Biology subjects

Du, Y.

Publications and source records attributed to Du, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

AI-enabled reconstruction of 3D spatial multi-omics at single-cell resolution

Three-dimensional (3D) spatial multi-omics provides unparalleled insights into biological activities, yet remains technically prohibitive. Here, we introduce Histo3D-MO, a hybrid experimental-computational pipeline for reconstructing single-cell-resolution 3D spatial multi-omics maps. Notably, Histo3D-MO integrates sparse, omics-disjoint spatial measurements with dense Hematoxylin and Eosin (H&E) histology through SPatial multi-Omics from h&E imaGEs (SPONGE), achieving cell-level 3D mapping across multiple omics layers. Validated using held-out slices, SPONGE substantially outperforms existing omics prediction methods. We further developed an algorithmic suite for 3D cell-type propagation and tissue-domain annotation, enabling whole-volume characterization of the tumor microenvironment. Applied to the in-house hepatocellular carcinoma data, Histo3D-MO revealed spatially organized patterns of translation efficiency, volumetric decoupling between malignant cells and monocytes, and depth-associated monocyte differentiation trajectories. Together, these results establish Histo3D-MO as a scalable framework for reconstructing single-cell-resolution 3D spatial multi-omics and interrogating tissue organization across complex biological systems.

bioinformatics↗

bioRxiv2026

Temporal decoding of blood flow derived mechanical cues driving liver regeneration

Liver regeneration is initiated by rapid vascular changes, yet how blood flow-derived mechanical cues are decoded by liver sinusoidal endothelial cells (LSECs) remains unclear. Here, we found that partial hepatectomy generates temporally distinct mechanical cues in vivo, with a transient rise in shear stress followed by progressive sinusoidal dilation and endothelial stretch. To dissect these forces, we developed a liver regeneration chip that reconstructs sinusoidal architecture and enables independent or coupled manipulation of shear stress and mechanical stretch. Shear-dominant, stretch-dominant, and coupled mechanical modalities induce divergent LSEC regenerative programs involving extracellular matrix remodeling, cell-cycle regulation, cytoskeletal organization, and angiocrine signaling. Mechanistically, force-specific pathways, including Wnt, HIF-1, NF-{kappa}B, and Piezo1-associated signaling, mediate these outputs. Inhibition of these pathways after partial hepatectomy impairs hepatocyte proliferation and survival. These findings reveal that LSECs temporally decode blood flow-derived mechanical forces into distinct regenerative outputs, establishing endothelial mechanotransduction as an upstream regulator of liver regeneration. HIGHLIGHTS{blacksquare} Partial hepatectomy decouples transient shear from progressive stretch in vivo. {blacksquare}A liver regeneration chip recreates structure and distinct mechanical modalities in sinusoids. {blacksquare}Distinct mechanical modalities encode divergent LSEC regenerative programs. {blacksquare}Force-specific LSEC mechanotransduction supports hepatocyte proliferation and survival.

bioengineering↗

bioRxiv2026

Lactylation of Influenza Virus Polymerase Acidic Protein Promotes Viral Replication and Pathogenicity

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

microbiology↗

bioRxiv2026

Computational Design Strategies for Nanoscale 3D Auxetic Metastructures from DNA

Auxetic metamaterials exhibit negative Poissons ratio behaviors due to their architecture of periodically arranged unit cells. Although mechanical metamaterials are well established at the macroscale, programmable auxetic units remain scarce at the nanoscale. DNA origami offers a promising platform to bridge this gap, but design principles for dynamically deformable 3D auxetic nanostructures remain largely unexplored. Here, we develop design strategies for such 3D auxetic metastructures built from wireframe DNA origami. As a model system, we use a 3D re-entrant triangular unit composed of double-stranded DNA (dsDNA) bundle edges connected by single-stranded DNA (ssDNA) joints. Using coarse-grained molecular dynamics (MD) and umbrella-sampling free-energy simulations, we examine how edge design and joint-connection scheme govern auxetic responses and the energetics of the structural transformation. Our results show that auxetic performance and deformation energetics emerge from the coupled effects of DNA bundle rigidity and connector mechanics at the joints. This study provides mechanistic insights and design guidelines for programmable auxetic motion and energetics in 3D DNA origami metamaterials, advancing the development of stimuli-responsive nanomechanical devices.

