ABSTRACT
Adaptive immune recognition is mediated by antigen receptors on B and T cells generated by somatic recombination during lineage development. The high level of diversity resulting from this process posed technical limitations that previously limited the comprehensive analysis of adaptive immune recognition. Advances over the last ten years have produced data and approaches allowing insights into how T cells develop, evolutionary signatures of recombination and selection, and the features of T cell receptors that mediate epitope-specific binding and T cell activation. The size and complexity of these data have necessitated the generation of novel computational and analytical approaches, which are transforming how T cell immunology is conducted. Here we review the development and application of novel biological, theoretical, and computational methods for understanding T cell recognition and discuss the potential for improved models of receptor:antigen interactions.
Subject(s)
Computational Biology/methods , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , Adaptive Immunity , Animals , Antigens/immunology , Antigens/metabolism , Cell Differentiation , Clonal Selection, Antigen-Mediated , Epitopes, T-Lymphocyte/metabolism , High-Throughput Nucleotide Sequencing , Humans , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolismABSTRACT
Genome technologies have defined a complex genetic architecture in major infectious, inflammatory, and autoimmune disorders. High density marker arrays and Immunochips have powered genome-wide association studies (GWAS) that have mapped nearly 450 genetic risk loci in 22 major inflammatory diseases, including a core of common genes that play a central role in pathological inflammation. Whole-exome and whole-genome sequencing have identified more than 265 genes in which mutations cause primary immunodeficiencies and rare forms of severe inflammatory bowel disease. Combined analysis of inflammatory disease GWAS and primary immunodeficiencies point to shared proteins and pathways that are required for immune cell development and protection against infections and are also associated with pathological inflammation. Finally, sequencing of chromatin immunoprecipitates containing specific transcription factors, with parallel RNA sequencing, has charted epigenetic regulation of gene expression by proinflammatory transcription factors in immune cells, providing complementary information to characterize morbid genes at infectious and inflammatory disease loci.
Subject(s)
Autoimmune Diseases/genetics , Immunologic Deficiency Syndromes/genetics , Infections/genetics , Inflammation/genetics , Vaccines/immunology , Animals , Epigenesis, Genetic , Exome/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Genome-Wide Association Study , High-Throughput Nucleotide Sequencing , Humans , Immunity/genetics , Infections/immunology , RiskABSTRACT
Transcriptomics, the high-throughput characterization of RNAs, has been instrumental in defining pathogenic signatures in human autoimmunity and autoinflammation. It enabled the identification of new therapeutic targets in IFN-, IL-1- and IL-17-mediated diseases. Applied to immunomonitoring, transcriptomics is starting to unravel diagnostic and prognostic signatures that stratify patients, track molecular changes associated with disease activity, define personalized treatment strategies, and generally inform clinical practice. Herein, we review the use of transcriptomics to define mechanistic, diagnostic, and predictive signatures in human autoimmunity and autoinflammation. We discuss some of the analytical approaches applied to extract biological knowledge from high-dimensional data sets. Finally, we touch upon emerging applications of transcriptomics to study eQTLs, B and T cell repertoire diversity, and isoform usage.
Subject(s)
Autoimmune Diseases/diagnosis , Inflammation/diagnosis , Transcriptome , Autoimmune Diseases/immunology , Datasets as Topic , High-Throughput Nucleotide Sequencing , Humans , Inflammation/immunology , Information Storage and Retrieval , Molecular Targeted Therapy , Monitoring, Immunologic , PrognosisABSTRACT
Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.
Subject(s)
CTLA-4 Antigen/genetics , Cation Transport Proteins/genetics , Genomics , Immune System Diseases/genetics , Mutation/genetics , Phosphatidylinositol 3-Kinases/genetics , X-Linked Combined Immunodeficiency Diseases/genetics , Animals , High-Throughput Nucleotide Sequencing , Humans , Immune System Diseases/therapy , Male , Molecular Targeted Therapy , X-Linked Combined Immunodeficiency Diseases/therapyABSTRACT
The gut microbiome modulates immune and metabolic health. Human microbiome data are biased toward industrialized populations, limiting our understanding of non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing on 351 fecal samples from the Hadza hunter-gatherers of Tanzania and comparative populations in Nepal and California. We recovered 91,662 genomes of bacteria, archaea, bacteriophages, and eukaryotes, 44% of which are absent from existing unified datasets. We identified 124 gut-resident species vanishing in industrialized populations and highlighted distinct aspects of the Hadza gut microbiome related to in situ replication rates, signatures of selection, and strain sharing. Industrialized gut microbes were found to be enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource, expands our understanding of microbes capable of colonizing the human gut, and clarifies the extensive perturbation induced by the industrialized lifestyle.
