A harmonized public resource of deeply sequenced diverse human genomes.

Underrepresented populations are often excluded from genomic studies due in part to a lack of resources supporting their analyses. The 1000 Genomes Project (1kGP) and Human Genome Diversity Project (HGDP), which have recently been sequenced to high coverage, are valuable genomic resources because of the global diversity they capture and their open data sharing policies. Here, we harmonized a high quality set of 4,094 whole genomes from HGDP and 1kGP with data from the Genome Aggregation Database (gnomAD) and identified over 153 million high-quality SNVs, indels, and SVs. We performed a detailed ancestry analysis of this cohort, characterizing population structure and patterns of admixture across populations, analyzing site frequency spectra, and measuring variant counts at global and subcontinental levels. We also demonstrate substantial added value from this dataset compared to the prior versions of the component resources, typically combined via liftover and variant intersection; for example, we catalog millions of new genetic variants, mostly rare, compared to previous releases. In addition to unrestricted individual-level public release, we provide detailed tutorials for conducting many of the most common quality control steps and analyses with these data in a scalable cloud-computing environment and publicly release this new phased joint callset for use as a haplotype resource in phasing and imputation pipelines. This jointly called reference panel will serve as a key resource to support research of diverse ancestry populations.

Systematic evaluation of genome sequencing for the diagnostic assessment of autism spectrum disorder and fetal structural anomalies.

Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.

Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens.

Most variants associated with complex traits and diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown effects. Using ancestrally diverse, biobank-scale GWAS data, massively parallel CRISPR screens, and single-cell transcriptomic and proteomic sequencing, we discovered 124 cis-target genes of 91 noncoding blood trait GWAS loci. Using precise variant insertion through base editing, we connected specific variants with gene expression changes. We also identified trans-effect networks of noncoding loci when cis target genes encoded transcription factors or microRNAs. Networks were themselves enriched for GWAS variants and demonstrated polygenic contributions to complex traits. This platform enables massively parallel characterization of the target genes and mechanisms of human noncoding variants in both cis and trans.

Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution.

Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility and other factors. Current methods for defining chromatin states cannot measure more than one aspect in a single experiment at single-cell resolution. Here we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), an assay capable of measuring the genome-wide presence of up to three histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq uses recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk-cell and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in human immune cells. We also extend NTT-seq to enable simultaneous profiling of cell surface protein expression and multimodal chromatin states to study cells of the immune system.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFß activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Rare coding variation provides insight into the genetic architecture and phenotypic context of autism.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.

A genome-scale screen for synthetic drivers of T cell proliferation.

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.

Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells.

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

Integrated analysis of multimodal single-cell data.

The simultaneous measurement of multiple modalities represents an exciting frontier for single-cell genomics and necessitates computational methods that can define cellular states based on multimodal data. Here, we introduce "weighted-nearest neighbor" analysis, an unsupervised framework to learn the relative utility of each data type in each cell, enabling an integrative analysis of multiple modalities. We apply our procedure to a CITE-seq dataset of 211,000 human peripheral blood mononuclear cells (PBMCs) with panels extending to 228 antibodies to construct a multimodal reference atlas of the circulating immune system. Multimodal analysis substantially improves our ability to resolve cell states, allowing us to identify and validate previously unreported lymphoid subpopulations. Moreover, we demonstrate how to leverage this reference to rapidly map new datasets and to interpret immune responses to vaccination and coronavirus disease 2019 (COVID-19). Our approach represents a broadly applicable strategy to analyze single-cell multimodal datasets and to look beyond the transcriptome toward a unified and multimodal definition of cellular identity.

Metagenomic next-generation sequencing of rectal swabs for the surveillance of antimicrobial-resistant organisms on the Illumina Miseq and Oxford MinION platforms.

