Biology subjects

Zhao, X.

Publications and source records attributed to Zhao, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

Ripk1 Deficiency or K376R Mutation in Dendritic Cells Promotes Potent Antitumor Immunity

Dendritic cells (DCs) are critical for initiating adaptive immunity through antigen presentation and T cell priming. While immunogenic cell death (ICD) enhances anti-tumor immunity, how programmed cell death in DCs shapes tumor immune responses remains unclear. Receptor-interacting protein kinase 1 (RIPK1) regulates apoptosis, necroptosis, and inflammation. Here, we show that mice with disrupted RIPK1 ubiquitination on K376 (Ripk1fl/K376R Cd11c-Cre, Ripk1DC-KO/K376R) develop inflammation and autoimmunity, phenocopying DC-specific RIPK1-deficient(Ripk1fl/fl Cd11c-Cre, Ripk1DC-KO) mice. These phenotypes were rescued by the genetic ablation of Ripk3 or Mlkl, establishing that RIPK1 ubiquitination is essential for suppressing DC necroptosis to maintain immune homeostasis. Remarkably, both Ripk1DC-KO and Ripk1DC-KO/K376R mice exhibit robust resistance to tumor growth, characterized by significantly enhanced T cell infiltration and activation. Mechanistically, we show that Ripk1 deficiency or the loss of its K376 ubiquitination augments antigen-presenting capacity of DCs through both necroptosis-dependent and -independent pathways. Furthermore, adoptive transfer of T cells from Ripk1DC-KO mice further amplifies anti-tumor immunity in recipient hosts. These results identify RIPK1 as a critical immunological checkpoint in DCs that constrains their immunogenic potential. Targeting this pathway represents a promising therapeutic strategy to potentiate T cell-mediated cancer immunotherapy.

immunology↗

bioRxiv2026

Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

The {gamma}- to {beta}-globin switch is intricately regulated during human ontogeny, and this process is manipulated for therapeutic approaches to treat {beta}-hemoglobinopathies by activating {gamma}-globin expression. Several genetic strategies to reactivate HbF have partially reversed the {gamma}- to {beta}-globin switch and ameliorated the clinical symptoms of {beta}-hemoglobinopathies. However, whether the {gamma}- to {beta}-globin switch can be completely reversed remains unknown. Completely reversing the {gamma}- to {beta}-globin switch requires a thorough redirection of the locus control region (LCR) from interacting with the {beta}-globin gene (HBB) to interacting with the {gamma}-globin gene (HBG). Here, we found that disrupting the KLF1-mediated HBB-LCR interaction by mutating the CACCC motif in HBB leads to the release of the LCR and its retargeting to other {beta}-like globin genes. Moreover, simultaneously disrupting the KLF1-mediated HBB-LCR interaction and the epigenetic repression of HBG by combined editing of the CACCC motif in HBB and the TGACCA motif in HBG reinforces the HBG-LCR interaction, resulting in almost exclusive {gamma}-globin expression while nearly absent {beta}-globin expression, achieving near complete reversal of the {gamma}- to {beta}-globin switch. This finding demonstrates the comprehensive regulation of the {gamma}- to {beta}-globin switch by gene competition and gene silencing mechanisms. This finding also suggests that silenced genes can be fully activated through the redirection of enhancer-promoter contacts and that the specificity of enhancer-promoter contact within chromosomal domains is achieved through the transcription factor clusters binding to enhancers and promoters. Combined editing of the CACCC&TGACCA motifs also offer a more optimal therapeutic strategy for {beta}-hemoglobinopathies.

molecular biology↗

bioRxiv2026

Prime-InDel version 1: A web tool providing InDel markers for targeted regions narrowing

