Biology subjects

Ge, C.

Publications and source records attributed to Ge, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Hippocampal extracellular matrix remodeling and temporally distinct glial reorganization following peripheral injury

Chronic pain is frequently accompanied by anxiety and other affective disturbances, but the molecular and cellular changes linking peripheral injury to persistent alterations in brain function remain incompletely defined. The hippocampal extracellular matrix (ECM) regulates synaptic plasticity, cellular signaling, and neuroimmune interactions and may therefore contribute to pain-related central plasticity. We used a tibial fracture/cast immobilization model to examine temporal changes in hippocampal ECM, glial morphology, and affective behavior following peripheral injury. Behavioral testing was performed at 3, 7, and 20 weeks after injury, while high-dimensional morphometric analysis of microglia and astrocytes was performed at 1, 3, 7, and 20 weeks. Hippocampal ECM associated molecular signals were mapped at the chronic stage using decellularization coupled with MALDI-TOF mass spectrometry imaging. Injured mice developed mechanical hypersensitivity and reduced exploration in two anxiety related paradigms during the early and chronic stages, with these behavioral phenotypes no longer detected at 20 weeks. At 7 weeks, injury was associated with changes in hippocampal ECM-associated peptide signals, including reduced signals corresponding to four ECM-associated proteins. Microglia and astrocytes exhibited distinct, time-dependent changes in density and multidimensional morphology, with some alterations persisting after behavioral measures returned toward control levels. In vitro, BV2 microglia exhibited rigidity-dependent changes in morphology, phagocytic behavior, and calcium signaling. Together, these findings define a temporally evolving hippocampal ECM and glial phenotype following peripheral injury and identify matrix biomechanics as a candidate regulator of microglial function.

neuroscience↗

bioRxiv2026

Piezo1 links cytoskeletal remodeling to differential YAP and β-catenin signaling in response to mechanical cues

Mechanical cues from the extracellular matrix regulate cancer cell behavior, but how these inputs are translated into distinct nuclear signaling responses remains incompletely understood. This study examined whether Piezo1, a mechanosensitive ion channel, contributes to cytoskeletal remodeling and differential regulation of YAP and {beta}-catenin localization in breast cancer cells exposed to defined mechanical cues. MDA-MB-231 breast cancer cells were cultured on substrates of defined stiffness and analyzed after Piezo1 knockdown or pharmacological modulation of Piezo1, Src signaling, myosin II activity, and actin polymerization. Nuclear localization of YAP and {beta}-catenin was assessed by immunofluorescence imaging, cytoskeletal organization was evaluated using filamentous and globular actin staining, protein phosphorylation was analyzed by capillary electrophoresis-based immunoblotting, and cell migration was assessed using a wound-healing assay. Piezo1 knockdown reduced YAP nuclear localization and increased {beta}-catenin nuclear localization, while Piezo1 activation partially reversed these localization changes. Piezo1 knockdown also disrupted filamentous actin organization, and pharmacological disruption of actin polymerization produced similar effects on YAP and {beta}-catenin localization. Piezo1 knockdown selectively reduced YAP tyrosine phosphorylation without altering canonical Hippo-associated YAP serine phosphorylation, and inhibition of Src signaling produced effects similar to Piezo1 knockdown. Functionally, Piezo1 knockdown impaired stiffness-dependent cell migration. These findings support a role for Piezo1 in linking extracellular mechanical cues to cytoskeletal organization and differential regulation of YAP and {beta}-catenin localization in breast cancer cells. This work provides a framework for understanding how mechanosensitive ion-channel signaling may contribute to context-dependent nuclear signaling responses during cancer cell mechanotransduction.

cell biology↗

bioRxiv2026

Robust causal gene network estimation for large-scale single-cell perturbation screens using reduced control function

