Biology subjects

Huang, J.

Publications and source records attributed to Huang, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Biosynthetic additive manufacturing through mycelium-bacteria consortia

Lignocellulosic biomass is the most abundant renewable raw material on Earth, yet its structural and carbon-storage potential remains unrealized: microbial decomposition returns stored carbon to the atmosphere before it can be harnessed as a durable material. Distinct microbial processes, such as biosynthesis and biomineralization, offer a natural counterpoint, converting organic carbon into mechanically robust, persistent materials such as polymers and minerals. Here, we establish a biosynthetic additive manufacturing strategy through mycelium-bacteria consortia, by coupling fungal self-assembly with bacterial biomineralization inside fabricated three-dimensional scaffolds derived from lignocellulosic biomass. Trametes versicolor forms a continuous living mycelial network that serves as both a structural template and a transport pathway, guiding the spatial colonization of Sporosarcina pasteurii and enabling volumetric calcium carbonate deposition throughout the scaffold interior, overcoming the surface-limited mineralization of conventional microbial systems. Integration with helicoidal Bouligand architectures translates microscale mineral reinforcement into macroscale performance, increasing compressive Young's modulus by 7-14-fold and ultimate strength by 5-fold relative to unmineralized conventional mycelium-based living composites. The living mycelial network also enables autonomous self-healing and modular self-assembly through hyphal bridging without external adhesives or energy input. This work establishes a scalable route for converting and upcycling transient biomass carbon into durable living structural materials for sustainable construction.

bioengineering↗

bioRxiv2026

Xenograft supervision and joint methylation-fragment-end encoding define a reusable molecular scoring layer for cell-free tumor DNA

Circulating tumor DNA is often scarce in plasma, and regional methylation summaries can obscure informative differences between individual molecules. Here we present ctDNA-BERT, a Transformer that scores individual bisulfite reads without predefined differentially methylated regions by jointly encoding CpG methylation state, nucleotide context and the fragment 3' end. Xenografts provide molecule-level tumor supervision. The resulting frozen scorer supported tumor-fraction estimation in synthetic and patient-derived xenograft mixtures and enriched tumor-concordant methylation and chromatin-accessibility signals in independent hepatocellular carcinoma and stage III pan-cancer plasma without cohort-specific retraining. Features derived from selected reads discriminated stage I cancers from healthy controls in a retrospective cohort of 1,477 specimens, achieving an out-of-fold area under the receiver operating characteristic curve of 0.884. By separating molecular scoring from sample-level inference, ctDNA-BERT provides a reusable read-level scoring layer for cell-free DNA methylation analysis.

bioinformatics↗

bioRxiv2026

ESX-1 substrates ESAT-6 and CFP-10 limit cGAS-STING activation via DNA and Zn2+ restriction

ESX-1-mediated phagosomal permeabilization exposes mycobacterial DNA to cytosolic cGAS-STING surveillance, yet the response is not overwhelming, suggesting additional layers of control exist. Here we show that the ESX-1 substrates ESAT-6 and CFP-10 constrain cGAS activation through complementary biochemical mechanisms. Purified Mycobacterium tuberculosis CFP-10 is a metal-dependent, 5'-3' exonuclease-like enzyme that preferentially degrades linear DNA, limiting the availability of stimulatory ligand. By contrast, purified M. tuberculosis ESAT-6 binds Zn2+ and limits the Zn2+-dependent conditions required for cGAS activation. During M. marinum infection, loss of these substrates augmented interferon-associated responses. Wild-type alleles restored immune suppression, whereas DNA-processing-defective CFP-10 or Zn2+-binding-defective ESAT-6 variants did not. These genotype-dependent differences were largely abolished in cGas-/- and Sting-/- macrophages. The double mutant was attenuated in zebrafish and mice, and its enhanced immune activation and attenuation were markedly diminished in cGas-/- mice. Prior exposure to the mutant improved zebrafish survival following wild-type challenge. Thus, beyond enabling mycobacterial DNA access to the cytosol, ESX-1 deploys its substrates to constrain cGAS-STING signaling by controlling activating-ligand availability and the metal-dependent environment required for sensor activation.

