In-depth analysis of human virus-specific CD8+ T cells delineates unique phenotypic signatures for T cell specificity prediction.

Following viral infection, the human immune system generates CD8+ T cell responses to virus antigens that differ in specificity, abundance, and phenotype. A characterization of virus-specific T cell responses allows one to assess infection history and to understand its contribution to protective immunity. Here, we perform in-depth profiling of CD8+ T cells binding to CMV-, EBV-, influenza-, and SARS-CoV-2-derived antigens in peripheral blood samples from 114 healthy donors and 55 cancer patients using high-dimensional mass cytometry and single-cell RNA sequencing. We analyze over 500 antigen-specific T cell responses across six different HLA alleles and observed unique phenotypes of T cells specific for antigens from different virus categories. Using machine learning, we extract phenotypic signatures of antigen-specific T cells, predict virus specificity for bulk CD8+ T cells, and validate these predictions, suggesting that machine learning can be used to accurately predict antigen specificity from T cell phenotypes.

Reproducible, scalable, and shareable analysis pipelines with bioinformatics workflow managers.

The rapid growth of high-throughput technologies has transformed biomedical research. With the increasing amount and complexity of data, scalability and reproducibility have become essential not just for experiments, but also for computational analysis. However, transforming data into information involves running a large number of tools, optimizing parameters, and integrating dynamically changing reference data. Workflow managers were developed in response to such challenges. They simplify pipeline development, optimize resource usage, handle software installation and versions, and run on different compute platforms, enabling workflow portability and sharing. In this Perspective, we highlight key features of workflow managers, compare commonly used approaches for bioinformatics workflows, and provide a guide for computational and noncomputational users. We outline community-curated pipeline initiatives that enable novice and experienced users to perform complex, best-practice analyses without having to manually assemble workflows. In sum, we illustrate how workflow managers contribute to making computational analysis in biomedical research shareable, scalable, and reproducible.

Sustainable data analysis with Snakemake.

Data analysis often entails a multitude of heterogeneous steps, from the application of various command line tools to the usage of scripting languages like R or Python for the generation of plots and tables. It is widely recognized that data analyses should ideally be conducted in a reproducible way. Reproducibility enables technical validation and regeneration of results on the original or even new data. However, reproducibility alone is by no means sufficient to deliver an analysis that is of lasting impact (i.e., sustainable) for the field, or even just one research group. We postulate that it is equally important to ensure adaptability and transparency. The former describes the ability to modify the analysis to answer extended or slightly different research questions. The latter describes the ability to understand the analysis in order to judge whether it is not only technically, but methodologically valid. Here, we analyze the properties needed for a data analysis to become reproducible, adaptable, and transparent. We show how the popular workflow management system Snakemake can be used to guarantee this, and how it enables an ergonomic, combined, unified representation of all steps involved in data analysis, ranging from raw data processing, to quality control and fine-grained, interactive exploration and plotting of final results.

Genomic landscape of lung adenocarcinoma in East Asians.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts.

Large-Scale Whole-Genome Sequencing of Three Diverse Asian Populations in Singapore.

Underrepresentation of Asian genomes has hindered population and medical genetics research on Asians, leading to population disparities in precision medicine. By whole-genome sequencing of 4,810 Singapore Chinese, Malays, and Indians, we found 98.3 million SNPs and small insertions or deletions, over half of which are novel. Population structure analysis demonstrated great representation of Asian genetic diversity by three ethnicities in Singapore and revealed a Malay-related novel ancestry component. Furthermore, demographic inference suggested that Malays split from Chinese ∼24,800 years ago and experienced significant admixture with East Asians ∼1,700 years ago, coinciding with the Austronesian expansion. Additionally, we identified 20 candidate loci for natural selection, 14 of which harbored robust associations with complex traits and diseases. Finally, we show that our data can substantially improve genotype imputation in diverse Asian and Oceanian populations. These results highlight the value of our data as a resource to empower human genetics discovery across broad geographic regions.

Structure mapping of dengue and Zika viruses reveals functional long-range interactions.

Dengue (DENV) and Zika (ZIKV) viruses are clinically important members of the Flaviviridae family with an 11 kb positive strand RNA genome that folds to enable virus function. Here, we perform structure and interaction mapping on four DENV and ZIKV strains inside virions and in infected cells. Comparative analysis of SHAPE reactivities across serotypes nominates potentially functional regions that are highly structured, conserved, and contain low synonymous mutation rates. Interaction mapping by SPLASH identifies many pair-wise interactions, 40% of which form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared interactions between serotypes reveals a conserved macro-organization whereby interactions can be preserved at physical locations beyond sequence identities. We further observe that longer-range interactions are preferentially disrupted inside cells, and show the importance of new interactions in virus fitness. These findings deepen our understanding of Flavivirus genome organization and serve as a resource for designing therapeutics in targeting RNA viruses.

Human pharyngeal microbiota in age-related macular degeneration.