Single-cell CRISPR perturbation screens provide a foundation for causal discovery in gene regulatory networks, but existing methods struggle with latent confounding, count-valued expression data, and high-multiplicity-of-infection (high-MOI) designs. We introduce RICE, a unified framework that addresses all three challenges. At its core is a reformulated control function tailored to the network-estimation setting, which restores robustness to exclusion-restriction violations that are pervasive in real CRISPR screens and that cause standard control function approaches to fail. RICE pairs this estimator with a constrained negative binomial model and a differentiable acyclicity penalty, accommodating hard and soft interventions and natively supporting high-MOI designs within a single, GPU-scalable model. Across extensive synthetic benchmarks, RICE consistently outperforms existing methods and remains stable under strong confounding, exclusion violations, and high-MOI conditions. Applied to CRISPRi screens, RICE achieves stronger causal-discovery performance on held-out data. RICE further reconstructs the canonical interferon (IFN)-{gamma} signaling pathway in melanoma cells upon immune stimulation, and nominates stable regulatory candidates that conventional significance tests may overlook due to limited statistical power. Together, these results establish RICE as a robust and scalable framework for causal discovery in single-cell perturbation genomics.

bioinformatics↗

bioRxiv2026

Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

BackgroundAtherosclerosis is the primary underlying cause of coronary artery disease (CAD). Leiomodin1 is a vascular smooth muscle cell (VSMC)-restricted CAD risk gene whose role in coronary artery pathophysiology is unknown. Global loss of Leiomodin1 causes lethal neonatal visceral myopathy, requiring unique approaches for study in VSMCs. MethodsSeveral distinct Leiomodin1 mutant mouse models were generated by clustered regularly interspaced short palindromic repeats (CRISPR). Control (Lmod1WT) and VSMC-restricted Lmod1 knockout (Lmod1SMKO) mice were subjected to various atherogenic regimens. Atherosclerosis and LMOD1 expression in mouse and human coronary arteries were assessed by histopathology and confocal immunofluorescence microscopy. Coronary arteries from Lmod1WT and Lmod1SMKO mice were analyzed with assorted stains and antibodies, immunogold lineage tracing, spatial metabolomics/transcriptomics, and single-cell RNA sequencing (scRNA-seq). Mouse aortic SMCs from Lmod1WT and Lmod1SMKO mice were subjected to lipid loading with lentiviruses expressing wild-type Lmod1, a nucleation deficient Leiomodin1 (Lmod1ND), or a short hairpin RNA (shRNA) targeting Thrombospondin (Thbs1). ResultsUnder atherogenic conditions, Lmod1SMKO mice displayed unremarkable vessels in several organs but developed diffuse and occlusive coronary atherosclerosis. No such disease was observed in Lmod1WT mice. Time-course studies documented lipid insudation and VSMC foam cell formation in the coronary arteries of Lmod1SMKO mice as early as six days post-regimen. Immunogold lineage tracing demonstrated 46% of coronary plaque cells being of VSMC origin, with most showing evidence of lipid uptake. An intronic deletion of Lmod1, containing a conserved region where the single nucleotide variant associated with CAD exists, showed attenuated LMOD1 expression; heterozygous Lmod1SMKO mice, with a similar reduction in LMOD1, showed no CAD. Spatial metabolomics uncovered multiple lipid species within coronary atheromata of Lmod1SMKO mice, and spatial/scRNA-seq of similar coronary lesions disclosed altered lipid pathways with a consistent elevation in Thbs1. In vitro mechanistic studies revealed lipid accumulation in Lmod1SMKO VSMCs that was rescued by Lmod1WT, Lmod1ND, and Thbs1 shRNA. VSMC-restricted expression of Lmod1ND in mice resulted in negligible coronary atherosclerosis. ConclusionsUnder proatherogenic conditions, Lmod1SMKO mice present with rapidly manifesting coronary atherosclerosis that appears to be independent of the actin nucleation function of LMOD1. Targeting Thbs1 represents a viable strategy to mitigate VSMC foam cell formation. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIVascular smooth muscle cell (VSMC) loss of Leiomodin1 (Lmod1) causes diffuse and occlusive coronary atherosclerosis in mice, with little or no such disease in other vascular beds. C_LIO_LIA novel immunogold lineage tracing assay shows VSMC migration to the intima as early as six days following an atherogenic regimen, and quantitative studies demonstrate that 46% of coronary plaque cells are of SMC origin. C_LIO_LIThe coronary phenotype appears to be independent of LMOD1s actin nucleation activity, but VSMC lipid uptake is thrombospondin-dependent. C_LI What are the clinical implications?O_LILMOD1 is an annotated smooth muscle cell-restricted risk allele for human coronary artery disease (CAD), offering new insight into the role of smooth muscle cells in atherogenesis. C_LIO_LIThe rapidly manifesting CAD phenotype in Lmod1 knockout mice enables expedited testing of novel therapeutics to mitigate disease progression. C_LIO_LINew insight into LMOD1 pathobiology will help inform further SNV interrogation of the LMOD1 locus for CAD risk in patients. C_LI