microbiology↗

bioRxiv2026

RAPPL-Seq Enables Rapid, High-Sensitivity Ribosome Profiling of Scarce Samples

Ribosome profiling is a powerful method for transcriptome-wide monitoring of translation activity in vivo, but its use is limited by lengthy protocols, reliance on specialized equipment and inefficient recovery of ribosome-protected fragments (RPFs). To overcome these challenges, we developed RAPPL-Seq, a magnetic bead-based affinity capture method that enables fast and efficient RPF isolation. RAPPL-Seq achieves high recovery efficiency, short processing time, and robust performance with limited input material. It is broadly applicable across multiple sample types, including cell lines, primary tissues, and human biospecimens. RAPPL-Seq can also be paired with translation inhibitors to characterize inhibitor- and tissue-specific mRNA translation. RAPPL-Seq efficiently captured translatomes of glioblastoma (GBM) and adjacent non-tumor brain tissues from patient biopsies. Together, these findings establish RAPPL-Seq as a straightforward, easily accessible, and highly sensitive approach that extends translatome studies to scarce experimental and clinical samples, opening new avenues for mechanistic insight and therapeutic discovery.

molecular biology↗

bioRxiv2026

Behavior-coupled single-cell transcriptomics reveals trafficking protein-dependent regulation of tumor cell migration

Aggressive cellular migration underlies cancer invasion and metastatic dissemination, yet the molecular programs defining highly migratory tumor cell states remain poorly characterized. In head and neck squamous cell carcinoma (HNSCC), metastatic progression is associated with poor clinical outcome, motivating method developments that link migratory behavior to underlying mechanisms. Here, we present behavior-coupled single-cell sequencing (BC-seq), a live-cell imaging-guided transcriptomic platform that enables real-time identification, isolation, and profiling of individual cells based on migratory behavior. Applying BC-seq to 10 HPV-negative oral squamous cell carcinoma (OSCC), the most common HNSCC subtype, we distinguished fast-migrating cells (FMCs) from slow-migrating cells (SMCs) and identified transcriptional programs associated with aggressive migration. Mechanistically, BC-seq uncovered cholesterol-regulated signaling pathways and associated trafficking proteins involving SREBP2, STARD4, Cavin1/3, and VEGFR2 that promotes actin cytoskeletal remodeling and cellular motility. Disruption of STARD4-mediated cholesterol trafficking or Cavin1/3-dependent VEGFR2 nuclear translocation significantly impaired cell migration, linking cholesterol metabolism to migratory machinery. We next examined whether molecular programs associated with aggressive migration are reflected in clinical outcomes. A 15-gene expression signature derived from FMCs predicted distant metastasis with 83.6% accuracy in an independent OSCC cohort, outperforming conventional clinicopathological risk stratification. Together, our study establishes BC-seq as an approach for integrating dynamic cellular behavior with single-cell transcriptomics to uncover mechanisms of aggressive migration and clinically relevant molecular signatures.

cancer biology↗

bioRxiv2026

Unifying N- and O-glycopeptide dissociation methods with expedient electron transfer dissociation