Subject(s)
Gastrointestinal Microbiome , Microbiota , Humans , Gastrointestinal Microbiome/genetics , Metagenome , Eukaryota , High-Throughput Nucleotide Sequencing , MetagenomicsABSTRACT
Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.
Subject(s)
Schizophrenia , Humans , Genetic Predisposition to Disease , Genome-Wide Association Study , Neurons/metabolism , Polymorphism, Single Nucleotide/genetics , Schizophrenia/genetics , Animals , Mice , High-Throughput Nucleotide SequencingABSTRACT
The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.
Subject(s)
Genome, Human , Whole Genome Sequencing , Female , High-Throughput Nucleotide Sequencing/methods , Humans , INDEL Mutation , Male , Polymorphism, Single NucleotideABSTRACT
In most sensory modalities, neuronal connectivity reflects behaviorally relevant stimulus features, such as spatial location, orientation, and sound frequency. By contrast, the prevailing view in the olfactory cortex, based on the reconstruction of dozens of neurons, is that connectivity is random. Here, we used high-throughput sequencing-based neuroanatomical techniques to analyze the projections of 5,309 mouse olfactory bulb and 30,433 piriform cortex output neurons at single-cell resolution. Surprisingly, statistical analysis of this much larger dataset revealed that the olfactory cortex connectivity is spatially structured. Single olfactory bulb neurons targeting a particular location along the anterior-posterior axis of piriform cortex also project to matched, functionally distinct, extra-piriform targets. Moreover, single neurons from the targeted piriform locus also project to the same matched extra-piriform targets, forming triadic circuit motifs. Thus, as in other sensory modalities, olfactory information is routed at early stages of processing to functionally diverse targets in a coordinated manner.
Subject(s)
Olfactory Cortex , Olfactory Pathways , Mice , Animals , Olfactory Bulb , Neurons/physiology , High-Throughput Nucleotide SequencingABSTRACT
Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.
Subject(s)
Genomics , High-Throughput Nucleotide Sequencing , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Cohort Studies , Female , Humans , Male , Organ Specificity/genetics , Prospective StudiesABSTRACT
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
Subject(s)
Gene Editing , Genetic Variation , High-Throughput Nucleotide Sequencing , Alleles , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Base Sequence , Catalytic Domain , Cell Line, Tumor , Humans , Loss of Function Mutation , Mutagenesis/genetics , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Point Mutation/genetics , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Reproducibility of Results , Selection, Genetic , bcl-X Protein/geneticsABSTRACT
Targeting cancer neoantigens generated by tumor-exclusive somatic mutations is an attractive yet challenging strategy for the robust and specific elimination of tumor cells by cellular immunotherapy. In this issue of Cell, Wells et al. describe a consortium-based approach to optimize bioinformatics pipelines to sensitively and accurately predict immunogenic neoantigens from next-generation sequencing data.
Subject(s)
Antigens, Neoplasm , Neoplasms , Antigens, Neoplasm/genetics , Epitopes , High-Throughput Nucleotide Sequencing , Humans , Immunotherapy , Neoplasms/genetics , Neoplasms/therapyABSTRACT
The generation of functional genomics datasets is surging, because they provide insight into gene regulation and organismal phenotypes (e.g., genes upregulated in cancer). The intent behind functional genomics experiments is not necessarily to study genetic variants, yet they pose privacy concerns due to their use of next-generation sequencing. Moreover, there is a great incentive to broadly share raw reads for better statistical power and general research reproducibility. Thus, we need new modes of sharing beyond traditional controlled-access models. Here, we develop a data-sanitization procedure allowing raw functional genomics reads to be shared while minimizing privacy leakage, enabling principled privacy-utility trade-offs. Our protocol works with traditional Illumina-based assays and newer technologies such as 10x single-cell RNA sequencing. It involves quantifying the privacy leakage in reads by statistically linking study participants to known individuals. We carried out these linkages using data from highly accurate reference genomes and more realistic environmental samples.
