RESUMEN
MOTIVATION: Phage genome annotation plays a key role in the design of phage therapy. To date, there have been various genome annotation tools for phages, but most of these tools focus on mono-functional annotation and have complex operational processes. Accordingly, comprehensive and user-friendly platforms for phage genome annotation are needed. RESULTS: Here, we propose PhaGAA, an online integrated platform for phage genome annotation and analysis. By incorporating several annotation tools, PhaGAA is constructed to annotate the prophage genome at DNA and protein levels and provide the analytical results. Furthermore, PhaGAA could mine and annotate phage genomes from bacterial genome or metagenome. In summary, PhaGAA will be a useful resource for experimental biologists and help advance the phage synthetic biology in basic and application research. AVAILABILITY AND IMPLEMENTATION: PhaGAA is freely available at http://phage.xialab.info/.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , Programas Informáticos , Computadores , Metagenoma , Genoma Bacteriano , Anotación de Secuencia MolecularRESUMEN
Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species-E. coli.
Asunto(s)
Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Enfermedades de los Porcinos , Escherichia coli Enterotoxigénica/virología , Escherichia coli Enterotoxigénica/genética , Animales , Porcinos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/veterinaria , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/virología , Especificidad del Huésped , Diarrea/microbiología , Diarrea/virología , Diarrea/veterinaria , Genoma Viral , Colifagos/genética , Colifagos/fisiología , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/aislamiento & purificación , Factores de Virulencia/genéticaRESUMEN
Plasmid fitness is directed by two orthogonal processes-vertical transfer through cell division and horizontal transfer through conjugation. When considered individually, improvements in either mode of transfer can promote how well a plasmid spreads and persists. Together, however, the metabolic cost of conjugation could create a tradeoff that constrains plasmid evolution. Here, we present evidence for the presence, consequences, and molecular basis of a conjugation-growth tradeoff across 40 plasmids derived from clinical Escherichia coli pathogens. We discover that most plasmids operate below a conjugation efficiency threshold for major growth effects, indicating strong natural selection for vertical transfer. Below this threshold, E. coli demonstrates a remarkable growth tolerance to over four orders of magnitude change in conjugation efficiency. This tolerance fades as nutrients become scarce and horizontal transfer attracts a greater share of host resources. Our results provide insight into evolutionary constraints directing plasmid fitness and strategies to combat the spread of antibiotic resistance.
Asunto(s)
Escherichia coli , Transferencia de Gen Horizontal , Escherichia coli/genética , Plásmidos/genética , Farmacorresistencia Microbiana , Antibacterianos/farmacologíaRESUMEN
In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism.
Asunto(s)
Enterobacteriaceae/crecimiento & desarrollo , Enterobacteriaceae/genética , Antibacterianos/farmacología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Microbiología Ambiental , Regulación Bacteriana de la Expresión Génica , Polimorfismo de Nucleótido Simple , Especificidad de la Especie , Factores de TiempoRESUMEN
Active matter comprises individual units that convert energy into mechanical motion. In many examples, such as bacterial systems and biofilament assays, constituent units are elongated and can give rise to local nematic orientational order. Such "active nematics" systems have attracted much attention from both theorists and experimentalists. However, despite intense research efforts, data-driven quantitative modeling has not been achieved, a situation mainly due to the lack of systematic experimental data and to the large number of parameters of current models. Here, we introduce an active nematics system made of swarming filamentous bacteria. We simultaneously measure orientation and velocity fields and show that the complex spatiotemporal dynamics of our system can be quantitatively reproduced by a type of microscopic model for active suspensions whose important parameters are all estimated from comprehensive experimental data. This provides unprecedented access to key effective parameters and mechanisms governing active nematics. Our approach is applicable to different types of dense suspensions and shows a path toward more quantitative active matter research.