While high-density SNP genotyping enables genome-wide assays in rice (Oryza sativa L.) and other crops, PCR-based markers--particularly those derived from insertion-deletion (InDel) variations--remain crucial for fine-mapping. Currently, readily available primer information for high-density InDel rice markers is still limited. We present the first version of PrimeInDel (https://rfgb.rmbreeding.cn/search/variation/primeIndel), an online tool integrating three sets of InDel markers: an 8K-set and a 316K-set from the 3K Rice Genomes (3K-RG) project, and a 22K-set compiled from published literatures. In user cases, primers from PrimeInDel proved highly effective, narrowing a cold-tolerance QTL (qSR2) from 2.7 Mb to 200 kb and a heading-date QTL (qDeh1.1) from 239.5 kb to 83.6 kb. Our work provides the community with a robust, PCR-based InDel primer, accessible via an online data set, which will facilitate germplasm genotyping and gene identification in rice breeding.

plant biology↗

bioRxiv2026

Global mRNA 3'UTR lengthening in small-cell neuroendocrine carcinoma

Small-cell neuroendocrine carcinoma (SCNC) is a rare but highly malignant tumor subtype that primarily arises in the lung, also rarely in other organs, and as a consequence of treatment induced lineage transdifferentiation of prostate adenocarcinomas. The molecular convergence of SCNC across diverse tissues enables its identification through conserved SCNC-specific molecular markers, facilitating tumor subtype classification. As a critical post-transcriptional regulatory mechanism, alternative polyadenylation (APA) modulates 3'UTR length and significantly impacts tumor progression. However, its role in SCNC remains largely unclear. Here, we report a global 3'UTR lengthening pattern driven by APA in SCNC. We identified a set of conserved 3'UTR lengthening events across SCNCs of different tissue origins, which are strongly associated with neural development and related signaling pathways. Furthermore, we developed a neural network-based prediction model to classify SCNC by leveraging these specific APA signatures. Our study provides new insights into the post-transcriptional landscape of SCNCs and highlights APA signatures as promising biomarkers for SCNC identification.

cancer biology↗

bioRxiv2026

Hyperactivated YAP1 Drives an Invasive EMT Subtype of Cervical Squamous Cell Carcinoma

Cervical cancer (CVC) is classically understood as an HPV-driven disease in which high-risk HPV infects cervical basal epithelial cells, induces neoplastic transformation, and drives upward epithelial expansion, pathogenic events underlying the success of cytology- and HPV-based screening. With widespread HPV vaccination and screening, the WHO has launched the CVC elimination initiative and believes that CVC will be the first cancer that would be eliminated as a public health problem. However, statistics showed that CVC remains the most common gynecologic malignancy worldwide, and in the United States, where HPV testing and Pap smears have reached near-maximal implementation, the CVC mortality rate has plateaued for more than two decades. These epidemiologic trends suggest the existence of a subset of CVC that can escape current screening strategies. Here, we demonstrate that hyperactivation of YAP1 caused by disruption of Hippo-YAP signaling is sufficient to induce a subtype of HPV-independent invasive CVC that lacks surface lesions and therefore evades HPV- and cytology-based detection. Using single-cell RNA sequencing combined with high-resolution spatial transcriptomics, we define the cellular architecture, molecular pathways, and immune microenvironment underlying this invasive subtype. We show that YAP1-driven tumors adopt an EMT-high transcriptional state and selectively recruit immunosuppressive myeloid-derived suppressor cells that functionally interact with cancer cells to promote invasion and progression. Our findings suggest that targeting the disrupted Hippo signaling may offer a new strategy to prevent the subset of invasive CVC that current HPV- and Pap-based programs cannot detect, an essential step toward achieving global CVC elimination.