Antimicrobial resistance (AMR) is a public health threat where efficient surveillance is needed to prevent outbreaks. Existing methods for detection of gastrointestinal colonization of multidrug-resistant organisms (MDRO) are limited to specific organisms or resistance mechanisms. Metagenomic next-generation sequencing (mNGS) is a more rapid and agnostic diagnostic approach for microbiome and resistome investigations. We determined if mNGS can detect MDRO from rectal swabs in concordance with standard microbiology results. We performed and compared mNGS performance on short-read Illumina MiSeq (N = 10) and long-read Nanopore MinION (N = 5) platforms directly from rectal swabs to detect vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative organisms (CRO). We detected Enterococcus faecium (N = 8) and Enterococcus faecalis (N = 2) with associated van genes (9/10) in concordance with VRE culture-based results. We studied the microbiome and identified CRO, Pseudomonas aeruginosa (N = 1), Enterobacter cloacae (N = 1), and KPC-producing Klebsiella pneumoniae (N = 1). Nanopore real-time analysis detected the blaKPC gene in 2.3 min and provided genetic context (blaKPC harbored on pKPC_Kp46 IncF plasmid). Illumina sequencing provided accurate allelic variant determination (i.e., blaKPC-2) and strain typing of the K. pneumoniae (ST-15). We demonstrated an agnostic approach for surveillance of MDRO, examining advantages of both short- and long-read mNGS methods for AMR detection.

Prediction of post-interventional physical function in diabetic foot ulcer patients using patient reported outcome measurement information system (PROMIS).

BACKGROUND: Infected diabetic foot ulcer (DFU) patients present with an impaired baseline physical function (PF) that can be further compromised by surgical intervention to treat the infection. The impact of surgical interventions on Patient Reported Outcomes Measurement Information System (PROMIS) PF within the DFU population has not been investigated. We hypothesize that preoperative PROMIS scores (PF, Pain Interference (PI), Depression) in combination with relevant clinical factors can be utilized to predict postoperative PF in DFU patients. METHODS: DFU patients from a single academic physician's practice between February 2015 and November 2018 were identified (n = 240). Ninety-two patients met inclusion criteria with complete follow-up and PROMIS computer adaptive testing records. Demographic and clinical factors, procedure performed, and wound healing status were collected. Spearman's rank correlation coefficient, Chi-Squared tests and multidimensional modelling were applied to all variables' pre- and postoperative values to assess patients' postoperative PF. RESULTS: The mean age was 60.5 (33-96) years and mean follow-up was 4.7 (3-12) months. Over 70 % of the patients' initial PF were 2-3 standard deviations below the US population (n = 49; 28). Preoperative PF (p < 0.01), PI (p < 0.01), Depression (p < 0.01), CRF (p < 0.02) and amputation level (p < 0.04) showed significant univariate correlation with postoperative PF. Multivariate model (r = 0.55) showed that the initial PF (p = 0.004), amputation level (p = 0.008), and wound healing status (p = 0.001) predicted postoperative PF. CONCLUSIONS: Majority of DFU patients present with poor baseline PF. Preoperative PROMIS scores (PF, PI, Depression) are predictive of postoperative PROMIS PF in DFU patients. Postoperative patient's physical function can be assessed by PFpostoperative = 29.42 + 0.34 (PFinitial) - 5.87 (Not Healed) - 2.63 (Amputation Category). This algorithm can serve as a valuable tool for predicting post-operative physical function and setting expectations.

Distinct vasculotropic versus osteotropic features of S. agalactiae versus S. aureus implant-associated bone infection in mice.