Subject(s)
Computer Security , Genomics , Privacy , Genome, Human , Genotype , High-Throughput Nucleotide Sequencing , Humans , Phenotype , Phylogeny , Reproducibility of Results , Sequence Analysis, RNA , Single-Cell AnalysisABSTRACT
The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , B-Lymphocytes/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Single-Cell Analysis , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , COVID-19 , Convalescence , High-Throughput Nucleotide Sequencing , Humans , Mice , Pandemics , Sequence Analysis, RNA , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , VDJ ExonsABSTRACT
Several general principles of global 3D genome organization have recently been established, including non-random positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, we systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intra-chromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. Our observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Chromatin/genetics , Genome Components/physiology , Cell Line , Cell Nucleus/genetics , Chromosomes , Fibroblasts/physiology , Genome/genetics , Genome Components/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Single-Cell AnalysisABSTRACT
The introduction of exome sequencing in the clinic has sparked tremendous optimism for the future of rare disease diagnosis, and there is exciting opportunity to further leverage these advances. To provide diagnostic clarity to all of these patients, however, there is a critical need for the field to develop and implement strategies to understand the mechanisms underlying all rare diseases and translate these to clinical care.
Subject(s)
Exome Sequencing/trends , Rare Diseases/diagnosis , Translational Research, Biomedical/methods , Exome , Genetic Testing , Genome, Human/genetics , High-Throughput Nucleotide Sequencing/trends , Humans , Rare Diseases/genetics , Sequence Analysis, DNA/methods , Exome Sequencing/methodsABSTRACT
Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
Subject(s)
DNA, Mitochondrial/genetics , Mitochondria/genetics , Base Sequence , Cell Lineage , Chromatin , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Genomics/methods , HEK293 Cells , Hematopoietic Stem Cells/physiology , High-Throughput Nucleotide Sequencing/methods , Humans , Mutation , Single-Cell Analysis , TransposasesABSTRACT
Affordable genome sequencing technologies promise to revolutionize the field of human genetics by enabling comprehensive studies that interrogate all classes of genome variation, genome-wide, across the entire allele frequency spectrum. Ongoing projects worldwide are sequencing many thousands-and soon millions-of human genomes as part of various gene mapping studies, biobanking efforts, and clinical programs. However, while genome sequencing data production has become routine, genome analysis and interpretation remain challenging endeavors with many limitations and caveats. Here, we review the current state of technologies for genetic variant discovery, genotyping, and functional interpretation and discuss the prospects for future advances. We focus on germline variants discovered by whole-genome sequencing, genome-wide functional genomic approaches for predicting and measuring variant functional effects, and implications for studies of common and rare human disease.
Subject(s)
Genetic Variation/genetics , Genome, Human/genetics , Sequence Analysis, DNA/trends , Biological Specimen Banks , Chromosome Mapping/methods , Genetic Predisposition to Disease/genetics , Genetic Testing/trends , Genome-Wide Association Study , Genomics/methods , Genomics/trends , High-Throughput Nucleotide Sequencing/methods , Human Genome Project , Humans , Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA/methods , Whole Genome Sequencing/methods , Whole Genome Sequencing/trendsABSTRACT
Circular RNAs (circRNAs) are an intriguing class of RNA due to their covalently closed structure, high stability, and implicated roles in gene regulation. Here, we used an exome capture RNA sequencing protocol to detect and characterize circRNAs across >2,000 cancer samples. When compared against Ribo-Zero and RNase R, capture sequencing significantly enhanced the enrichment of circRNAs and preserved accurate circular-to-linear ratios. Using capture sequencing, we built the most comprehensive catalog of circRNA species to date: MiOncoCirc, the first database to be composed primarily of circRNAs directly detected in tumor tissues. Using MiOncoCirc, we identified candidate circRNAs to serve as biomarkers for prostate cancer and were able to detect circRNAs in urine. We further detected a novel class of circular transcripts, termed read-through circRNAs, that involved exons originating from different genes. MiOncoCirc will serve as a valuable resource for the development of circRNAs as diagnostic or therapeutic targets across cancer types.
Subject(s)
Gene Expression Profiling/methods , Neoplasms/genetics , RNA/genetics , Biomarkers, Tumor/genetics , Databases, Genetic , Gene Expression Regulation, Neoplastic/genetics , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/genetics , RNA/metabolism , RNA, Circular , Sequence Analysis, RNA/methods , Exome Sequencing/methodsABSTRACT
Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in â¼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.
Subject(s)
Epigenomics/methods , Gene Regulatory Networks/genetics , Single-Cell Analysis/methods , Chromatin/genetics , Chromatin/metabolism , Chromatin Assembly and Disassembly/physiology , Clustered Regularly Interspaced Short Palindromic Repeats/physiology , Gene Regulatory Networks/physiology , High-Throughput Nucleotide Sequencing/methods , Humans , Sequence Analysis, DNA/methods , Transcription Factors/metabolismABSTRACT
Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.