cancer biology↗

bioRxiv2026

TcrDesign: De novo design of epitope specific full-length T cell receptors

T cell receptors (TCRs) are essential for adaptive immune recognition. Recently computational tools have been developed to predict the interactions between TCR and epitope, and artificial intelligence models have been proposed to generate the complementarity determining region 3 (CDR3) region of {beta} chain TCR. However, de novo design of experimentally validated, functional, full-length epitope-specific TCRs remains a significant challenge. Here we developed TcrDesign, a deep learning framework based on large-scale unlabeled TCR and epitope datasets to generate epitope-specific, full-length TCRs. TcrDesign comprises two modules: TcrDesign-B for TCR-pMHC binding prediction with state-of-the-art accuracy, and TcrDesign-G for functional full-length TCR sequence generation. Pre-trained on large-scale unlabeled datasets using transformer-based architectures, TcrDesign achieves state-of-the-art performance in both TCR-epitope binding prediction and de novo TCR sequence generation. Furthermore, epitope-major histocompatibility complex (MHC) binding and functional activation of TcrDesign-generated TCRs were experimentally validated. TcrDesign provides an efficient and modular approach for designing epitope-specific full-length TCRs, with experimental validation confirming its utility.

bioinformatics↗

bioRxiv2026

Subchondral H-type Blood Vessel Formation Aggravates Articular Cartilage Degeneration through LEP-LEPR Axis

BackgroundOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage deterioration that has resulted in severe physical and economic costs for society. Risk factors for osteoarthritis encompass genetic predisposition, mechanical influences, obesity, inflammation, and metabolic problems. Recent reports indicate that the morphological alterations of subchondral bone evolve dynamically with the advancement of osteoarthritis. A significant alteration in subchondral bone is the development of aberrant blood vessels, particularly H-type vessels. H-type arteries are essential for preserving subchondral bone homeostasis. They support nutrients and oxygen to bone tissue and eliminate waste, thereby supporting the metabolic activities of osteoblasts and osteoclasts. However, the upstream factors governing H-type arteries are yet unknown. MethodsThe whole transcriptome analysis was performed by informatics which is further verified in clinical samples. Using LEP and LEPR knockout mice, the correlation between abnormal subchondral bone metabolism and LEP, LEPR, and H-type vessel formation were uncovered. ConclusionsThis study reveals that the lipid metabolism pathway was highly enriched on the OA lesion, and LEP and LEPR were markedly underexpressed in the subchondral bone on the side with severe OA lesions. The causal relationship of the LEP-LEPR-CD31 signalling pathway in aging and osteoarthritis models is revealed at the overall and molecular levels. This project will provide a new theoretical basis for the pathogenesis of early OA and new ideas and targets for the clinical treatment of OA by exploring the LEP-LEPR-CD31 signalling in osteoarthritis.

pathology↗

bioRxiv2026

Benchmarking algorithms for RNA velocity inference

RNA velocity is a computational framework for single-cell RNA sequencing (scRNA-seq) that estimates the future transcriptional state of individual cells, thereby capturing the direction and rate of cell state transitions rather than providing a purely static snapshot. Since its introduction in 2018, multiple RNA velocity methods have been developed, differing in their modeling assumptions, required inputs, computational complexity, and robustness. However, there remains limited consensus on how best to evaluate these methods or on which tools are most reliable under specific biological and technical settings. Here, we perform a systematic comparison of 29 velocity inference algorithms across 114 simulated datasets with known ground-truth cell dynamics and 62 real scRNA-seq datasets, and we extend the evaluation to spatial and multi-omics levels where velocity is increasingly applied. We benchmark RNA velocity methods using a unified framework that decomposes performance into four practical dimensions: accuracy, scalability, stability, and usability. Our results show that performance rankings vary substantially across metrics and datasets, indicating no single method is uniformly optimal and that practical deployment is often constrained by feasibility and robustness as much as by accuracy. Based on these results, we provide actionable guidance for selecting RNA velocity tools according to data modality, available priors, and computational constraints. Finally, we identify key bottlenecks that currently limit RNA velocity development and deployment, including scalability to large size of datasets, sensitivity to gene selection, and the lack of genuinely multimodal and spatially explicit velocity models for spot-based technologies.

