Biology subjects

Cheng, A.

Publications and source records attributed to Cheng, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the alpha-herpesviruses HSV-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting involvement of an immediate early or early (IE-E) viral protein. In support of this mechanism, cycloheximide treatment of HCMV-infected cells prevents the expression of viral proteins and simultaneously blocks APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which is a viral DNA synthesis inhibitor affecting late protein expression, still permits A3B relocalization. These results combine to show that the beta-herpesvirus HCMV uses a fundamentally different, RNR-independent molecular mechanism to antagonize APOBEC3B. ImportanceHuman cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses in order to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

microbiology↗

bioRxiv2022

Ras-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy

RIT1 belongs to the family of Ras guanosine triphosphatases (GTPases) that regulate many aspects of signal transduction and are drivers of cancer and congenital disorders. RIT1 gain-of-function mutations are found in lung cancer, leukemia, and in the germline of Noonan syndrome individuals with an increased prevalence of cardiac hypertrophy and other congenital heart defects. Pathogenic RIT1 proteins evade proteasomal degradation and promote MEK/ERK mitogen-activated protein kinase (MAPK) hyperactivation, yet the mechanism remains poorly understood. Here we show that RAF kinases are putative mutant RIT1 effectors necessary for MAPK activation and characterize RIT1 association with plasma membrane lipids and interaction with RAF kinases. We identify critical residues present in the RIT1 hypervariable region that facilitate interaction with negatively charged membrane lipids and show that these are necessary for association with RAF kinases. Although mutant RIT1 binds to RAF kinases directly, it fails to activate RAF-MAPK signaling in the absence of classical Ras proteins. Consistent with aberrant RAF/MEK/ERK activation as a driver of disease, we show that MEK inhibition alleviates cardiac hypertrophy in a mouse model of RIT1-mutant Noonan syndrome. These data shed light on pathogenic RIT1 function and identify avenues for therapeutic intervention. One Sentence SummaryElectrostatic plasma membrane association facilitates RIT1-mediated Ras-dependent RAF kinase activation to promote pathogenic MAPK signaling.

biochemistry↗

bioRxiv2022

Identification and structural modeling of the chlamydial RNA polymerase omega subunit

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing {sigma} factor. RNAP core enzyme comprises two subunits, one {beta} subunit, one {beta} s subunit, and one {omega} (omega) subunit. Across multiple bacterial taxa, the RNAP {omega} subunit plays critical roles in the assembly of RNAP core enzyme and in other cellular functions, including regulation of bacterial growth, stress response, and biofilm formation. However, for several intracellular bacterium, including the obligate intracellular bacterium Chlamydia, no RNAP {omega} subunit previously has been identified. Here, we report the identification of Chlamydia trachomatis hypothetical protein CTL0286 as the chlamydial RNAP {omega} ortholog, based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. We conclude that CTL0286 functions as the previously missing chlamydial {omega} ortholog. Extensions of our analysis indicate that all obligate intracellular bacteria have {omega} orthologs. IMPORTANCEChlamydiae are common mammalian pathogens. Chlamydiae have a unique developmental cycle characterized with an infectious but nondividing elementary body (EB), which can temporarily survive outside host cells, and a noninfectious reticulate body (RB), which replicates only intracellularly. Chlamydial development inside host cells can be arrested during persistence in response to adverse environmental conditions. Transcription plays a central role in the progression of the chlamydial developmental cycle as well as entry into and recovery from persistence. The identification of the elusive {omega} subunit of chlamydial RNAP makes possible future study of its regulatory roles in gene expression during chlamydial growth, development, and stress responses. This discovery also paves the way to prepare and study the intact chlamydial RNAP and its interactions with inhibitors in vitro.

molecular biology↗

bioRxiv2022

Lung Tumor Microphysiological System with 3D Endothelium to Evaluate Modulators of T-Cell Infiltration

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor on chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells infiltrate; and a chemoattractant/tumor compartment. Here, activated T-cells extravasate and infiltrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips, increases T-cell expression of CXCR3 and CXCR4 receptors, and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose a translational strategy by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

bioengineering↗

bioRxiv2022

Inferring Single-Molecule Chromatin Interactions via Online Convex Network Dictionary Learning