Osteomyelitis is a devastating complication of orthopaedic surgery and commonly caused by Staphylococcus aureus (S. aureus) and Group B Streptococcus (GBS, S. agalactiae). Clinically, S. aureus osteomyelitis is associated with local inflammation, abscesses, aggressive osteolysis, and septic implant loosening. In contrast, S. agalactiae orthopaedic infections generally involve soft tissue, with acute life-threatening vascular spread. While preclinical models that recapitulate the clinical features of S. aureus bone infection have proven useful for research, no animal models of S. agalactiae osteomyelitis exist. Here, we compared the pathology caused by these bacteria in an established murine model of implant-associated osteomyelitis. In vitro scanning electron microscopy and CFU quantification confirmed similar implant inocula for both pathogens (~105 CFU/pin). Assessment of mice at 14 days post-infection demonstrated increased S. aureus virulence, as S. agalactiae infected mice had significantly greater body weight, and fewer CFU on the implant and in bone and adjacent soft tissue (p < 0.05). X-ray, µCT, and histologic analyses showed that S. agalactiae induced significantly less osteolysis and implant loosening, and fewer large TRAP+ osteoclasts than S. aureus without inducing intraosseous abscess formation. Most notably, transmission electron microscopy revealed that although both bacteria are capable of digesting cortical bone, S. agalactiae have a predilection for colonizing blood vessels embedded within cortical bone while S. aureus primarily colonizes the osteocyte lacuno-canalicular network. This study establishes the first quantitative animal model of S. agalactiae osteomyelitis, and demonstrates a vasculotropic mode of S. agalactiae infection, in contrast to the osteotropic behavior of S. aureus osteomyelitis.

Species-Specific Immunoassay Aids Identification of Pathogen and Tracks Infectivity in Foot Infection.

BACKGROUND: Conventional bacterial cultures frequently fail to identify the dominant pathogen in polymicrobial foot infections, in which Staphylococcus aureus is the most common infecting pathogen. Previous work has shown that species-specific immunoassays may be able to identify the main pathogen in musculoskeletal infections. We sought to investigate the clinical applicability of a S. aureus immunoassay to accurately identify the infecting pathogen and monitor its infectivity longitudinally in foot infection. We hypothesized that this species-specific immunoassay could aid in the diagnosis of S. aureus and track the therapeutic response in foot infections. METHODS: From July 2015 to July 2019, 83 infected foot ulcer patients undergoing surgical intervention (debridement or amputation) were recruited and blood was drawn at 0, 4, 8, and 12 weeks. Whole blood was analyzed for S. aureus-specific serum antibodies (mix of historic and new antibodies) and plasmablasts were isolated and cultured to quantify titers of newly synthesized antibodies (NSAs). Anti-S. aureus antibody titers were compared with culture results to assess their concordance in identifying S. aureus as the pathogen. The NSA titer changes at follow-ups were compared with wound healing status to evaluate concordance between evolving host immune response and clinically resolving or relapsing infection. RESULTS: Analysis of serum for anti-S. aureus antibodies showed significantly increased titers of 3 different anti-S. aureus antibodies, IsdH (P = .037), ClfB (P = .025), and SCIN (P = .005), in S. aureus culture-positive patients compared with culture-negative patients. Comparative analysis of combining antigens for S. aureus infection diagnosis increased the concordance further. During follow-up, changes of NSA titers against a single or combination of S. aureus antigens significantly correlated with clinically resolving or recurring infection represented by wound healing status. CONCLUSION: In the management of foot infection, the use of S. aureus-specific immunoassay may aid in diagnosis of the dominant pathogen and monitoring of the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay could detect recurrent foot infection, which may guide a surgeon's decision to intervene. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Operative Intervention Does Not Change Pain Perception in Patients With Diabetic Foot Ulcers.

Researchers investigated pain perception in patients with diabetic foot ulcers (DFUs) by analyzing pre- and postoperative physical function (PF), pain interference (PI), and depression domains of the Patient-Reported Outcome Measurement Information System (PROMIS). They hypothesized that 1) because of painful diabetic peripheral neuropathy (DPN), a majority of patients with DFUs would have high PROMIS PI scores unchanged by operative intervention, and 2) the initially assessed PI, PF, and depression levels would be correlated with final outcomes. Seventy-five percent of patients with DFUs reported pain, most likely because of painful DPN. Those who reported high PI and low PF were likely to report depression. PF, PI, and depression levels were unchanged after operative intervention or healing of DFUs.