bioinformatics↗

bioRxiv2025

COMPARATIVE METAGENOMIC ASSESSMENT OF SHORT- AND LONG-READ SEQUENCING TECHNOLOGIES REVEALS UNKNOWN MICROBIAL INFORMATION IN A COMPLEX ENVIRONMENTAL SAMPLE

Metagenomics enables comprehensive exploration of microbial communities but is influenced by library preparation and sequencing technologies, affecting recovery of microbial genomes and proteins. Here, we benchmarked six Illumina short-read library kits at 2x150 bp and 2x250 bp read lengths alongside PacBio HiFi long-read sequencing using a complex environmental sample. Longer short reads (2x250 bp) combined with optimal library preparation approaches notably improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results similar to those of long-read sequencing. Although long reads yield more contiguous and complete genomes, longer short reads offer a cost-effective, scalable alternative for uncovering microbial and functional diversity. These findings provide critical guidance for metagenomic experimental design, demonstrating the importance of strategic selection of library preparation chemistry and sequencing parameters in revealing unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCEMetagenomic outcomes are strongly influenced by library preparation and sequencing strategies, yet their combined effects in complex environmental samples remain poorly defined. Here, we provide the first direct comparison of Illumina NovaSeq short-read metagenomic sequencing at 2x150 bp and 2x250 bp across multiple library preparation kits, alongside PacBio long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. Optimized short-read sequencing at 2x250 bp recovered high-quality MAGs approaching those obtained with long-read technologies while substantially improving protein discovery compared to 2x150 bp at the same sequencing depth. Together, these results provide actionable guidance for experimental design and reveal how sequencing read length can be leveraged to improve the recovery of microbial and functional diversity in complex biomes.

genomics↗

bioRxiv2025

Temporal boundary gating of auditory sensitivity

Perception unfolds over time, but whether continuous sounds are sampled with uniform sensitivity is unknown. We combine human psychophysics, EEG and rodent neurophysiology to show that local auditory change detection is strongly gated by stimulus boundaries. In humans, brief perturbations inserted at different temporal positions within 0.5-1-s tones revealed an inverted U-shaped sensitivity profile: detection was attenuated near sound onset and offset and maximal mid-epoch, with EEG change responses showing a closely matching dependence on change timing. Electrocorticography in awake rats exhibited homologous temporal weighting, and analogous profiles for amplitude changes and visual motion demonstrated cross-feature and cross-modal generality. To uncover circuit mechanisms, we recorded single units along the inferior colliculus-medial geniculate body-auditory cortex pathway together with laminar local field potentials in A1. Onset-locked suppression of change responses emerged in midbrain and was progressively amplified in thalamus and cortex, whereas the full start-end profile was expressed in granular-layer alpha-gamma power. A simple biophysically grounded model in which onset responses saturate and cortical populations integrate over finite temporal windows recapitulates this pattern, explaining how stimulus boundaries disrupt prospective and retrospective integration and thereby degrade change detection near the beginning and end of sounds.

neuroscience↗

bioRxiv2025

TUPAD: A Tn5-Mediated, Pre-amplification/Adapter-Dimer-Free Platform for Ultra-Uniform, One-Step PGT Profiling with High Stability and Broad Compatibility