MotivationGenomes of multicellular systems are compartmentalized and dynamically folded within the three-dimensional (3D) confines of the nucleus in order to facilitate gene regulation. Among the 3D-genome mapping technologies currently in use, droplet-based, barcode-linked sequencing (ChIA-Drop) has the unique capability to capture complex multi-way chromatin interactions at the single-molecule level. ChIA-Drop data gives rise to higher-order interaction networks in which nodes represent genomic fragments while (hyper)edges capture observed physical contacts. The problem of interest is to use this data to create a "dictionary" of interaction patterns (subnetworks) that accurately describe all global chromatin structures and associate dictionary elements with cellular functions. ResultsTo construct interpretable chromatin dictionaries, we introduce a new algorithm termed online convex network dictionary learning (online cvxNDL). Unlike classical dictionary learning for image or text processing, online cvxNDL uses special subgraph sampling methods and produces interpretable subnetwork representatives corresponding to "convex mixtures" of patterns observed in real data. To demonstrate the utility of the method, we perform an in-depth study of RNAPII-enriched ChIA-Drop data from Drosophila Melanogaster S2 cell lines. Our results are two-fold: First, we show that online cvxNDL allows for accurate reconstruction of the original interaction network data using only a collection of roughly 25 dictionary elements and their "representatives" directly observed in the data. Second, we identify collections of interaction patterns of chromatin elements shared by related processes on different chromosomes and those unique to certain chromosomes. This is accomplished through Gene Ontology (GO) enrichment analysis that allows us to associate dictionary element representatives with functional properties of their corresponding chromatin region and in the process, determine what we call the "span" and "density" of chromatin interaction patterns. Availability and ImplementationThe code and dataset are available at: https://github.com/jianhao2016/online_cvxNDL/ Contactmilenkov@illinois.edu

bioinformatics↗

bioRxiv2022

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by focusing on a functional unit within the community, the metabolic niche that controls bile acid 7-dehydroxylation. By constructing variants of a complex defined community in which we drop out strains that occupy this niche, we explore how interactions within and between niches shape community-level metabolism. Omitting both members of the niche, Clostridium scindens (Cs) and Clostridium hylemonae (Ch), eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains go up or down in relative abundance by >100-fold, while the remaining strains are largely unaffected. In single-strain dropout communities (i.e., a strain swap within the niche), Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, the effect on strains in other niches differs markedly: Clostridium sporogenes increases >1000-fold in the {Delta}Cs but not {Delta}Ch dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Mice colonized by the {Delta}Cs{Delta}Ch community show decreased liver steatosis relative to those colonized by the {Delta}Ch community, demonstrating that a single strain from the microbiome can have a substantive impact on host physiology. Our work opens the door to the mechanistic studies of the role of an individual strain on community ecology and host physiology.

microbiology↗

bioRxiv2022

Fully Automated Multi-Grid Cryo-EM Screening using Smart Leginon

Single particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized over several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness, and particle behaviors. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel, purpose-built image processing and microscope-handling algorithms are incorporated into the automated data collection software, Leginon, to provide an open-source solution for fully automated, high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid Autoscreen session and conventional manual screening by five expert microscope operators are presented. On average, Autoscreen reduces operator time from [~]6 hours to <10 minutes and provides a comparable percentage of suitable images for evaluation as the best operator. Smart Leginons ability to target holes that are particularly difficult to identify is analyzed. Finally, Smart Leginons utility is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. Smart Leginons fully automated functionality significantly reduces the burden on operator screening time, improves the throughput of screening, and recovers idle microscope time, thereby improving availability of cryoEM services.

biophysics↗

bioRxiv2022

ct2vl: Converting Ct Values to Viral Loads for SARS-CoV-2 RT-qPCR Test Results

RT-qPCR is the de facto reference method for detecting the presence of SARS-CoV-2 genomic material in infected individuals (1). Although RT-qPCR is inherently quantitative and despite SARS-CoV-2 viral loads varying by 10 orders of magnitude and therefore being potentially highly clinically informative, in practice SARS-CoV-2 RT-qPCR results are usually reported qualitatively as simply positive or negative. This is both because of the mathematical complexity of converting from Ct values to viral loads and because the same Ct value can correspond to orders-of-magnitude differences in viral load depending on the testing platform (2, 3, 4). To address this problem, here we present ct2vl, a Python package designed to help individual clinical laboratories, investigators, and test developers convert from Ct values to viral loads on their own platforms, using only the data generated during validation of those platforms. It allows any user to convert Ct values to viral loads and is readily applicable to other RT-qPCR tests. ct2vl is open source, has 100% code coverage, and is freely available via the Python Package Index (PyPI). IMPORTANCEUp to now, COVID-19 test results have been reported as positive vs. negative, even though "positive" can mean anywhere from 1 copy of SARS-CoV-2 virus per milliliter of transport media to over 1 billion copies/mL, with attendant clinical consequences. Democratizing access to this quantitative data is the first step toward its eventual incorporation into test development, the research literature, and clinical care.

