 50 million) in non-human primates. Only escalating dose (ED) priming immunization using the saponin adjuvant SMNP elicited detectable BG18-like cells in germinal centers (GCs). All groups had strong GC responses, but only ED+SMNP and bolus+SMNP induced BG18-like memory B cells in >50% of animals. One group had vaccine-specific GC responses equivalent to ED+SMNP, but BG18-like memory B cells were rarely detected. Following homologous boosting, BG18-like memory B cells were more frequent in a bolus priming group, but had lower somatic hypermutation and affinities. This outcome was inversely associated with post-prime antibody titers, suggesting antibody feedback can significantly influence rare precursor B cell responses.

immunology↗

bioRxiv2024

Addressing technical pitfalls in pursuit of molecular factors that mediate immunoglobulin gene regulation

The expressed antibody repertoire is a critical determinant of immune-related phenotypes. Antibody-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human antibodies are composed of two identical heavy and light chain polypeptides derived from genes in the immunoglobulin heavy chain (IGH) locus and one of two light chain loci. The combinatorial diversity that results from antibody gene rearrangement and the pairing of different heavy and light chains contributes to the immense diversity of the baseline antibody repertoire. During rearrangement, antibody gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the antibody repertoire associates with germline variation in IGH, implicating polymorphism in antibody gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. Here, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype-resolution. Using simulated and ChIP-seq reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed antibody repertoires.

genomics↗

bioRxiv2023

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of IG loci has hindered effective use of standard high- throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n=36), representing the first comprehensive description of IGK haplotype variation at population-scale. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and a common novel [~]24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 of the IGKV genes, including alleles unique to specific populations within this dataset. Finally, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

immunology↗

bioRxiv2023

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), has been shown to be of critical importance for immune responses to pathogens and vaccines. In recent years, B cell and T cell receptor repertoire sequencing (Rep-Seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci in different populations. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference and genotyping allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput genotyping and novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software (https://mixcr.com) and can be incorporated into any pipeline utilizing upstream processing with MiXCR. We demonstrate the accuracy of this approach using Rep-Seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) Rep-Seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) Rep-Seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity of genes within the IGH, TRA and TRB loci in different populations and demonstrate the connection between antibody repertoire gene usage and the number of allelic variants present in the population. Finally we established a database of allelic variants of V and J genes inferred from Rep-Seq data and their population frequencies with free public access at https://vdj.online.

immunology↗

bioRxiv2023

AIRR-C Human IG Reference Sets: curated sets of immunoglobulin heavy and light chain germline genes

Analysis of an individuals immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene Reference Sets. The Adaptive Immune Receptor Repertoire-Community (AIRR-C) Reference Sets have been developed to include only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. By including only those alleles with a high level of support, including some new sequences that currently lack official names, AIRR-seq analysis will have greater accuracy and studies of the evolution of immunoglobulin genes, their allelic variants and the expressed immune repertoire will be facilitated. Although containing less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), the Reference Sets eliminated erroneous calls and provided excellent coverage when tested on a set of repertoires from 99 individuals comprising over 4 million V(D)J rearrangements. To improve AIRR-seq analysis, some alleles have been extended to deal with short 3 or 5 truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata. The Reference Sets also include novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. The version-tracked AIRR-C Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly-observed and previously-reported sequences that can be confirmed by new high-quality data.

genomics↗

bioRxiv2022

IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data

In adaptive immune receptor repertoire analysis, determining the germline variable (V) allele associated with each T- and B-cell receptor sequence is a crucial step. This process is highly impacted by allele annotations. Aligning sequences, assigning them to specific germline alleles, and inferring individual genotypes are challenging when the repertoire is highly mutated, or sequence reads do not cover the whole V region. Here, we propose an alternative naming scheme for the V alleles as well as a novel method to infer individual genotypes. We demonstrate the strength of the two by comparing their outcomes to other genotype inference methods and validated the genotype approach with independent genomic long read data. The naming scheme is compatible with current annotation tools and pipelines. Analysis results can be converted from the proposed naming scheme to the nomenclature determined by the International Union of Immunological Societies (IUIS). Both the naming scheme and the genotype procedure are implemented in a freely available R package (PIgLET). To allow researchers to explore further the approach on real data and to adapt it for their future uses, we also created an interactive website (https://yaarilab.github.io/IGHV_reference_book).

immunology↗

bioRxiv2022

FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires

Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve expressed antibody (Ab) transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted amplification by 5 rapid amplification of cDNA ends (RACE), combined with single molecule, real-time sequencing to generate highly accurate (>Q40, 99.99%) IG heavy chain transcripts. FLAIRR-seq was benchmarked by comparing IG heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) gene usage, complementarity-determining region 3 (CDR3) length, and somatic hypermutation to matched datasets generated with standard 5 RACE AIRR-seq and full-length isoform sequencing. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving novel IG heavy chain constant (IGHC) gene features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class-switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk expressed Ab repertoires to date.

immunology↗

bioRxiv2022

Characterization of the immunoglobulin lambda chain locus from diverse populations reveals extensive genetic variation