pathology↗

bioRxiv2025

Predation limits the abundance of {triangleup}gacA cheaters in Pseudomonas protegens populations

In bacteria, defense against predators and competitors often requires cooperation within large clonal bacterial populations. Mutations that inactivate biosynthesis of costly molecules involved in cooperative defense can increase the growth rate of the mutants, while mutant cells continue to benefit from the efforts of wild type cells. The GacA-GacS signaling pathway regulates biosynthesis and secretion of toxic secondary metabolites and proteins that play crucial roles in bacterial defense in Pseudomonas species, including resistance to the protozoan predator Dictyostelium discoideum. A P. protegens {Delta}gacA mutant is vulnerable to predation but can benefit from protection provided by wild type cells in mixed populations. Wild type P. protegens sporadically infects D. discoideum fruiting bodies which may aid in bacterial dispersal to new environments. We found that the {Delta}gacA mutant does not infect fruiting bodies and that the wild type does not infect when at low abundance in the population. The P. protegens {Delta}gacA mutant is a cheater whose fitness under predation depends on its relative abundance. Protozoan predators can limit the abundance of {Delta}gacA mutants in Pseudomonas populations, preventing selective sweeps that could lead to the loss of the important but costly Gac regulon.

microbiology↗

bioRxiv2025

De Novo Negatively Charged Binders Targeting MMLVRT Nucleic Acid Binding Sites Overcome Stability-Activity Trade-offs

The limited thermostability and storage-induced inactivation of Moloney murine leukemia virus reverse transcriptase (MMLV RT) have constrained its applications. In this study, a high-stability mutant, MMLV RT-SV, was generated through a multi-site mutagenesis strategy targeting the nucleic acid-binding pocket. Through de novo protein design, three highly negatively charged binders with affinities of 45 nM, 362 nM, and 829 nM were developed to specifically target the positively charged nucleic acid-binding region on the surface of MMLV RT-SV. Experimental results demonstrated that these binders formed complexes with the enzyme via electrostatic interaction, significantly enhancing the thermostability and long-term storage stability of MMLV RT-SV while not affecting its RNA- dependent DNA polymerase function. This work pioneers the rational design of negatively charged binders targeting strongly positively charged nucleic acid-binding sites, overcoming the traditional stability-activity trade-off inherent in site-directed mutagenesis and directed evolution. The proposed strategy not only provides an innovative solution to address the thermal sensitivity and storage instability of reverse transcriptase but also establishes a novel paradigm for De Novo-based precision engineering of enzyme functions, demonstrating significant potential in molecular diagnostics, gene editing, and related fields.

bioengineering↗

bioRxiv2024

Deep Ensemble Model for Quantitative Optical Property and Chromophore Concentration Images of Biological Tissues

The ability to quantify widefield tissue optical properties (OPs, i.e., absorption and scattering) has major implications on the characterization of various physiological and disease processes. However, conventional image processing methods for tissue optical properties are either limited to qualitative analysis, or have tradeoffs in speed and accuracy. The key to quantification of optical properties is the extraction of amplitude maps from reflectance images under sinusoidal illumination of different spatial frequencies. Conventional three-phase demodulation (TPD) method has been demonstrated for the mapping of OPs, but it requires as many as 14 measurement images for accurate OP extraction, which leads to limited throughput and hinders practical translation. Although single-phase demodulation (SPD) method has been proposed to map OPs with a single measurement image, it is typically subject to image artifacts and decreased measurement accuracy. To tackle those challenges, here we develop a deep ensemble model (DEM) that can map tissue optical properties with high accuracy in a single snapshot, increasing the measurement speed by 14x compared to conventional TPD method. The proposed method was validated with measurements on an array of optical phantoms, ex vivo tissues, and in vivo tissues. The errors for OP extraction were 0.83 {+/-} 5.0% for absorption and 0.40 {+/-} 1.9% for reduced scattering, dramatically lower than that of the state-of-the-art SPD method (2.5 {+/-} 15% for absorption and -1.2 {+/-} 11% for reduced scattering). It was further demonstrated that while trained with data from a single wavelength, the DEM can be directly applied to other wavelengths and effectively obtain optical property and chromophore concentration images of biological tissues. Together, these results highlight the potential of DEM to enable new capabilities for quantitative monitoring of tissue physiological and disease processes.