microbiology↗

bioRxiv2022

Epsin2, a novel target for multiple system atrophy therapy via regulating α-synuclein propagation associated with FABP7

Multiple system atrophy (MSA) is a neurodegenerative disease showing accumulation of misfolded -synuclein and myelin disruption. However, the mechanism how -synuclein (-syn) accumulate in MSA brain remains unclear. Here, we identify the protein epsin-2 as a novel target for MSA therapy via controlling -synuclein accumulation. In MSA mouse model, PLP-hSyn transgenic mice and FABP7/-syn hetero-aggregates injected mice, we firstly found that fatty acid-binding protein 7 (FABP7) related to MSA development and formed hetero-aggregates with -syn, which exhibited stronger toxicity than -syn aggregates. Furthermore, injected FABP7/-syn hetero-aggregates in mice selectively accumulated in oligodendrocytes and Purkinje neurons and cause cerebellar dysfunction. By bioinformatic analyses, the protein epsin-2 expresses in both oligodendrocyte and Purkinje cells was found as a potential target to regulate FABP7/-syn hetero-aggregates propagation via clathrin-dependent endocytosis. The AAV5-dependent epsin-2 knock-down mice exhibited decreased levels of aggregates accumulation in Purkinje neurons and oligodendrocytes as well as performed improved myelin levels and Purkinje neurons in cerebellum and motor functions. Thus, we propose epsin-2 as a novel and therapeutic candidate for MSA.

pathology↗

bioRxiv2022

Mapping the T cell repertoire to a complex gut bacterial community

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with a complex defined community (97 or 112 bacterial strains) and profile T cell responses to each strain individually. Unexpectedly, the pattern of T cell responses suggests that many T cells in the gut repertoire recognize multiple bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all of the bacteria-specific TCRs exhibit a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that are polyspecific for 18 Firmicutes in the community. By screening three pooled bacterial genomic libraries against 13 pooled hybridomas, we discover that they share a single target: a conserved substrate-binding protein (SBP) from an ABC transport system. Treg and Th17 cells specific for an epitope from this protein are abundant in community-colonized and specific-pathogen-free mice. Our work reveals that T cell recognition of Firmicutes is focused on a widely conserved cell-surface antigen, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

immunology↗

bioRxiv2022

Molecular differences in APOBEC3 counteraction by primate herpesviruses reveal an evolutionary link between the genesis of APOBEC3B and novel viral ribonucleotide reductase function

Viruses have evolved diverse mechanisms to antagonize host immunity such as direct inhibition and relocalization of cellular APOBEC3B (A3B) by the ribonucleotide reductase (RNR) of Epstein-Barr virus. Here, we investigate the mechanistic conservation and evolutionary origin of this innate immune counteraction strategy. First, we find that human gamma-herpesvirus RNRs engage A3B via largely distinct surfaces. Second, we show that RNR-mediated enzymatic inhibition and relocalization of A3B depend upon binding to different regions of the catalytic domain. Third, we show that the capability of viral RNRs to antagonize A3B is conserved among gamma-herpesviruses that infect humans and Old World monkeys that encode this enzyme but absent in homologous viruses that infect New World monkeys that naturally lack the A3B gene. Finally, we reconstruct the ancestral primate A3B protein and demonstrate that it is active and similarly engaged by the RNRs from viruses that infect humans and Old World monkeys but not by the RNRs from viruses that infect New World monkeys. These results combine to indicate that the birth of A3B at a critical branchpoint in primate evolution may have been a driving force in selecting for an ancestral gamma-herpesvirus with an expanded RNR functionality through counteraction of this antiviral enzyme.

microbiology↗

bioRxiv2022

Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Copy number variations (CNV) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

neuroscience↗

bioRxiv2022

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

genetics↗

bioRxiv2021

Identification of a promiscuous conserved CTL epitope within the SARS-CoV-2 spike protein

The COVID-19 disease caused by infection with SARS-CoV-2 and its variants is devastating to the global public health and economy. To date, over a hundred COVID-19 vaccines are known to be under development and the few that have been approved to fight the disease are using the spike protein as the primary target antigen. Although virus neutralizing epitopes are mainly located within the RBD of the spike protein, the presence of T cell epitopes, particularly the CTL epitopes that are likely to be needed for killing infected cells, has received comparatively little attention. In this study, we predicted several potential T cell epitopes with web-based analytic tools, and narrowed them down from several potential MHC-I and MHC-II epitopes by ELIspot and cytolytic assays to a conserved MHC-I epitope. The epitope is highly conserved in current viral variants including the most recent Omicron and compatible with presentation by most HLA alleles worldwide. In conclusion, we identified a CTL epitope suitable for evaluating the CD8+ T cell-mediated cellular response and potentially for addition into future COVID-19 vaccine candidates to maximize CTL responses against SARS-CoV-2.