The glycoproteome is highly heterogeneous, where each glycosite can be modified by potentially hundreds of glycan compositions. This complexity necessitates methods that can provide glycosite-specific characterization in a high-throughput manner. Electron transfer dissociation (ETD) is particularly valuable for glycopeptide analysis, where it is critical for glycosite localization in O-glycopeptides and often produces higher quality spectra than higher energy collisional dissociation (HCD) for N-glycopeptides. Despite this, lower quality HCD spectra are often favored in high-throughput glycoproteomics experiments because the near instantaneous fragmentation of HCD results in faster MS/MS spectral acquisition relative to ETD. In short, overhead times for ETD reactions limit acquisition rates and sensitivity that can outweigh its advantages. Here we implement changes to ion handling, scan parallelization, and ion-ion reaction conditions to increase ETD MS/MS acquisition rates on the Orbitrap Tribrid MS platform. We explore fundamental parameters of ETD ion-ion reactions, including reaction q-values (i.e., Mathieu q-values) and ion-ion reaction time, showing that we can reduce the overhead of ion-ion reaction scan functions and improve the overall synchronization of data acquisition. These optimizations lead to faster spectral acquisition times for ETD MS/MS spectra while maintaining high spectral quality. Ultimately, these improvements allow the ETD scan functions to be nested within ion accumulation times needed per MS/MS spectrum, enabling ETD spectral acquisition to be parallelized within the duty cycle of the instrument and removing the major time cost to doing ETD-based fragmentation. We argue that this expedient implementation of ETD makes ETD-based fragmentation a universally beneficial dissociation method for both N- and O-glycopeptides.

systems biology↗

bioRxiv2026

FLASH X-rays preserve neutrophil chromatin to boost antitumor immunity

Ultrahigh dose-rate FLASH radiotherapy spares normal tissue while preserving antitumor activity, positioning it as a promising technology in radiation oncology, but its immunomodulatory mechanisms remain elusive. Here, using a bilateral subcutaneous tumor model in mice, we show that 8-Gy FLASH X-rays, versus an equal dose of conventional dose-rate (CONV) X-rays, elicit a robust abscopal antitumor effect with anti-PD-1 therapy. Single-cell transcriptome and T cell receptor (TCR) profiling reveal that FLASH X-rays plus anti-PD-1 enhance antitumor immune infiltration and TCR clonal expansion in both irradiated and abscopal tumor lesions. Mechanistically, the ultrashort temporal continuity of FLASH dose delivery conserves chromatin compaction in neutrophils during exposure and inhibits neutrophil extracellular trap (NET) formation, thereby dismantling NET-mediated CD8+ T-cell exclusion within primary tumors. Conversely, CONV or split-dose FLASH X-rays induce marked chromatin modifications prior to dose completion, leading to chromatin hyper-relaxation which promotes NETosis and consequently abrogates systemic antitumor immunity. Notably, this detrimental effect can be reversed by pharmacologically suppressing PARylation-mediated chromatin modification and decondensation in neutrophils. These findings identify radiation-induced aberrant chromatin relaxation in neutrophils as a critical determinant of radioimmunotherapy outcomes, providing a rationale for redefining the FLASH effect and advancing its clinical translation.

cancer biology↗

bioRxiv2026

Epithelial Self-Organization Patterns the Tadpole Oral Disc

Vertebrate epidermal organs are generally patterned through epithelial-mesenchymal interactions, but whether such interactions are essential for all epidermal organs remains unclear. Here we show that the elaborate oral disc of anuran tadpoles arises by partitioning a continuous oral epithelium into developmentally distinct jaw, tooth-row and papilla modules, in the absence of a mesenchymal organizer. Spatially restricted epithelial programs specify, maintain and differentiate these modules, enabling their semi-independent development. Perturbation of lef1, hoxc13, sox7 and pitx2 selectively disrupts individual modules. The resulting module-specific reductions, expansions and rearrangements mirror oral-disc configurations that have evolves repeatedly across anurans. These findings indicate that differential deployment of shared epithelial programs can generates selectable variation in the tadpole oral disc, allowing transient larval feeding structures to diversify independently of adult ecology and enabling tadpoles to exploit diverse ecological niches. Epidermal self-organization can therefore generate structural complexity, promote functional diversification and enhance evolvability in transient vertebrate organs.