Asunto(s)
Hidrodinámica , Modelos Teóricos , Serratia marcescensRESUMEN
Identifying active prophages is critical for studying coevolution of phage and bacteria, investigating phage physiology and biochemistry, and engineering designer phages for diverse applications. We present Prophage Hunter, a tool aimed at hunting for active prophages from whole genome assembly of bacteria. Combining sequence similarity-based matching and genetic features-based machine learning classification, we developed a novel scoring system that exhibits higher accuracy than current tools in predicting active prophages on the validation datasets. The option of skipping similarity matching is also available so that there's higher chance for novel phages to be discovered. Prophage Hunter provides a one-stop web service to extract prophage genomes from bacterial genomes, evaluate the activity of the prophages, identify phylogenetically related phages, and annotate the function of phage proteins. Prophage Hunter is freely available at https://pro-hunter.bgi.com/.
Asunto(s)
Bacteriófagos/genética , Genoma Bacteriano/genética , Profagos/genética , Programas Informáticos , Internet , FilogeniaRESUMEN
Numerous studies have reported critical roles for the gut microbiota in obesity. However, the specific microbes that causally contribute to obesity and the underlying mechanisms remain undetermined. Here, we conducted shotgun metagenomic sequencing in a Chinese cohort of 631 obese subjects and 374 normal-weight controls and identified a Megamonas-dominated, enterotype-like cluster enriched in obese subjects. Among this cohort, the presence of Megamonas and polygenic risk exhibited an additive impact on obesity. Megamonas rupellensis possessed genes for myo-inositol degradation, as demonstrated in vitro and in vivo, and the addition of myo-inositol effectively inhibited fatty acid absorption in intestinal organoids. Furthermore, mice colonized with M. rupellensis or E. coli heterologously expressing the myo-inositol-degrading iolG gene exhibited enhanced intestinal lipid absorption, thereby leading to obesity. Altogether, our findings uncover roles for M. rupellensis as a myo-inositol degrader that enhances lipid absorption and obesity, suggesting potential strategies for future obesity management.
Asunto(s)
Microbioma Gastrointestinal , Inositol , Obesidad , Inositol/metabolismo , Obesidad/microbiología , Obesidad/metabolismo , Animales , Humanos , Ratones , Masculino , Metabolismo de los Lípidos , Femenino , Absorción Intestinal , Ratones Endogámicos C57BL , Metagenómica , Persona de Mediana Edad , Adulto , Ácidos Grasos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
BACKGROUND: Zoonotic viruses cause substantial public health and socioeconomic problems worldwide. Understanding how viruses evolve and spread within and among wildlife species is a critical step when aiming for proactive identification of viral threats to prevent future pandemics. Despite the many proposed factors influencing viral diversity, the genomic diversity and structure of viral communities in East Africa are largely unknown. RESULTS: Using 38.3 Tb of metatranscriptomic data obtained via ultradeep sequencing, we screened vertebrate-associated viromes from 844 bats and 250 rodents from Kenya and Uganda collected from the wild. The 251 vertebrate-associated viral genomes of bats (212) and rodents (39) revealed the vast diversity, host-related variability, and high geographic specificity of viruses in East Africa. Among the surveyed viral families, Coronaviridae and Circoviridae showed low host specificity, high conservation of replication-associated proteins, high divergence among viral entry proteins, and frequent recombination. Despite major dispersal limitations, recurrent mutations, cocirculation, and occasional gene flow contribute to the high local diversity of viral genomes. CONCLUSIONS: The present study not only shows the landscape of bat and rodent viromes in this zoonotic hotspot but also reveals genomic signatures driven by the evolution and dispersal of the viral community, laying solid groundwork for future proactive surveillance of emerging zoonotic pathogens in wildlife. Video Abstract.