biophysics↗

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2026

An Integrated muti-omics cell atlas of the human trabecular meshwork and ciliary body

The trabecular meshwork (TM) and ciliary body (CB) regulate aqueous humor dynamics and intraocular pressure (IOP), and TM/Schlemms canal (SC) dysfunction underlies glaucoma. Here, we present a spatially resolved multi-omics atlas of human TM and CB, integrating snRNA-seq, scRNA-seq, and snATAC-seq from over one million cells and nuclei across 112 donors with Xenium spatial transcriptomics. We identified 9 major cell classes and 21 cell types, revealing heterogeneity, including undercharacterized fibroblast and epithelial subpopulations. Spatial mapping supported TM fibroblast zonation and CB epithelial organization. Regulatory analyses identified cell type-specific programs, including OTX/PAX networks in CB epithelium and SMAD3/TGF-{beta} signaling in fibroblasts. Integration with glaucoma loci showed enrichment of non-coding variants in regulatory elements associated with POAG and PACG. Age- and ancestry-associated remodeling revealed divergent fibroblast aging with increased PIEZO1, suggesting impaired outflow and elevated IOP. Together, this high-resolution atlas links cellular, regulatory, and genetic variation to anterior segment function and glaucoma susceptibility.

genomics↗

bioRxiv2026

Entanglement-governed protein networks enable mechanically adaptive artificial skin for transplantation-scale skin replacement

Artificial skin substitutes that simultaneously achieve mechanical robustness, regenerative bioactivity, and transplantation-scale tissue integration remain challenging to engineer. Here we report a mechanically adaptive bilayer artificial skin based on entanglement-mediated protein networks. By integrating protein chain entanglement, flexible molecular linkers, and photo-triggered intermolecular crosslinking, we establish a hierarchically organized protein matrix with enhanced toughness, structural adaptability, and regenerative compatibility. Spatial biofunctionalization further enables integration of an antibacterial Zn{superscript 2}-coordinated epidermal layer and a regenerative CLP-EGF-functionalized dermal layer within a unified construct. The engineered skin promotes cellular proliferation through PI3K-AKT-mTOR activation, exhibits sustained antibacterial activity, and supports large-area full-thickness skin replacement covering approximately 40% of the dorsal skin surface in mice. The construct further accelerates diabetic wound repair and extracellular matrix remodeling in vivo. These findings establish entanglement-mediated protein engineering as a strategy for mechanically adaptive regenerative biomaterials and provide a platform for transplantation-scale skin regeneration.

bioengineering↗

bioRxiv2026

Robust integration of weakly anchored spatial multi-omics

Spatial multi-omics holds great promise for dissecting complex biological processes, though inherent technical constraints continue to limit its widespread adoption. Currently, most studies therefore measure distinct omics features on separate tissue sections, necessitating spatial diagonal integration. An emerging practical solution is to leverage hematoxylin and eosin (H&E) images as an integration anchor, given their ubiquity, low cost, and compatibility across tissue preparations. However, this anchor is frequently compromised in real-world settings by variations in H&E staining style, absence of reliable histological landmarks, and mismatches in spatial resolutions across omics modalities. To address this, we introduce SpaWeaver, a computational framework that couples a pathology foundation model with a graph Transformer and a latent feature aligner module, providing a highly robust solution for weakly anchored spatial omics data diagonal integration. Extensive experiments demonstrate that SpaWeaver exhibits superior robustness against isolated or synergistic weak-anchoring factors. The spatial multi-omics profiles generated by SpaWeaver link molecular features originally separated on two sections, unlocking diverse downstream analyses once exclusive to co-assayed spatial multi-omics data, including niche-aware cell-cell communication inference and multi-omics resolved cell state. In this study, it unveils tumor-distance-dependent fibroblast-CD4{square}T-cell signaling in human colon adenocarcinoma and identifies a hypoxic glycolytic tumor state with pyknotic nuclei in human ovarian cancer. Overall, our approach bridges readily accessible single-omics measurements across weakly anchored tissue sections, enabling unified spatial multi-omics characterization and system-level tissue analysis.