High-throughput sequencing stands as a cornerstone of clinical molecular diagnostics, particularly in preimplantation genetic testing (PGT)--a key application where the selection of chromosomally normal embryos is critical to enhancing in vitro fertilization (IVF) success rates. A fundamental challenge inherent to PGT, however, lies in the ultra-low gDNA input (typically <100 pg) from embryo biopsies. To mitigate this limitation, traditional workflows rely on whole genome amplification (WGA) technologies, such as MDA, PicoPlex, and MALBAC. Yet these WGA-based protocols inherently entail pre-amplification of gDNA prior to standard library construction---a step that introduces significant coverage bias and compromised uniformity. These are intrinsic flaws irreparable by downstream library preparation or bioinformatics analysis, ultimately leading to clinical uncertainty in the interpretation of copy number variations (CNVs). In this study, we developed TUPAD (Tn5-Mediated, Uniform Pre-amplification-free and Adapter-Dimer-Free Library preparation), a transformative technology that bypasses the limitations of traditional WGA. By engineering a novel adapter-dimer-free transposon system, TUPAD enables high-efficiency library construction from as little as 1 picogram (pg) of gDNA. This breakthrough allows for a pre-amplification-free workflow, effectively eliminating the primary source of amplification bias. Compared to existing WGA standards, these advantages are fully retained in embryo biopsy and positive sample testing, TUPAD consistently generates highly uniform library data, significantly enhancing the detection limit and accuracy of CNVs and other genomic variants. Beyond these performance advantages, TUPAD also cuts wet-lab processing time by 50%. Practical validation confirms TUPAD results are stable, reliable, with proven practical utility in PGT testing.

molecular biology↗

bioRxiv2025

Microgradients in porosity and canal diameter in femur bone

Bone exhibits hierarchical structural gradients that optimize mechanical performance and regenerative potential. Longitudinal and radial variations in cortical porosity and canal architecture influence load distribution, vascularization, and remodeling. Understanding these gradients is essential for designing scaffolds and implants that mimic native bone structure and function. This study quantified longitudinal and radial microgradients in cortical porosity and canal diameter along the rabbit femur and explored their implications for bone regeneration and repair implant design. Rabbit femora were divided into proximal, mid-shaft, and distal regions. High-resolution micro-computed tomography quantified cortical thickness, porosity, and canal diameter along radial and longitudinal axes in micron-scale resolutions. Compressive mechanical testing of cortical slices determined local moduli, which were correlated with microstructural parameters to establish structure-function relationships. Cortical thickness peaked at the mid-shaft and decreased toward both ends. Porosity and canal diameter increased radially toward the medullary cavity and longitudinally toward the bone ends. Upto 500 micron cortical thickness from the outer surface toward modullary cavity, porosity and canal diameter ranged, respectively, from ~5% and 40 {micro}m at the mid-shaft to ~40% and 110 {micro}m at the ends. At 750 micron cortical thickness, porosity and canal diameter ranged, respectively, from ~5% and 50 {micro}m at the mid-shaft to ~80% and 200 {micro}m at the ends. As expected, compressive moduli declined linearly with increasing porosity and canal size. The mid-shaft, with the lowest porosity and smallest canals, exhibited the highest modulus of around 15{square}MPa, which decreased to 5{square}MPa toward the ends. The rabbit femur displays distinct longitudinal and radial microgradients in porosity and canal architecture that govern local stiffness. These gradients define structural benchmarks for designing functionally graded tissue engineering scaffolds and bone implants that replicate native tissue structure and stiffness transitions to promote osteoconduction, osteoinduction, osteogenesis in bone regeneration and improve osseointegration of bone implants.

bioengineering↗

bioRxiv2025

Echoes as Signal, Not Noise: Reverberation Sharpens Sensitivity to Temporal Structure

Sensitivity to temporal structure is fundamental to hearing, yet how natural reverberation shapes this sensitivity is unclear. Here we identify echo-facilitated temporal sensitivity (EFTS), a principle whereby the auditory system exploits echoes to selectively enhance responses to rapid temporal changes. Using transitional click trains with subtle inter-click-interval (ICI) shifts, we first show that increasing echo strength systematically enlarges the sound-level step at ICI transitions in both recorded and simulated stimuli, sharpening the physical boundary between segments of distinct temporal structure. In humans, psychophysical detection of ICI changes improves monotonically with echo level in both simulated and real free-field environments, while a late EEG change response scales with echo strength as onset responses remain comparatively stable. In awake rats, electrocorticography over auditory cortex reveals parallel echo-dependent enhancement of transition-evoked activity. Single units exhibit echo-level-dependent amplification of change-related firing and improved neurometric discriminability. Laminar local field potential and current source density analyses further show that echo-dependent divergence emerges first in granular and infragranular layers before propagating to supragranular cortex, consistent with thalamocortical drive followed by intracortical amplification. Together, these findings establish EFTS as a cross-species mechanism that reframes echoes as structured signals the brain uses to sharpen temporal integration in everyday listening.