developmental biology↗

bioRxiv2026

High temperature ligation enables in vitro assembly of eukaryotic genes

Our ability to efficiently construct synthetic DNA products is essential to studying and engineering biology. There is an abundance of widely adopted DNA assembly technologies suitable for generating synthetic constructs from diverse DNA inputs, but these techniques suffer from low specificity of connection during assembly, which decreases the accuracy, efficiency, and throughput of construction. Here we propose a broadly applicable paradigm for DNA assembly, High Temperature (HighT) assembly, that lowers misconnection rates by ligating complementary single strand DNA overhangs at reaction temperatures significantly higher than their predicted melting temperatures. By expanding the concept of melting temperature to demarcate smaller association fractions of DNA duplexes, we characterize HighT and demonstrate 40 percentage points increase in accuracy, and 4 orders of magnitude increase in yield of transformants when applying the technique to conventional assembly technologies. HighT is then applied to develop cloning pipelines for the development of efficient assembly-to-integration protocols, the construction of specific low-abundance isoforms of medically relevant proteins from human gDNA and oligos, and the de novo construction of a 10 kb, GC rich genomic region from the functionally extinct Northern White Rhinoceros created entirely in vitro.

molecular biology↗

bioRxiv2026

Structural basis for phosphorylation-induced inactivation of the rice ammonium transporter AMT1;1

Ammonium (NH4+) uptake and transport in plants are mediated by ammonium transporters (AMTs), which are tightly regulated by phosphorylation to prevent excess ammonium influx and toxic accumulation. However, the mechanism of phosphorylation-mediated inactivation is unique and remains unclear. Here, we report cryo-EM structures of the rice (Oryza sativa) ammonium transporter OsAMT1;1 in its wild-type and phosphomimetic variant T452D states. OsAMT1;1 forms a stable homotrimer, with conserved motifs affirming a shared transport mechanism within the AMT/MEP/Rh superfamily. Our structural analysis reveals that T452D phosphomimetic stabilizes an intracellular salt-bridge network and maintains the channel in a closed state, in which the ICL5 loop positions the hydrophobic plug residue Leu360 near the cytoplasmic exit to occlude the channel exit and abolish complementation capacity. Our findings elucidate phosphorylation-induced inactivation mechanism for the plant AMT1 subfamily, providing a structural paradigm for the post-translational regulation of nutrient uptake.

biophysics↗

bioRxiv2026

A direct RNA sequencing workflow for simultaneous profiling of tRNA abundance and modifications in mouse liver

Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [≥] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [≥]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [≥]50%, coverage [≥]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.

molecular biology↗

bioRxiv2026

Hypomagnetic field inhibits seed germination via ROS signaling and reprogramming transcriptome in Arabidopsis thaliana

The geomagnetic field (GMF) is a ubiquitous yet poorly understood environmental factor that affects plant growth and development. It has been suggested that the blue-light photoreceptor cryptochrome (CRY) functions as a critical magnetic-field sensor in birds and insects through the radical-pair mechanism (RPM). However, whether CRY acts as a bona fide transducer of GMF signals and what are the downstream cascades remain largely elusive in plants. To address these key issues, we generated hypomagnetic field (HMF) via Helmholtz coils and passive magnetic shielding to investigate the GMF deprivation effects in Arabidopsis thaliana. HMF significantly delays the germination speed without altering the final germination rate, suggesting that HMF specifically modulates germination kinetics rather than seed viability. Time-resolved transcriptomic profiling revealed a coordinated transcriptional shift characterized by downregulation of growth-promoting genes and concurrent upregulation of defense-related genes under HMF. This biphasic transcriptional reprogramming coincides with a significant increase of reactive oxygen species (ROS), linking redox perturbation to the germination delay. Consistently, supplementation with ROS-scavenging antioxidants (e.g., reduced glutathione and ascorbic acid) rescues both germination speed and the misregulated expression of a subset of HMF-responsive genes, suggesting the central role of ROS in mediating HMF bioeffects. Genetic analysis using cry1 cry2 double mutants further indicated that HMF-induced germination delay operates through both CRY-dependent and -independent signaling pathways, suggesting the involvement of additional magnetosensitive modules beyond the canonical CRY-based RPM. These findings suggest that the GMF acts as a positive environmental cue that fine tunes the growth-defense trade-off through a redox-dependent signaling.

plant biology↗

bioRxiv2026

Generative Access to Make-on-Demand Chemical Space Enables Ultra-Large Virtual Screening