bioinformatics↗

bioRxiv2026

HOMED enables hierarchical and multimodal optimization of DNA methylation deconvolution across tissues

Cellular heterogeneity is a major confounder in bulk DNA methylation data for epigenome-wide association studies. Existing reference-based DNAm deconvolution methods often ignore hierarchies among related cell types and may generalize poorly across datasets due to limited variability in reference profiles. We developed HOMED (Hierarchically Optimized Methylation Deconvolution), a framework that integrates cell-lineage hierarchies, single-cell RNA sequencing-guided deconvolution, and paired bulk RNA-seq/DNAm data for CpG signature optimization. Across simulated and real peripheral blood mononuclear cell, lung, and placental datasets, HOMED consistently yielded the highest PCCs and lowest RMSEs, outperforming existing scRNA-seq-guided DNAm deconvolution methods, improving accuracy, resolution, and cross-tissue generalizability.

bioinformatics↗

bioRxiv2026

Chronic Stress Exacerbates Long-term Microvascular Network Dysfunction Following Brain Trauma

BackgroundPreexisting factors are among the strongest predictors of recovery following traumatic brain injury (TBI), with chronic stress closely associated with permanent disability and worse long-term outcomes. While chronic cerebrovascular dysfunction is linked to these poor trajectories, as impaired blood flow regulation drives secondary disease progression, the mechanisms regulating this vascular failure remain incompletely understood. Crucially, how premorbid chronic stress and TBI disrupt the fundamental coordination of the cerebrovascular network post-injury remains unknown. MethodsTo elucidate this coordination at rest and in response to increased neuronal activity, we used a model of three repeated moderate closed cortical impacts comorbid with chronic stress induced by social isolation (SI) post-weaning. Our previously developed vascular analysis pipeline (NOVAS3D) was employed to estimate changes in vascular radii across cerebrovasculature proximal to neuronal activation. Arteries and veins were annotated in the imaged volumes to allow for blood flow simulations. ResultsUsing graph-based network analysis, we demonstrate that TBI, when compounded with chronic stress, critically disrupts the long-range coordination of the capillary network. Specifically, the functional coordination of radius changes between nearby, non-adjacent capillaries was reduced by 40{+/-}20% in TBI+SI mice relative to controls. Consequently, simulations estimated that the vascular networks in TBI+SI mice experienced a 68{+/-}7% reduction in arterial red blood cell velocity (VRBC) responses to neuronal activation. These network-wide impairments were fundamentally driven by severely blunted vessel reactivity, including a 28{+/-}7% decrease in the magnitude of arteriolar dilations and a 47{+/-}7% decrease in the magnitude of arteriolar constrictions. ConclusionThese findings provide a mechanistic foundation for worse clinical outcomes seen in TBI patients with comorbid chronic stress, identifying arteriolar reactivity and long-range capillary coordination as critical therapeutic targets to mitigate secondary injury and improve long-term recovery.

neuroscience↗

bioRxiv2026

Cortical Organoid Model of PPP2R5D Genetic Intellectual Disability Models Disease Severity Phenotype