biophysics↗

bioRxiv2024

RCANE: A Deep Learning Algorithm for Whole-genome Pan-Cancer Somatic Copy Number Aberration Prediction using RNA-seq Data

Transcriptome sequencing (RNA-seq) is widely used in cancer research to study the transcriptome and its role in disease progression. Somatic copy number aberrations (SCNAs) are key drivers of cancer development, and inferring SCNAs from RNA-seq data can provide critical insights for disease classification and treatment prediction. We introduce RCANE, a deep learning-based method designed to predict genome-wide SCNAs across various cancer types using RNA-seq data. RCANE is trained on data from The Cancer Genome Atlas (TCGA) and DepMap cancer cell lines, demonstrating superior performance compared to existing methods. This scalable approach offers a robust solution for improving SCNA prediction in cancer diagnostics and treatment.

bioinformatics↗

bioRxiv2024

Peripheral Inflammation is accompanied by Cerebral Hypoperfusion in Mice

IntroductionChronic pain is a disabling condition that is accompanied by neuropsychiatric comorbidities such as anxiety, depression, and cognitive decline. While the peripheral alterations are well-studied, we lack an understanding of how these peripheral changes can result in long-lasting brain alterations and the ensuing behavioral phenotypes. This study aims to quantify changes in cerebral blood perfusion using laser speckle contrast imaging (LSCI) in the murine Complete Freunds adjuvant (CFA) model of unilateral peripheral inflammation. MethodsTwenty female and 24 male adult C57BL/6 mice were randomly assigned to control (0.05ml saline) or 1 of 3 experimental groups receiving CFA (0.01ml, 0.05ml, and 0.1ml) on the right hindpaw. Three days after the intraplantar injections, animals were assessed for signs of pain (von Frey), working memory (y-maze), and anxiety (zero maze and open field), and subjected to craniotomy and in vivo LSCI of the parietal-temporal lobes. ResultsUnilateral administration of CFA resulted in signs of local inflammation, decreased mechanical thresholds on the affected hindpaw, signs of anxiety in the zero maze, as well as cerebral hypoperfusion in dose-dependent manner. DiscussionTo our knowledge, this is the first study using laser speckle contrast imaging to examine the effects of CFA-induced peripheral inflammation on cerebral blood perfusion. It serves as a first step in delineating the path by which insult to peripheral tissues can cause long-lasting brain plasticity via vascular mechanisms.

neuroscience↗

bioRxiv2024

Comparative and population genomics analyses of eared pheasants inhabiting highly varying altitudes

Oxygen pressure varies dramatically with altitudes on Earth; however, humans and animals thrive at almost all altitudes. To better understand genetic basis underlying adaptation of closely related species to varying altitudes, we annotated and compared the genome of a white eared pheasant (WT) (Crossoptilon crossoptilon) inhabiting high altitudes and the genome of a brown eared pheasant (BR) (C. mantchuricum) inhabiting low altitudes. Moreover, we compared genetic variations in populations of WT and BR as well as of blue eared pheasants (BL) (C. auritum) inhabiting intermediate altitudes, and identified thousands of selective sweeps in each species. Intriguingly, the unique genes and pseudogenes in the two genomes converge on the same set of altitude adaptation-related pathways of four functional categories as genes in selective sweeps in each species. Thus, these species appear to adapt to highly varying altitudes by diverging selection on the same traits via loss-of-function mutations and fine-tuning genes in common pathways.