BACKGROUND: While the aetiology of age-related macular degeneration (AMD)-a major blinding disease-remains unknown, the disease is strongly associated with variants in the complement factor H (CFH) gene. CFH variants also confer susceptibility to invasive infection with several bacterial colonizers of the nasopharyngeal mucosa. This shared susceptibility locus implicates complement deregulation as a common disease mechanism, and suggests the possibility that microbial interactions with host complement may trigger AMD. In this study, we address this possibility by testing the hypothesis that AMD is associated with specific microbial colonization of the human nasopharynx. RESULTS: High-throughput Illumina sequencing of the V3-V6 region of the microbial 16S ribosomal RNA gene was used to comprehensively and accurately describe the human pharyngeal microbiome, at genus level, in 245 AMD patients and 386 controls. Based on mean and differential microbial abundance analyses, we determined an overview of the pharyngeal microbiota, as well as candidate genera (Prevotella and Gemella) suggesting an association towards AMD health and disease conditions. CONCLUSIONS: Utilizing an extensive study population from Singapore, our results provided an accurate description of the pharyngeal microbiota profiles in AMD health and disease conditions. Through identification of candidate genera that are different between conditions, we provide preliminary evidence for the existence of microbial triggers for AMD. Ethical approval for this study was obtained through the Singapore Health Clinical Institutional Review Board, reference numbers R799/63/2010 and 2010/585/A.

Single-virion sequencing of lamivudine-treated HBV populations reveal population evolution dynamics and demographic history.

BACKGROUND: Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients - once before antiviral treatment and once after viral rebound due to resistance. RESULTS: With single-virion sequencing, we obtained 248-8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing. CONCLUSIONS: Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history.

Whole metagenome profiling reveals skin microbiome-dependent susceptibility to atopic dermatitis flare.

Whole metagenome analysis has the potential to reveal functional triggers of skin diseases, but issues of cost, robustness and sampling efficacy have limited its application. Here, we have established an alternative, clinically practical and robust metagenomic analysis protocol and applied it to 80 skin microbiome samples epidemiologically stratified for atopic dermatitis (AD). We have identified distinct non-flare, baseline skin microbiome signatures enriched for Streptococcus and Gemella but depleted for Dermacoccus in AD-prone versus normal healthy skin. Bacterial challenge assays using keratinocytes and monocyte-derived dendritic cells established distinct IL-1-mediated, innate and Th1-mediated adaptive immune responses with Staphylococcus aureus and Staphylococcus epidermidis. Bacterial differences were complemented by perturbations in the eukaryotic community and functional shifts in the microbiome-wide gene repertoire, which could exacerbate a dry and alkaline phenotype primed for pathogen growth and inflammation in AD-susceptible skin. These findings provide insights into how the skin microbial community, skin surface microenvironment and immune system cross-modulate each other, escalating the destructive feedback cycle between them that leads to AD flare.

INC-Seq: accurate single molecule reads using nanopore sequencing.

BACKGROUND: Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionize genomics. However, several applications are limited by relatively high single-read error rates (>10 %), including RNA-seq, haplotype sequencing and 16S sequencing. RESULTS: We developed the Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) as a strategy for obtaining long and accurate nanopore reads, starting with low input DNA. Applying INC-Seq for 16S rRNA-based bacterial profiling generated full-length amplicon sequences with a median accuracy >97 %. CONCLUSIONS: INC-Seq reads enabled accurate species-level classification, identification of species at 0.1 % abundance and robust quantification of relative abundances, providing a cheap and effective approach for pathogen detection and microbiome profiling on the MinION system.

Tissue Microbiome Profiling Identifies an Enrichment of Specific Enteric Bacteria in Opisthorchis viverrini Associated Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the primary cancer of the bile duct system. The role of bile duct tissue microbiomes in CCA tumorigenesis is unestablished. To address this, sixty primary CCA tumors and matched normals, from both liver fluke (Opisthorchis viverrini) associated (OVa, n=28) and non-O. viverrini associated (non-OVa, n=32) cancers, were profiled using high-throughput 16S rRNA sequencing. A distinct, tissue-specific microbiome dominated by the bacterial families Dietziaceae, Pseudomonadaceae and Oxalobacteraceae was observed in bile duct tissues. Systemic perturbation of the microbiome was noted in tumor and paired normal samples (vs non-cancer normals) for several bacterial families with a significant increase in Stenotrophomonas species distinguishing tumors vs paired normals. Comparison of parasite associated (OVa) vs non-associated (non-OVa) groups identified enrichment for specific enteric bacteria (Bifidobacteriaceae, Enterobacteriaceae and Enterococcaceae). One of the enriched families, Bifidobacteriaceae, was found to be dominant in the O. viverrini microbiome, providing a mechanistic link to the parasite. Functional analysis and comparison of CCA microbiomes revealed higher potential for producing bile acids and ammonia in OVa tissues, linking the altered microbiota to carcinogenesis. These results define how the unique microbial communities resident in the bile duct, parasitic infections and the tissue microenvironment can influence each other, and contribute to cancer.

In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation.

Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra- and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the underexplored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.

Fast and sensitive mapping of nanopore sequencing reads with GraphMap.