Jordans Syndrome (JS) is a rare, neurodevelopmental disorder caused by de novo missense mutations in protein phosphatase 2 regulatory subunit Bdelta (PPP2R5D). JS is characterized by severe neurological impairments starting in early life. PPP2R5D encodes for B56{delta}, one of the regulatory subunits of protein phosphatase 2A (PP2A). PP2A is a heterotrimeric protein serine/threonine phosphatase that is highly expressed in the brain and the liver. Past studies have focused on PP2As role in liver and little is known about the holoenzymes behavior in neuronal cells. Although B56{delta} is known to play an important role in the substrate specificity of PP2A, the identification of validated downstream substrates in JS remains unclear. To better understand how the mutations affect neuronal cells, we developed cerebral cortical-like organoids from an engineered allele series of the most common JS mutations to characterize the physiological changes throughout different stages of neurodevelopment. Organoids were assessed for transcriptomic, protein, and electrophysiological changes utilizing bulk RNA sequencing, immunocytochemistry, Western Blot, and high-density MicroElectrode Array. The results identify differentially expressed genes and translated proteins, potential neuronal substrates, and significant electrophysiological signatures that suggest mutations in B56{delta} lead to variant-specific dysfunction of PP2A. Overexpression of PPP2R5D through AAV transduction of organoids rescued several phenotypes in the variants, suggesting different pathogenetic etiology underneath. Our findings successfully characterized cerebral cortical-like organoids in JS cell lines and demonstrated its potential as a model for studying neurodevelopmental disorder and for screening therapeutic approaches.

cell biology↗

bioRxiv2026

Programmable assembly of higher-order DNA nanostructures from microbial ssDNA staple libraries

Bottom-up manufacturing of structural DNA nanotechnology requires a long single-stranded DNA (ssDNA) scaffold and hundreds of short ([~]30 nt) ssDNA staples. However, scaling production remains bottlenecked by the high economic cost and environmental footprint of solid-phase chemical staple synthesis. To address these limitations, a phage-free, biological nanomanufacturing platform engineered in Escherichia coli is developed here. Two intracellular strategies for producing programmable ssDNA were systematically evaluated: retron-based multicopy ssDNA (msDNA) synthesis via the Ec67 system and plasmid-encoded rolling circle replication (RCR). While native structural topology constraints within the retron (msd) cassette limit its sequence-design flexibility, the alternative RCR-driven engine successfully decouples ssDNA replication from sequence secondary structures, enabling the synthesis of arbitrary staples. This RCR platform reliably generates long circular ssDNA (cssDNA) precursors of at least 1.8 kb with exceptional sequence fidelity (>99%). Integrating programmable BseGI cleavage sites allows targeted strand-selective enzymatic processing to cleanly release stoichiometric, origami-grade pools of 32-nt staple strands. Atomic force microscopy (AFM) confirms that these biologically produced staples successfully drive the high-fidelity self-assembly of complex DNA tiles and hollow tubules. Crucially, robust structural folding is demonstrated directly within crude, unpurified cellular lysates, establishing a green, cost-effective framework for the one-pot fabrication of advanced DNA-based nanomaterials.

synthetic biology↗

bioRxiv2026

Drosophila-virus genotype interactions dominate transmission, virulence and load, eroding additive fitness variance

Host-parasite coevolution is expected to generate strong selection for susceptibility and infectivity that has the potential to erode genetic variation. Despite this, natural populations often retain extensive genetic variation in these traits. Negative frequency-dependent selection could explain the maintenance of variation since it results in genotype-by-genotype interactions on fitness that, unlike additive genetic variance among hosts and pathogens, is masked from selection and so can remain cryptic. Here we combine coevolutionary modelling with large-scale experimental infection assays to quantify how host-pathogen interactions structure fitness variance in a vertically transmitted virus system, Drosophila melanogaster and sigma virus. Simulations show that coevolution typically erodes additive genetic variance in host and pathogen fitness, concentrating variance in host-virus interaction terms. Consistent with these predictions, experiments spanning 90 host-virus genotype combinations reveal that transmission, viral load and virulence are overwhelmingly governed by host-virus genetic interactions rather than host or virus main (i.e., additive) effects. As a result, neither host resistance alleles nor viral genotypes confer consistently higher fitness across genetic backgrounds. Interactions tend to be sex-specific, further limiting heritable fitness variation. Our results demonstrate that coevolution can substantially mask heritable genetic variation from selection by rendering fitness context dependent. This extensive cryptic genetic variation may be revealed either when ecological or evolutionary conditions shift, or when the process of coevolution itself alters the direction of selection. This demonstrates the need to account for genotype-specific interactions when forecasting evolutionary responses.