Asunto(s)
Quirópteros , Virus , Animales , Animales Salvajes , Genoma Viral/genética , Filogenia , Recombinación Genética , Roedores , Uganda/epidemiologíaRESUMEN
Phages are the functional viruses that infect bacteria and they play important roles in microbial communities and ecosystems. Phage research has attracted great attention due to the wide applications of phage therapy in treating bacterial infection in recent years. Metagenomics sequencing technique can sequence microbial communities directly from an environmental sample. Identifying phage sequences from metagenomic data is a vital step in the downstream of phage analysis. However, the existing methods for phage identification suffer from some limitations in the utilization of the phage feature for prediction, and therefore their prediction performance still need to be improved further. In this article, we propose a novel deep neural network (called MetaPhaPred) for identifying phages from metagenomic data. In MetaPhaPred, we first use a word embedding technique to encode the metagenomic sequences into word vectors, extracting the latent feature vectors of DNA words. Then, we design a deep neural network with a convolutional neural network (CNN) to capture the feature maps in sequences, and with a bi-directional long short-term memory network (Bi-LSTM) to capture the long-term dependencies between features from both forward and backward directions. The feature map consists of a set of feature patterns, each of which is the weighted feature extracted by a convolution filter with convolution kernels in the CNN slide along the input feature vectors. Next, an attention mechanism is used to enhance contributions of important features. Experimental results on both simulated and real metagenomic data with different lengths demonstrate the superiority of the proposed MetaPhaPred over the state-of-the-art methods in identifying phage sequences.
Asunto(s)
Bacteriófagos , Microbiota , Bacteriófagos/genética , Redes Neurales de la Computación , Algoritmos , Metagenoma/genéticaRESUMEN
Phages widely exist in numerous environments from wastewater to deep ocean, representing a huge virus diversity, yet remain poorly characterized. Among them, jumbo phages are of particular interests due to their large genome (>200 kb) and unusual biology. To date, only six strains of jumbo phages infecting Klebsiella pneumoniae have been described. Here, we report the isolation and characterization of two jumbo phages from hospital wastewater representing the sixth genus: φKp5130 and φKp9438. Both phages showed lytic activity against broad range of clinical antibiotic-resistant K. pneumoniae strains and distinct physiology including long latent period, small burst size, and high resistance to thermal and pH stress. The treatment of sewage water with the phages cocktail resulted in dramatic decline in K. pneumoniae population. Overall, this study provides detailed molecular and genomics characterization of two novel jumbo phages, expands viral diversity, and provides novel candidate phages to facilitate environmental wastewater treatment.
RESUMEN
Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6-18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrAB-TolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is up-regulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ΔmdtEF cells. Moreover, ΔmdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells.
Asunto(s)
Daño del ADN/fisiología , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Anaerobiosis/fisiología , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Fumaratos/metabolismo , Genoma Bacteriano/fisiología , Estudio de Asociación del Genoma Completo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Nitratos/metabolismoRESUMEN
The human gut virome, which is often referred to as the "dark matter" of the gut microbiome, remains understudied. A better understanding of the composition and variations of the gut virome across populations is critical for exploring its impact on diseases and health. A series of advances in the characterization of human gut virome have unveiled high genetic diversity and various functional potentials of gut viruses. Here, we summarize the recently available human gut virome databases and discuss their features, procedures, and challenges with the intention to provide a reference to researchers to use while choosing a profiling database. We also propose a "best practice" for cataloging the viral population.
Asunto(s)
Bacteriófagos , Microbioma Gastrointestinal , Virus , Bacteriófagos/genética , Catalogación , Microbioma Gastrointestinal/genética , Humanos , Viroma/genética , Virus/genéticaRESUMEN
With the number of phage genomes increasing, it is urgent to develop new bioinformatics methods for phage genome annotation. Promoter, a DNA region, is important for gene transcriptional regulation. In the era of post-genomics, the availability of data makes it possible to establish computational models for promoter identification with robustness. In this work, we introduce DPProm, a two-layer model composed of DPProm-1L and DPProm-2L, to predict promoters and their types for phages. On the first layer, as a dual-channel deep neural network ensemble method fusing multi-view features (sequence feature and handcrafted feature), the model DPProm-1L is proposed to identify whether a DNA sequence is a promoter or non-promoter. The sequence feature is extracted with convolutional neural network (CNN). And the handcrafted feature is the combination of free energy, GC content, cumulative skew, and Z curve features. On the second layer, DPProm-2L based on CNN is trained to predict the promoters' types (host or phage). For the realization of prediction on the whole genomes, the model DPProm, combines with a novel sequence data processing workflow, which contains sliding window and merging sequences modules. Experimental results show that DPProm outperforms the state-of-the-art methods, and decreases the false positive rate effectively on whole genome prediction. Furthermore, we provide a user-friendly web at http://bioinfo.ahu.edu.cn/DPProm. We expect that DPProm can serve as a useful tool for identification of promoters and their types.