neuroscience↗

bioRxiv2025

Post-weaning social isolation impairs the orbitofrontal cortical circuit subserving contagious pain and prosocial behaviors

Empathic behaviors are sensitive to environmental factors like post-weaning social isolation (SI), yet the mechanisms by which SI affects empathy remain unclear. Here, we show that mice subjected to SI exhibit marked impairments in contagious pain and prosocial behaviors, including allo-grooming and allo-licking toward cagemates experiencing inflammatory pain. Mechanistically, we identify the glutamatergic projection from the ventromedial thalamic nucleus (VM) to the orbitofrontal cortex (OFC) as critical for these empathy-like responses. SI induces hypoexcitability of OFC glutamatergic neurons and attenuates excitatory synaptic transmission within the VM[->]OFC pathway. Remarkably, chemogenetic activation of OFC neurons or the VM[->]OFC projection restores empathic behaviors in SI mice. Furthermore, we uncover a molecular basis for SI-induced OFC hypoexcitability: the downregulation of Grik3, encoding a kainate-type glutamate receptor subunit. These findings reveal a previously uncharacterized thalamocortical mechanism through which early-life social deprivation disrupts empathic behaviors, offering insights into the underpinnings of social-affective dysfunction.

neuroscience↗

bioRxiv2025

A Population-Scale Single-Cell Atlas of the Human Heart Reveals Cellular Remodeling and Cell-Cell Communication in Aging and Cardiac Disease

Previous single-cell and single-nucleus heart atlases, often limited by small sample sizes, lack the statistical power needed for phenotype association analysis, particularly for cardiovascular diseases and cardiac aging. To address this, we integrated data from 436 samples across 12 single-cell studies, harmonized the corresponding sample metadata, and constructed a comprehensive heart atlas comprising 355,762 cells and 1,436,719 nuclei. Consensus annotation identified 10 broad cell types and 54 fine-grained subsets. Associating gene expression patterns and cell type proportions with phenotypic data, we identified NRG1-expressing endocardial cells linked to multiple cardiac diseases and found that interferon (IFN) response signatures mark aging in multiple heart cell types. Importantly, we also developed PopComm, a novel computational method for inferring ligand-receptor (LR) interactions from population-scale single-cell data and quantifying interaction strength for individual samples. Using PopComm, we revealed a close association between the IFN response state and altered cell-cell communication during cardiac aging.

bioinformatics↗

bioRxiv2025

Energy Landscape Analysis Reveals Thalamic Modulation of Brain State Transitions During Movie Watching

Understanding how large-scale brain networks dynamically coordinate during complex, real-world experiences remains a crucial topic in contemporary neuroscience. Leveraging naturalistic paradigm, this study employed energy landscape modeling to characterize the temporal dynamics of brain state transitions and their functional significance. Using the Sherlock fMRI dataset, we identified two dominant brain states: a perception- and attention-oriented state, and an introspective, integrative state, each associated with distinct canonical brain networks. State transitions were differentially associated with the recruitment of these networks and manifested as either the "easy" or "hard" route, depending on the energy required for the transition. Notably, we demonstrated that the probability of specific state transitions robustly predicted inter-subject correlation within three clusters: subcortical-rich, salience/attention-rich, and DMN-rich regions. This indicates that flexible reorganization of cortical networks underlies shared neural engagement during ecologically valid experiences. Critically, the thalamus emerged as a central modulator, displaying activity changes and dynamic nucleus-specific thalamocortical connectivity changes tied to distinct transition profiles. Together, our findings shed light on the energetic and network mechanisms that orchestrate brain dynamics during naturalistic cognition and underscore the pivotal role of cortico-thalamo-cortical circuits in governing flexible, collective neural responses to richly structured real-life stimuli.