High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange.

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.

A framework for assessing 16S rRNA marker-gene survey data analysis methods using mixtures.

BACKGROUND: There are a variety of bioinformatic pipelines and downstream analysis methods for analyzing 16S rRNA marker-gene surveys. However, appropriate assessment datasets and metrics are needed as there is limited guidance to decide between available analysis methods. Mixtures of environmental samples are useful for assessing analysis methods as one can evaluate methods based on calculated expected values using unmixed sample measurements and the mixture design. Previous studies have used mixtures of environmental samples to assess other sequencing methods such as RNAseq. But no studies have used mixtures of environmental to assess 16S rRNA sequencing. RESULTS: We developed a framework for assessing 16S rRNA sequencing analysis methods which utilizes a novel two-sample titration mixture dataset and metrics to evaluate qualitative and quantitative characteristics of count tables. Our qualitative assessment evaluates feature presence/absence exploiting features only present in unmixed samples or titrations by testing if random sampling can account for their observed relative abundance. Our quantitative assessment evaluates feature relative and differential abundance by comparing observed and expected values. We demonstrated the framework by evaluating count tables generated with three commonly used bioinformatic pipelines: (i) DADA2 a sequence inference method, (ii) Mothur a de novo clustering method, and (iii) QIIME an open-reference clustering method. The qualitative assessment results indicated that the majority of Mothur and QIIME features only present in unmixed samples or titrations were accounted for by random sampling alone, but this was not the case for DADA2 features. Combined with count table sparsity (proportion of zero-valued cells in a count table), these results indicate DADA2 has a higher false-negative rate whereas Mothur and QIIME have higher false-positive rates. The quantitative assessment results indicated the observed relative abundance and differential abundance values were consistent with expected values for all three pipelines. CONCLUSIONS: We developed a novel framework for assessing 16S rRNA marker-gene survey methods and demonstrated the framework by evaluating count tables generated with three bioinformatic pipelines. This framework is a valuable community resource for assessing 16S rRNA marker-gene survey bioinformatic methods and will help scientists identify appropriate analysis methods for their marker-gene surveys.

Evolving concepts in bone infection: redefining "biofilm", "acute vs. chronic osteomyelitis", "the immune proteome" and "local antibiotic therapy".

Osteomyelitis is a devastating disease caused by microbial infection of bone. While the frequency of infection following elective orthopedic surgery is low, rates of reinfection are disturbingly high. Staphylococcus aureus is responsible for the majority of chronic osteomyelitis cases and is often considered to be incurable due to bacterial persistence deep within bone. Unfortunately, there is no consensus on clinical classifications of osteomyelitis and the ensuing treatment algorithm. Given the high patient morbidity, mortality, and economic burden caused by osteomyelitis, it is important to elucidate mechanisms of bone infection to inform novel strategies for prevention and curative treatment. Recent discoveries in this field have identified three distinct reservoirs of bacterial biofilm including: Staphylococcal abscess communities in the local soft tissue and bone marrow, glycocalyx formation on implant hardware and necrotic tissue, and colonization of the osteocyte-lacuno canalicular network (OLCN) of cortical bone. In contrast, S. aureus intracellular persistence in bone cells has not been substantiated in vivo, which challenges this mode of chronic osteomyelitis. There have also been major advances in our understanding of the immune proteome against S. aureus, from clinical studies of serum antibodies and media enriched for newly synthesized antibodies (MENSA), which may provide new opportunities for osteomyelitis diagnosis, prognosis, and vaccine development. Finally, novel therapies such as antimicrobial implant coatings and antibiotic impregnated 3D-printed scaffolds represent promising strategies for preventing and managing this devastating disease. Here, we review these recent advances and highlight translational opportunities towards a cure.