immunology↗

bioRxiv2021

System Environmental Metrics Collector for EM facilities

Recent developments in cryo-electron microscopy (cryoEM) have led to the routine determination of structures at near atomic resolution and greatly increased the number of biomedical researchers wanting access to high-end cryoEM instrumentation. The high costs and long wait times for gaining access encourages facilities to maximize instrument uptime for data collection. To support these goals, we developed a System Environmental Metrics Collector for facilities (SEMCf) that serves as a laboratory performance and management tool. SEMCf consists of an architecture of automated and robust sensors that track, organize and report key facility metrics. The individual sensors are connected to Raspberry Pi (RPi) single board computers installed in close proximity to the input metrics being measured. The system is controlled by a central server that may be installed on a RPi or existing microscope support PC. Tracking the system and the environment provides feedback of imminent issues, suggestions for interventions that are needed to optimize data production, and indications as to when preventative maintenance should be scheduled. The sensor components are relatively inexpensive and widely commercially available, and the open-source design and software enables straightforward implementation, customization, and optimization by any facility that would benefit from real time environmental monitoring and reporting.

biophysics↗

bioRxiv2021

CryoEM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition

Viruses use a plethora of mechanisms to evade immune responses. A new example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000 [A]2 binding surface comprised of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. An atomic understanding of this novel pathogen-host interaction may contribute to the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. One-Sentence SummaryThese studies show how a conserved viral nucleotide metabolism protein is repurposed to inhibit a potent antiviral factor.

microbiology↗

bioRxiv2021

Genetic loci of the R. anatipestifer serotype discovered by Pan-GWAS and its application for the development of a multiplex PCR serotyping method

The disease caused by Riemerella anatipestifer (R. anatipestifer) causes large economic losses to the global duck industry every year. Serotype-related genomic variation (such as in O-antigen and capsular polysaccharide gene clusters) has been widely used for the serotyping in many gram-negative bacteria. To date, there have been few studies focused on genetic basis of serotypes in R. anatipestifer. Here, we used pan-genome-wide association studies (Pan-GWAS) to identify the serotype-specific genetic loci of 38 R. anatipestifers strain. Analyses of the loci of 11 serotypes showed that the loci could be well mapped with the serotypes of the corresponding strains. We constructed the knockout strain for the wzy gene at the locus, and the results showed that the mutant lost the agglutination characteristics to positive antisera. Based on the of Pan-GWAS results, we developed a multiple PCR method to identify serotypes 1, 2, and 11 of R. anatipestifer. Our study provides a precedent for systematically analysing the genetic basis of the R anatipestifer serotypes and establishing a complete serotyping system in the future. HighlightsO_LIR. anatipestifer serotype-specific locus was identified by Pan-GWAS for the first time. C_LIO_LIMolecular serotyping multiplex PCR was developed based on O-antigen biosynthesis gene clusters C_LI

genomics↗

bioRxiv2021

DNA methylation calling tools for Oxford Nanopore sequencing: a survey and human epigenome-wide evaluation

BackgroundNanopore long-read sequencing technology greatly expands the capacity of long-range single-molecule DNA-modification detection. A growing number of analytical tools have been actively developed to detect DNA methylation from Nanopore sequencing reads. Here, we examine the performance of different methylation calling tools to provide a systematic evaluation to guide practitioners for human epigenome-wide research. ResultsWe compare five analytic frameworks for detecting DNA modification from Nanopore long-read sequencing data. We evaluate the association between genomic context, CpG methylation-detection accuracy, CpG sites coverage, and running time using Nanopore sequencing data from natural human DNA. Furthermore, we provide an online DNA methylation database (https://nanome.jax.org) with which to display genomic regions that exhibit differences in DNA-modification detection power among different methylation calling algorithms for nanopore sequencing data. ConclusionsOur study is the first benchmark of computational methods for mammalian whole genome DNA-modification detection in Nanopore sequencing. We provide a broad foundation for cross-platform standardization, and an evaluation of analytical tools designed for genome-scale modified-base detection using Nanopore sequencing.

bioinformatics↗