evolutionary biology↗

bioRxiv2026

CRISPR/Cas9-based knockout screening revealed GSK3β as a key regulator for structural plasticity of axon initial segment

The axon initial segment (AIS) undergoes structural plasticity to tune neuronal excitability, yet the underlying molecular mechanisms remain unclear. Here we developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to identify molecules that regulate developmental AIS shortening in the chicken nucleus magnocellularis. We targeted fourteen molecules associated with microtubules and found that knockout of either glycogen synthase kinase 3{beta} (GSK3{beta}) or Tau impaired the AIS shortening. Conversely, overexpression of a constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This GSK3{beta}-induced shortening was reproduced in slice cultures and suppressed by microtubule stabilization. Together, these findings identify GSK3{beta}-dependent microtubule remodeling as a mechanism underlying developmental AIS shortening and establish an in vivo genetic approach for molecular screening in chick embryos.

neuroscience↗

bioRxiv2026

Novel tool use does not depend on mechanical reasoning: evidence from apraxia

A hallmark of human intelligence is the ability to use tools. Yet the cognitive processes supporting this ability remain debated. One contemporary view holds that mechanical reasoning is central for tool use, especially in the case of tools with which we have no prior experience. However, previous support for the role of mechanical reasoning often relies on circular logic, wherein poor performance on novel tool-use tasks is taken as evidence that impaired mechanical reasoning causes tool-use deficits in limb apraxia. To address this limitation, we independently assessed mechanical reasoning and novel tool use in separate tasks in individuals with limb apraxia, and compared their performance to individuals without apraxia. We also examined whether these two abilities are similarly associated with other cognitive abilities including motor imagery, mental rotation of non-body objects, general reasoning, and spatial working memory. Finally, we explored brain-behavior relationships using support vector regression lesion-symptom mapping. Our behavioral and imaging data together showed that mechanical reasoning does not underlie novel tool-use deficits in apraxia. Graphical analysis further revealed that novel tool use and mechanical reasoning loaded onto distinct latent clusters: novel tool use was strongly associated with other praxis abilities yet separable from cognitive abilities that require reasoning and mental simulation, whereas mechanical reasoning was primarily linked to other high-level reasoning abilities but not tool use. These findings challenge the notion that mechanical reasoning is central to tool-use ability, and instead suggest that tool use is more likely to be an intuitive or automatic process.

neuroscience↗

bioRxiv2026

MurineCyto-Det: A High-Resolution Murine BALF Cytology Dataset for Leukocyte Segmentation and Detection

Automated analysis of murine bronchoalveolar lavage fluid (BALF) cytology is important for preclinical respiratory research, yet progress has been limited by the lack of publicly available, well-annotated mouse BALF image datasets. We present MurineCyto-Det, a high-resolution murine BALF cytology dataset comprising 333 image tiles of size 1024x1024 pixels, annotated across five cytological categories with both pixel-level segmentation masks and one-to-one matched bounding boxes. The dataset contains 14,551 annotated cell instances and supports two complementary analysis tasks: morphology-oriented cell segmentation and object-level cell detection. To establish reproducible benchmark baselines, we evaluated representative segmentation and detection models. The results demonstrate the practical utility of MurineCyto-Det while highlighting realistic challenges arising from class imbalance, small object size, irregular cell morphology, and ambiguous debris-like structures. MurineCyto-Det provides a standardized resource for developing, evaluating, and comparing automated methods for murine BALF cytology analysis. The dataset is publicly available at https://doi.org/10.5281/zenodo.17608677.

bioinformatics↗

bioRxiv2026

Dual regulation of chemical stress-induced DDI2/3 expression by a transcription factor Fzf1 and nucleosome in Saccharomyces cerevisiae