bioinformatics↗

bioRxiv2024

Large scale loss-of-function mutations during chicken evolution and domestication

Evolution and domestication are often driven by genetic innovations, yet the role of gene loss remains debated. Here, we present comparative genomic analyses of four indigenous chicken breeds from Yunnan Province, China and a reference red jungle fowl genome (GRCg6a). We identify extensive gene presence-absence variation and large numbers of pseudogenes, revealing highly dynamic gene repertoires among closely related chickens. By reconstructing ancestral gene content, we estimate that the common ancestor harbored at least 21,972 genes, of which 7,993 are dispensable. Each lineage has independently lost thousands of genes through both complete gene loss and pseudogenization. These loss-of-function events are non-random: pseudogenization mutations are biased toward gene termini, frequently fixed in populations, and enriched in specific biological pathways. Notably, patterns of gene loss recapitulate phylogenetic relationships, suggesting that loss-of-function mutations are shaped by selection rather than neutral drift. Analysis of four other chicken genomes assembled using PacBio HiFi reads draws the same conclusion. Thus, our results support a model in which large-scale loss-of-function mutations are a major driver of chicken evolution and domestication, consistent with the "less-is-more" hypothesis of adaptive evolution.

bioinformatics↗

bioRxiv2024

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in sub-telomeric regions with high G/C contents

BackgroundAlthough multiple chicken genomes have been assembled and annotated, the number of protein-coding genes in chicken genomes is still uncertain due to the low quality of these genome assemblies and limited resources used in gene annotations. ResultsTo fill the gap, we annotated our four recently assembled high-quality genomes of four indigenous chickens with distinct traits using a combination of RNA-seq- and homology-based approach. Our annotated genes in the four chickens recovered 51 of the 274 "missing" genes in birds in general and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in each chicken breed, we found a total of 1,420 new protein-coding genes in the four chicken genomes, which were missed in the reference chicken genome annotations. These newly annotated genes (NAGs) tend to have high G/C contents and be located in sub-telomeric regions of almost all assembled chromosomes and some unplaced contigs. The NAGs showed tissue-specific expression and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. We found that most of the NAGs also are encoded in previously assembled chicken genomes. The NAGs form functional modules with homology-supported genes that are involved in many important biological pathways. We also identified numerous unique genes in each indigenous chicken genome that might be related to the unique traits of each breed. ConclusionThe ubiquitous presence of the NAGs in various chicken genomes indicate that they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

bioinformatics↗

bioRxiv2023

High-quality genome assembly of a white eared pheasant individual and related functional and genetics data resources

White eared pheasant (WT), (Crossoptilon crossoptilon), inhibiting at high altitudes (3000[~]4,300 m), is a Galliformes bird native to the Qinghai, Sichuan, Yunnan and Tibet Province of China. Due to the difficulty of sequencing the precious species, there is no high-quality genome assembly for the species, hampering the understanding of their genetic mechanisms. To fill the gap, we sequenced and assembled a WT individual using Illumina short reads, PacBio long reads and Hi-C reads. With a contig N50 of 19.63 Mb, scaffold N50 of 29.59 Mb, total length of 1.02 Gb and BUSCO completeness of 97.2%, the assembly is highly complete. Evaluation shows that the assembly is at chromosome-level with only six gaps. Thus, our assembly provides a valuable genetic resource for the crossoptilon species. To further provide resources for gene annotation and population genetics analysis, we also sequenced transcriptomes of 20 tissues of the WT individual and re-sequenced another 10 individuals of WT. Our assembled WT genome and the sequencing data can be valuable resources to study the crossoptilon species.

bioinformatics↗

bioRxiv2023

Arrestin-3-assisted activation of JNK3 mediates dopaminergic behavioral and signaling plasticity in vivo.