neuroscience↗

bioRxiv2025

Selective induction by statins of FAM134B-mediated sarcoplasmic reticulum (SR)-phagy degrades the SR calcium pump SERCA1 and contributes to myopathy

Statins, HMG-CoA reductase inhibitors, are the most prescribed medication for lowering low-density lipoprotein cholesterol. Although statin intolerance due to statin-associated muscle symptoms (SAMS) has been a clinically important issue for decades, the molecular mechanisms of statin myopathy remain incompletely understood. To seek to clarify how statins induce myopathy, here we perform transcriptome analysis and identify FAM134B short isoform (FAM134B-S), an endoplasmic reticulum (ER)-phagy receptor, as a statin-inducible gene. FAM134B-S expression is regulated by the mevalonate pathway and sterol regulatory element-binding proteins. We further show that FAM134B plays a critical role in the regulation of skeletal muscle integrity both in the steady-state and statin-treated conditions in human iPS cell-derived myocytes and mice. Mechanistically, FAM134B-S interacts with the sarcoplasmic reticulum (SR) calcium pump SERCA1 and promotes its autophagic degradation. Collectively, our work reveals a hitherto undisclosed mechanism of statin myopathy; blocking the mevalonate pathway induces SERCA1 degradation through FAM134B-mediated SR-phagy, exerting myotoxicity.

pharmacology and toxicology↗

bioRxiv2025

Adapting Epigenetic Clocks for Cell-Free DNA High-Throughput Sequencing Data

Background.Circulating cell-free DNA (cfDNA or ccfDNA) methylation sequencing holds promise for developing epigenetic aging clocks in minimally invasive aging assessment applications. However, current clocks--primarily trained on array-based data--do not readily generalize to methylation profiles of cfDNA, due to stochasticity and uncertainty arising from the limited input amounts of cfDNA and technical characteristics of high-throughput sequencing (HTS) platform. Due to lack of training to correct this uncertainty, direct application of legacy clocks to HTS data will inevitably produce unreliable estimates, introduce predictive discordance and undermine the trustworthiness of epigenetic biomarkers. Despite this urgent challenge, a systematic, model-agnostic framework for adapting existing epigenetic clocks to HTS-based cfDNA data remains lacking. Methods.Here, we generated a dedicated benchmark dataset comprising paired cfDNA and genomic DNA (gDNA), which was replicated and profiled across two methylation arrays and two targeted HTS platforms. We evaluated 53 epigenetic clocks for CpG coverage, reproducibility, cross-platform consistency, and age prediction accuracy. Further, we systematically explored and tested multiple adaptation strategies including depth filtering, beta-value imputation, and transfer learning via model distillation aiming at improving performance and clinical concordance of legacy epigenetic clocks on cfDNA HTS datasets. Results.Our results show that inherent technical noise of HTS platforms compromises diagnostic precision, presenting as a significant burden for applying legacy clocks on cfDNA HTS data. However, this technical limitation can be systematically neutralized. By enforcing stringent sequencing depth protocols ([≥]10x ideally 20x), employing robust algorithmic stabilization (L2-heavy clocks and imputation) and transfer learning, we can effectively isolate genuine physiological aging signals from technical artifacts. Ultimately, an adaptation pipeline incorporating all these methods effectively improved age prediction accuracy and, closed the gap between epigenetically induced aging status and clinically assessed realities. Besides, the pipeline demonstrated improved diagnostic sensitivity for neurodegenerative conditions such as amyotrophic lateral sclerosis, establishing a reliable foundation for non-invasive clinical monitoring. Conclusions.This study provided a comprehensive framework and practical guidelines for adapting epigenetic clocks to HTS-based cfDNA data, paved the path for more general application of cfDNA-based aging assessment and liquid biopsies.

bioinformatics↗