Immunoglobulins (IGs), crucial components of the adaptive immune system, are encoded by three genomic loci. However, the complexity of the IG loci severely limits the effective use of short read sequencing, limiting our knowledge of population diversity in these loci. We leveraged existing long read whole-genome sequencing (WGS) data, fosmid technology, and IG targeted single-molecule, real-time (SMRT) long-read sequencing (IG-Cap) to create haplotype-resolved assemblies of the IG Lambda (IGL) locus from 6 ethnically diverse individuals. In addition, we generated 10 diploid assemblies of IGL from a diverse cohort of individuals utilizing IG-cap. From these 16 individuals, we identified significant allelic diversity, including 37 novel IGLV alleles. In addition, we observed highly elevated single nucleotide variation (SNV) in IGLV genes relative to IGL intergenic and genomic background SNV density. By comparing SNV calls between our high quality assemblies and existing short read datasets from the same individuals, we show a high propensity for false-positives in the short read datasets. Finally, for the first time, we nucleotide-resolved common 5-10 Kb duplications in the IGLC region that contain functional IGLJ and IGLC genes. Together these data represent a significant advancement in our understanding of genetic variation and population diversity in the IGL locus.

genetics↗

bioRxiv2022

Antibody repertoire gene usage is explained by common genetic variants in the immunoglobulin heavy chain locus

Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) significantly impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function and dynamics of the antibody response in disease.

genomics↗

bioRxiv2022

Targeted long-read sequencing facilitates phased diploid assembly and genotyping of the human T cell receptor alpha, delta and beta loci

T cell receptors (TCRs) recognize peptide fragments presented by the major histocompatibility complex (MHC) and are critical to T cell mediated immunity. Early studies demonstrated an enrichment of polymorphisms within TCR-encoding (TR) gene loci. However, more recent data indicate that variation in these loci are underexplored, limiting understanding of the impact of TR polymorphism on TCR function in disease, even though: (i) TCR repertoire signatures are heritable and (ii) associate with disease phenotypes. TR variant discovery and curation has been difficult using standard high-throughput methods. To address this, we expanded our published targeted long-read sequencing approach to generate highly accurate haplotype resolved assemblies of the human TR beta (TRB) and alpha/delta (TRA/D) loci, facilitating the detection and genotyping of single nucleotide polymorphisms (SNPs), insertion-deletions (indels), structural variants (SVs) and TR genes. We validate our approach using two mother-father-child trios and 5 unrelated donors representing multiple populations. Comparisons of long-read derived variants to short-read datasets revealed improved genotyping accuracy, and TR gene annotation led to the discovery of 79 previously undocumented V, D, and J alleles. This demonstrates the utility of this framework to resolve the TR loci, and ultimately our understanding of TCR function in disease.

genomics↗

bioRxiv2021

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n=2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions. Author summaryLymphoblastoid cell lines (LCLs) are cells that have been manipulated to proliferate indefinitely in order to provide a replenishable source of DNA. However, because these cell lines are derived from B cells which have undergone V(D)J recombination they contain somatic deletions within regions of the genome that encode antibody genes. Although several large collaborative projects have utilized DNA from LCLs to generate invaluable genomic resources for the scientific community, the negative impacts of cell line artifacts in these regions of the genome have not been fully appreciated. In this study, we used newly released sequencing data from a large collection of LCLs to determine that the non-inherited artificial deletions within the antibody gene loci can have detrimental effects on downstream genetic analyses.

genomics↗

bioRxiv2020

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

An incomplete ascertainment of genetic variation within the highly polymorphic immunoglobulin heavy chain locus (IGH) has hindered our ability to define genetic factors that influence antibody and B cell mediated processes. To date, methods for locus-wide genotyping of all IGH variant types do not exist. Here, we combine targeted long-read sequencing with a novel bioinformatics tool, IGenotyper, to fully characterize genetic variation within IGH in a haplotype-specific manner. We apply this approach to eight human samples, including a haploid cell line and two mother-father-child trios, and demonstrate the ability to generate high-quality assemblies (>98% complete and >99% accurate), genotypes, and gene annotations, including 2 novel structural variants and 16 novel gene alleles. We show that multiplexing allows for scaling of the approach without impacting data quality, and that our genotype call sets are more accurate than short-read (>35% increase in true positives and >97% decrease in false-positives) and array/imputation-based datasets. This framework establishes a foundation for leveraging IG genomic data to study population-level variation in the antibody response.

genomics↗

bioRxiv2019

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls [≥]50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

genomics↗

bioRxiv2018

Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance

The observation that humans can produce broadly neutralizing antibodies (bnAbs) against HIV-1 has generated enthusiasm about the potential for a bnAb vaccine against HIV-1. Conventional immunization strategies will likely be insufficient for the development of a bnAb HIV vaccine and vaccines to other difficult pathogens, due to the significant immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. Using longitudinal lymph node fine needle aspirates, we found that two independent methods of slow delivery immunization of rhesus macaques (RM) resulted in larger GCs, more robust and sustained GC Tfh cell responses, and GC B cells with improved Env-binding, which correlated with the development of ~20 to 30-fold higher titers of tier 2 HIV-1 nAbs. Using a new RM genomic immunoglobulin loci reference sequence, we identified differential IgV gene usage between slow delivery immunized and conventional bolus immunized animals. The most immunodominant IgV gene used by conventionally immunized animals was associated with many GC B cell lineages. Ab mapping of those GC B cell specificities demonstrated targeting of an immunodominant non-neutralizing trimer base epitope, while that response was muted in slow delivery immunized animals. Thus, alternative immunization strategies appear to enhance nAb development by altering GCs and modulating immunodominance of non-neutralizing epitopes.

immunology↗