In rodents with unilateral ablation of the substantia nigra neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA or dopamine agonists induces a progressive increase of behavioral responses, a process known as behavioral sensitization. The sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of arrestin-3 knockout mice, we found that the restoration of arrestin-3 fully rescued behavioral sensitization, whereas its mutant defective in JNK activation did not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in the direct pathway striatal neurons, fully rescued sensitization, whereas an inactive homologous arrestin-2-derived peptide did not. Behavioral rescue was accompanied by the restoration of JNK3 activity and of JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-dependent JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization.

neuroscience↗

bioRxiv2023

Timing-dependent modulation of working memory by VTA dopamine release in medial prefrontal cortex

Dopamine significantly modulates working memory (WM)1-8, a fundamental cognitive function that maintains information during a brief delay period9-11. The temporal precision of dopaminergic modulation in WM and related neural mechanisms remain elusive. Here we unveiled the pivotal role of dopamine timing in WM performance with head-fixed mice engaged in learning an olfactory WM task. Through electrophysiology and optogenetics, we found that dopaminergic neurons in the ventral tegmental area (VTA) could encode WM information during the delay period, and manipulating dopaminergic neuronal activity bidirectionally modulated WM performance in an inverted U-shaped manner. Optogenetic manipulation of VTA activity also induced bidirectional changes in WM-related neural activity in the medial prefrontal cortex (mPFC). Imaging with a dopamine-sensitive GPCR-activation-based sensor (GRABDA)12,13 revealed transient dopamine peaks in the mPFC specifically during the early-delay period. Optogenetic stimulation of VTA-to-mPFC dopaminergic projections during the early- and late-delay period enhanced and impaired WM performance, respectively. Manipulations outside these periods or involving the nucleus accumbens had no effect. Single-unit recordings demonstrated that optogenetic excitation of VTA-to-mPFC projections could modulate mPFC neuronal activity in a manner consistent with behavioral modulation. Thus, the timing of dopamine release is critical in modulation of neuronal activity and behavior.

neuroscience↗

bioRxiv2023

Artificial selection footprints in domestic chicken genomes

Accurate and low-cost next generation sequencing technologies make re-sequencing of large populations of a species possible. Although many studies related to artificial selection signatures of commercial and indigenous chickens have been carried out, quite a small number of genes have been found to be under selection. In this study, we re-sequenced 85 individuals of five indigenous chicken breeds with distinct traits from Yunnan, a southwest province of China. By analyzing these indigenous chickens together with 116 individuals of commercial chickens (broilers and layers) and 35 individuals of red jungle fowl, we find a substantially large number of selective sweeps and affected genes for each chicken breed using a rigorous statistic model than previously reported. We confirm most of previously identified selective sweeps and affected genes. Meanwhile the vast majority ([~]98.3%) of our identified selective sweeps overlap known chicken quantitative trait loci. Thus, our predictions are highly reliable. For each breed, we also identify candidate genes and selective sweeps that might be related to the unique traits of the chickens.

bioinformatics↗

bioRxiv2022

Tumor microenvironments impair T cell receptor affinity and function

CD8+ T cells underpin effective anti-tumor immune responses in melanoma; however, their functions are attenuated due to various immunosuppressive factors in the tumor microenvironment (TME), resulting in disease progression. T cell function is elicited by the T cell receptor (TCR), which recognizes antigen peptide-major histocompatibility complex (pMHC) expressed on tumor cells via direct physical contact, i.e., two-dimensional (2D) interaction. TCR-pMHC 2D affinity plays a central role in antigen recognition and discrimination, and is sensitive to both the conditions of the T cell and the microenvironment in which it resides. Herein, we demonstrate that CD8+ T cells residing in TME have lower 2D TCR-pMHC bimolecular affinity and TCR-pMHC-CD8 trimolecular avidity, pull fewer TCR-pMHC bonds by endogenous forces, flux lower level of intracellular calcium in response to antigen stimulation, exhibit impaired in vivo activation, and show diminished anti-tumor effector function. These detrimental effects are localized in the tumor and tumor draining lymph node (TdLN), and affect both antigen-inexperienced and antigen-experienced CD8+ T cells irrespective of their TCR specificities. These findings implicate impaired antigen recognition as a mechanism of T cell dysfunction in the TME.

immunology↗

bioRxiv2022

Subtyping evaluation of Salmonella Enteritidis using SNP and core genome MLST with nanopore reads

Whole genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing such as Oxford Nanopore Technologies (ONT) presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. However, evaluation, standardization and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of ONT for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths under seven hours of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms.

microbiology↗