Biology subjects

Watson, C. T.

Publications and source records attributed to Watson, C. T..

30 records · Page 2Linked to original sources

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↗

bioRxiv2022

Characterization of Extensive Diversity In Immunoglobulin Light Chain Variable Germline Genes Across Biomedically Important Mouse Strains

The light chain immunoglobulin genes of biomedically relevant mouse strains are poorly documented in current germline gene databases. We previously showed that IGH loci of wild-derived mouse strains representing the major mouse subspecies contained 247 germline IGHV sequences not curated in the international ImMunoGeneTics (IMGT) information system, which is the most commonly used database that curates the germline repertoires used for sequence alignment in AIRR-seq analysis. Despite containing levels of polymorphism similar to the IGH locus, the germline gene content and diversity of the light chain loci have not been comprehensively cataloged. To explore the extent of germline light chain repertoire diversity across mouse strains commonly used in the biomedical sciences, we performed AIRR-seq analysis and germline gene inference for 18 inbred mouse strains, including the four wild-derived strains with diverse sub-species origins. We inferred 1582 IGKV and 63 IGLV sequences, representing 459 and 22 unique IGKV and IGLV sequences. Of the unique inferred germline IGKV and IGLV sequences, 67.8% and 59%, respectively, were undocumented in IMGT. Across strains we observed germline IGKV sequences shared by three distinct IGK haplotypes and a more conserved IGLV germline repertoire. In addition, J gene inference indicated a novel IGK2 allele shared between PWD/PhJ and MSM/MsJ and a novel IGLJ1 allele for LEWES/EiJ and IGLJ2 allele for MSM/MsJ. Finally, a combined IGHV, IGKV, and IGLV phylogenetic analysis of wild-derived germline repertoires displayed reduced germline diversity for the light chain repertoire compared to the heavy chain repertoire, suggesting potential evolutionary differences between the two chains.

immunology↗

bioRxiv2022

A BALB/c IGHV Reference Set, defined by haplotype analysis of long-read VDJ-C sequences from F1 (BALB/c / C57BL/6) mice

The immunoglobulin genes of inbred mouse strains that are commonly used in models of antibody-mediated human diseases are poorly characterized. This compromises data analysis. To infer the immunoglobulin genes of BALB/c mice, we used long-read SMRT sequencing to amplify VDJ-C sequences from F1 (BALB/c x C57BL/6) hybrid animals. Previously unreported strain variations were identified in the Ighm and Ighg2b genes, and analysis of VDJ rearrangements led to the inference of 278 germline IGHV alleles. 169 alleles are not present in the C57BL/6 genome reference sequence. To establish a set of expressed BALB/c IGHV germline gene sequences, we computationally retrieved IGHV haplotypes from the IgM dataset. Haplotyping led to the confirmation of 162 BALB/c IGHV gene sequences. A musIGHV398 pseudogene variant also appears to be present in the BALB/cByJ substrain, while a functional musIGHV398 gene is highly expressed in the BALB/cJ substrain. Only four of the BALB/c alleles were also observed in the C57BL/6 haplotype. The full set of inferred BALB/c sequences has been used to establish a BALB/c IGHV reference set, hosted at https://ogrdb.airr-community.org. We assessed whether assemblies from the Mouse Genome Project (MGP) are suitable for the determination of the genes of the IGH loci. Only 37 (43.5%) of the 85 confirmed IMGT-named BALB/c IGHV and 33 (42.9%) of the 77 confirmed non-IMGT IGHV were found in a search of the MGP BALB/cJ genome assembly. This suggests that Adaptive Immune Receptor Repertoire sequencing (AIRR-Seq) data, but not currently-available genome assemblies, are suited to the documentation of germline IGHV genes.

immunology↗

bioRxiv2021

Revealing how variations in antibody repertoires correlate with vaccine responses

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold a key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated dataset, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study revealed that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

immunology↗

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↗

bioRxiv2021

T Cell Receptor Beta (TRB) Germline Variability is Revealed by Inference From Repertoire Data