Realizing the democratic promise of nanopore sequencing requires the development of new bioinformatics approaches to deal with its specific error characteristics. Here we present GraphMap, a mapping algorithm designed to analyse nanopore sequencing reads, which progressively refines candidate alignments to robustly handle potentially high-error rates and a fast graph traversal to align long reads with speed and high precision (>95%). Evaluation on MinION sequencing data sets against short- and long-read mappers indicates that GraphMap increases mapping sensitivity by 10-80% and maps >95% of bases. GraphMap alignments enabled single-nucleotide variant calling on the human genome with increased sensitivity (15%) over the next best mapper, precise detection of structural variants from length 100 bp to 4 kbp, and species and strain-specific identification of pathogens using MinION reads. GraphMap is available open source under the MIT license at https://github.com/isovic/graphmap.

Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution.

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children ,2 years of age. Little is known about RSV intra-host genetic diversity over the course of infection or about the immune pressures that drive RSV molecular evolution. We performed whole-genome deep-sequencing on 53 RSV-positive samples (37 RSV subgroup A and 16 RSV subgroup B) collected from the upper airways of hospitalized children in southern Vietnam over two consecutive seasons. RSV A NA1 and RSV B BA9 were the predominant genotypes found in our samples, consistent with other reports on global RSV circulation during the same period. For both RSV A and B, the M gene was the most conserved, confirming its potential as a target for novel therapeutics. The G gene was the most variable and was the only gene under detectable positive selection. Further, positively selected sites inG were found in close proximity to and in some cases overlapped with predicted glycosylation motifs, suggesting that selection on amino acid glycosylation may drive viral genetic diversity. We further identified hotspots and coldspots of intra-host genetic diversity in the RSV genome, some of which may highlight previously unknown regions of functional importance.

Tracking Dengue Virus Intra-host Genetic Diversity during Human-to-Mosquito Transmission.

Dengue virus (DENV) infection of an individual human or mosquito host produces a dynamic population of closely-related sequences. This intra-host genetic diversity is thought to offer an advantage for arboviruses to adapt as they cycle between two very different host species, but it remains poorly characterized. To track changes in viral intra-host genetic diversity during horizontal transmission, we infected Aedes aegypti mosquitoes by allowing them to feed on DENV2-infected patients. We then performed whole-genome deep-sequencing of human- and matched mosquito-derived DENV samples on the Illumina platform and used a sensitive variant-caller to detect single nucleotide variants (SNVs) within each sample. >90% of SNVs were lost upon transition from human to mosquito, as well as from mosquito abdomen to salivary glands. Levels of viral diversity were maintained, however, by the regeneration of new SNVs at each stage of transmission. We further show that SNVs maintained across transmission stages were transmitted as a unit of two at maximum, suggesting the presence of numerous variant genomes carrying only one or two SNVs each. We also present evidence for differences in selection pressures between human and mosquito hosts, particularly on the structural and NS1 genes. This analysis provides insights into how population drops during transmission shape RNA virus genetic diversity, has direct implications for virus evolution, and illustrates the value of high-coverage, whole-genome next-generation sequencing for understanding viral intra-host genetic diversity.

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints.

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients' sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.

BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

Draft Genome Sequence of Polychlorinated Biphenyl-Dechlorinating Dehalococcoides mccartyi Strain SG1, Which Carries a Circular Putative Plasmid.

Dehalococcoides mccartyi strain SG1, isolated from digester sludge, dechlorinates polychlorinated biphenyls (PCBs) to lower congeners. Here we report the draft genome sequence of SG1, which carries a 22.65 kbp circular putative plasmid.

Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls.

Fastidious anaerobic bacteria play critical roles in environmental bioremediation of halogenated compounds. However, their characterization and application have been largely impeded by difficulties in growing them in pure culture. Thus far, no pure culture has been reported to respire on the notorious polychlorinated biphenyls (PCBs), and functional genes responsible for PCB detoxification remain unknown due to the extremely slow growth of PCB-respiring bacteria. Here we report the successful isolation and characterization of three Dehalococcoides mccartyi strains that respire on commercial PCBs. Using high-throughput metagenomic analysis, combined with traditional culture techniques, tetrachloroethene (PCE) was identified as a feasible alternative to PCBs to isolate PCB-respiring Dehalococcoides from PCB-enriched cultures. With PCE as an alternative electron acceptor, the PCB-respiring Dehalococcoides were boosted to a higher cell density (1.2 × 10(8) to 1.3 × 10(8) cells per mL on PCE vs. 5.9 × 10(6) to 10.4 × 10(6) cells per mL on PCBs) with a shorter culturing time (30 d on PCE vs. 150 d on PCBs). The transcriptomic profiles illustrated that the distinct PCB dechlorination profile of each strain was predominantly mediated by a single, novel reductive dehalogenase (RDase) catalyzing chlorine removal from both PCBs and PCE. The transcription levels of PCB-RDase genes are 5-60 times higher than the genome-wide average. The cultivation of PCB-respiring Dehalococcoides in pure culture and the identification of PCB-RDase genes deepen our understanding of organohalide respiration of PCBs and shed light on in situ PCB bioremediation.