Asunto(s)
Bacteriófagos , Aprendizaje Profundo , Bacteriófagos/genética , ADN , Genómica/métodos , Humanos , Regiones Promotoras Genéticas/genéticaRESUMEN
Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , Genómica , Edición Génica , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/genéticaRESUMEN
Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.
Asunto(s)
Bacteriófagos/genética , Genoma Viral , Klebsiella pneumoniae/virología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , Genómica , Filogenia , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
Increasing evidences indicate that high-risk HPV variants are heterogeneous in carcinogenicity and ethnic dispersion. In this work, we identified genetic signatures for convenient determination of lineage/sublineage of HPV16, 18, 52 and 58 variants. Using publicly available genomes, we found that E2 of HPV16, L2 of HPV18, L1 and LCR of HPV52, and L2, LCR and E1 of HPV58 contain the proper genetic signature for lineage/sublineage classification. Sets of hierarchical signature nucleotide positions were further confirmed for high accuracy (>95%) by classifying HPV genomes obtained from Chinese females, which included 117 HPV16 variants, 48 HPV18 variants, 117 HPV52 variants and 89 HPV58 variants. The circulation of HPV variants posing higher cancer risk in Eastern China, such as HPV16 A4 and HPV58 A3, calls for continuous surveillance in this region. The marker genes and signature nucleotide positions may facilitate cost-effective diagnostic detections of HPV variants in clinical settings.
Asunto(s)
Alphapapillomavirus/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Infecciones por Papillomavirus/virología , Alphapapillomavirus/clasificación , Alphapapillomavirus/aislamiento & purificación , China , Femenino , Variación Genética , Genoma Viral , Papillomavirus Humano 16/clasificación , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/clasificación , Papillomavirus Humano 18/aislamiento & purificación , Humanos , Nucleótidos , Filogenia , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. METHODS: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). RESULTS: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. CONCLUSIONS: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.
Asunto(s)
COVID-19/prevención & control , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Polimorfismo de Nucleótido Simple , SARS-CoV-2/genética , COVID-19/virología , Frecuencia de los Genes , Genotipo , Haplotipos , Interacciones Huésped-Patógeno , Humanos , Filogenia , SARS-CoV-2/clasificación , SARS-CoV-2/fisiologíaRESUMEN
The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.
RESUMEN
Plasmids are key vehicles of horizontal gene transfer (HGT), mobilizing antibiotic resistance, virulence, and other traits among bacterial populations. The environmental and genetic forces that drive plasmid transfer are poorly understood, however, due to the lack of definitive quantification coupled with genomic analysis. Here, we integrate conjugative phenotype with plasmid genotype to provide quantitative analysis of HGT in clinical Escherichia coli pathogens. We find a substantial proportion of these pathogens (>25%) able to readily spread resistance to the most common classes of antibiotics. Antibiotics of varied modes of action had less than a 5-fold effect on conjugation efficiency in general, with one exception displaying 31-fold promotion upon exposure to macrolides and chloramphenicol. In contrast, genome sequencing reveals plasmid incompatibility group strongly correlates with transfer efficiency. Our findings offer new insights into the determinants of plasmid mobility and have implications for the development of treatments that target HGT.
Asunto(s)
Antibacterianos/farmacología , Conjugación Genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Transferencia de Gen Horizontal , Genoma Bacteriano , Plásmidos/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Secuenciación Completa del GenomaRESUMEN
BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.