DDI2 and DDI3 (DDI2/3) are duplicated genes in Saccharomyces cerevisiae that exhibit strong induction by a transcription factor Fzf1 in response to chemical treatments like cyanamide (CY) and methyl methanesulfonate (MMS). Although, like DDI2/3, SSU1, YHB1 and YNR064C also contain an Fzf1-binding consensus sequence CS2 and are coordinately regulated by Fzf1, these genes are only modestly induced by CY and MMS. To identify additional cis-acting elements in the DDI2/3 promoter, we made DDI2/3 promoter deletions in a reporter system and identified upstream repressing sequences (URS) spanning 480 nucleotides. To test a hypothesis that the chromatin structure constitutes the URS, we utilized a yeast strain capable of histone H3/H4 depletion by shifting carbon sources. Following histone depletion, DDI2/3 were strongly induced in an Fzf1 dependent manner, while YHB1 was repressed. Interestingly, under histone depletion conditions, CY or MMS treatment further increased expression of all Fzf1-regulated genes to comparable levels in an Fzf1 dependent manner. A genome-wide MNase-seq analysis showed that CY treatment reduced the nucleosome occupancy at the mapped DDI2/3 URS region in wild-type cells, but not in in fzf1{Delta} cells. These findings collectively indicate that Fzf1 plays dual roles in regulating the DDI2/3 response to CY. Firstly, it binds CS2 and serves as a transcription activator. Secondly, it is required for the chromatin remodeling at URS. This two-tier regulation at the DDI2/3 promoter helps to explain why DDI2/3 achieve much higher fold induction by CY and MMS than other Fzf1-regulated genes, suggesting Fzf1 to be a candidate pioneer transcription factor.

molecular biology↗

bioRxiv2026

Mitag4taxa: Extracting SSU rRNA Illumina reads from metagenomes for taxonomic classification

The prevailing taxonomic profiling methods for an environmental sample rely heavily on PCR amplification of SSU ribosomal RNA (rRNA) genes and genome-based reference databases. Identification and extraction of Illumina metagenomics sequencing data are PCR independent but technically challenging in recognition of the SSU rRNA fragments. Here we present Mitag4taxa, a computational pipeline designed for taxonomic profiling of microbial communities from metagenomic Illumina sequencing reads containing rRNA tags (mitag). A Hidden Markov Model (HMM) of SSU rRNA genes and those for the V4 region of 16S rRNA and the V9 region of 18S rRNA genes were created, respectively, using the representative sequences of different families and corresponding hypervariable regions in the SILVA database. The pipeline identifies and extracts 16S and 18S rRNA gene fragments along with the quality score from metagenomic or metatranscriptomic datasets using HMM search integrated with the models. The hypervariable regions, including the V4 region of 16S rRNA and the V9 region of 18S rRNA genes, can be further scanned and recruited for taxonomic classification and biodiversity estimate. To demonstrate its high reliability, the performance of Mitag4taxa was evaluated using both real and simulated datasets. In human gut metagenomic assessments, taxonomic profiles derived from Mitag4taxa showed high consistency with those based on conventional 16S rRNA gene amplicons, identifying dominant families such as Bacteroidaceae and Prevotellaceae with similar relative abundances. Statistical analyses confirmed highly significant positive correlations between Mitag4taxa and amplicon-based community structures. The 18S V9 module was further validated using shotgun metagenomic data from deep-sea sediment cores, successfully recovering key eukaryotic taxa such as Collodaria and Leotiomycetes. Furthermore, benchmarking against the RiboTagger software using CAMI marine simulated datasets revealed that Mitag4taxa achieved a higher average F1 score and lower error metrics. Overall, Mitag4taxa provides a complementary rRNA gene amplicon- and genome-independent strategy for microbial community profiling, enabling improved detection of both prokaryotic and eukaryotic taxa from metagenomic and metatranscriptomic sequencing data.

bioinformatics↗