BackgroundT and B cell receptor (TCR, BCR) repertoires constitute the foundation of adaptive immunity. Adaptive immune receptor repertoire sequencing (AIRR-seq) is a common approach to study immune system dynamics. Understanding the genetic factors influencing the composition and dynamics of these repertoires is of major scientific and clinical importance. The chromosomal loci encoding for the variable regions of TCRs and BCRs are challenging to decipher due to repetitive elements and undocumented structural variants. MethodsTo confront this challenge, AIRR-seq-based methods have recently been developed for B cells, enabling genotype and haplotype inference and discovery of undocumented alleles. However, this approach relies on complete coverage of the receptors variable regions, whereas most T cell studies sequence a small fraction of that region. Here, we adapted a B cell pipeline for undocumented alleles, genotype, and haplotype inference for full and partial TCR sequences. The pipeline also deals with gene assignment ambiguities, which is especially important in the analysis of data-sets of partial sequences. ResultsWe identified 39 undocumented polymorphisms in T cell receptor Beta V (TRBV) and 31 undocumented 5 UTR sequences. A subset of these inferences was also observed using independent genomic approaches. We found that a single nucleotide polymorphism differentiating between the two documented T cell receptor Beta D2 (TRBD2) alleles is strongly associated with dramatic changes in the expressed repertoire. ConclusionsWe reveal a rich picture of germline variability, and demonstrate how a single nucleotide polymorphism dramatically affects the composition of the whole repertoire. Our findings provide a basis for annotation of TCR repertoires for future basic and clinical studies.

immunology↗

bioRxiv2021

Vaccine genetics of IGHV1-2 VRC01-class broadly neutralizing antibody precursor naive human B cells

A successful HIV vaccine must overcome the hurdle of being able to activate naive precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether broadly neutralizing antibody (bnAb) precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naive VRC01-class B cells. The results indicate that IGHV1-2 alleles incompatible with VRC01-class responses are relatively common in various human populations, and germline variation within IGHV1-2 associates with gene usage frequencies in the naive BCR repertoire.

immunology↗

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

Germline murine immunoglobulin IGHV genes in wild-derived and classical inbred strains: a comparison

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflect differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ), and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ, and PWD/PhJ), and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the International Immunogenetics Information System. In contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. In contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand inter-strain variation in models of antibody-mediated disease.

immunology↗

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↗

bioRxiv2018

Identification of rare de novo epigenetic variations in congenital disorders

Certain human traits such as neurodevelopmental disorders (NDs) and congenital anomalies (CAs) are believed to be primarily genetic in origin. With recent dramatic advances in genomic technologies, genome-wide surveys of cohorts of patients with ND/CAs for point mutations and structural variations have greatly advanced our understanding of their genetic etiologies1,2. However, even after whole genome sequencing (WGS), a substantial fraction of such disorders remain unexplained3. In contrast, the possibility that constitutive epigenetic variations (epivariations) might underlie such traits has not been well explored. We hypothesized that some cases of ND/CA are caused by aberrations of DNA methylation that lead to a dysregulation of normal genome function. By comparing DNA methylation profiles from 489 individuals with ND/CAs against 1,534 population controls, we identified epivariations as a frequent occurrence in the human genome. De novo epivariations were significantly enriched in cases when compared to controls. RNAseq data from population studies showed that epivariations often have an impact on gene expression comparable to loss-of-function mutations. Additionally, we detected and replicated an enrichment of rare sequence mutations overlapping CTCF binding sites close to epivariations. Thus, some epivariations occur secondary to cis-linked mutations in regulatory regions, providing a rationale for interpreting non-coding genetic variation. We propose that epivariations likely represent the causative genomic defect in 5-10% of patients with unexplained ND/CAs. This constitutes a yield comparable to CNV microarrays, and as such has significant diagnostic relevance.

genomics↗