Ultra-large-scale virtual screening (ULVS) increasingly relies on make-on-demand chemical spaces, but efficient access to these spaces remains challenging because their scale far exceeds what can be exhaustively enumerated and docked. Here, we present REAL-SWIT, a generative ULVS framework that learns Enamine REAL Space as a synthesizable molecular distribution and couples this generator with a target-specific scoring model to enable docking-guided exploration of REAL Space at the level of complete molecule. The generative model learned transferable features of the REAL Space distribution: approximately 96% of generated molecules were found in REAL Space, and a subset of generated molecules absent from the training release appeared in later expanded releases. In computational benchmarks, REAL-SWIT identified more molecules with favorable docking scores than representative fragment-based search strategies, including cooperative building-block combinations that fragment-level prioritization tended to miss. Experimental validation for ROCK1 yielded six biochemical inhibitors among 23 synthesized compounds, including RX-3 with an IC50 of 0.17 M. These results establish generative access to make-on-demand chemical space as a practical strategy for ULVS.

bioinformatics↗

bioRxiv2026

RNA Editing of DEGS1 Links Genetic Risk to Ceramide Dysregulation and Astrocyte Toxicity in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with ~90% of cases being sporadic. Although genome-wide association studies have identified numerous genetic risk loci, these variants account for only a portion of ALS heritability, suggesting that additional genetic and regulatory mechanisms contribute to disease risk. Furthermore, the mechanisms linking common variants to disease pathogenesis remain poorly understood. Adenosine-to-inosine (A-to-I) RNA editing is dysregulated in ALS, yet systematic identification of disease-relevant editing events and their functional consequences has been limited. We performed comprehensive RNA editing analysis across eight central nervous system regions from a Target ALS cohort, identifying 752 differentially edited sites in 304 genes. Global RNA editing was significantly reduced in ALS tissues, particularly in spinal cord and choroid plexus, with altered editing in genes involved in immune signaling, stress response and RNA processing. Through genetic colocalization analysis integrating editing quantitative trait loci (edQTLs) with ALS GWAS data, we identified common variants at the DEGS1 locus associated with both increased 3' UTR editing and elevated ALS risk (posterior probability >0.88). DEGS1 encodes dihydroceramide desaturase 1, a key enzyme converting dihydroceramide to ceramide, the precursor to all sphingolipid species. Using CRISPR-generated DEGS1 knockout iPSCs and lipidomic profiling, we demonstrated that DEGS1 is essential for ceramide synthesis and downstream sphingolipid production in astrocytes. Functional validation revealed that ALS-associated editing of the DEGS1 3' UTR significantly increased DEGS1 protein expression. Critically, astrocytes expressing the fully edited DEGS1 3' UTR increased ceramide levels and reduced cell viability compared to those expressing the unedited construct. These findings establish a novel regulatory link between RNA editing, sphingolipid metabolism and ALS pathogenesis. Our results demonstrate that common genetic variants can influence disease risk through post-transcriptional mechanisms that alter lipid homeostasis in astrocytes, a cell type increasingly recognized as central to ALS progression.

neuroscience↗

bioRxiv2026

SWELL1 channel-mediated D-2HG export promotes immune evasion and metabolic fitness in IDH-mutant gliomas

Isocitrate dehydrogenase (IDH) is the most frequently mutated metabolic enzyme in human cancers. Mutant IDH produces the oncometabolite D-2-hydroxyglutarate (D-2HG), which promotes tumorigenesis in part through epigenetic alterations. Beyond its cell-autonomous effects, tumor-derived D-2HG acts as a potent immunosuppressant that establishes an immune-cold tumor microenvironment. However, the mechanism by which D-2HG is released from tumors remains largely unknown. Here, we identify the SWELL1 (LRRC8A)/LRRC8C-containing volume-regulated anion channel (VRAC) as a principal pathway for D-2HG efflux from IDH-mutant cells. Genetic deletion of SWELL1, the essential VRAC subunit, markedly reduces D-2HG release and reverses associated immunosuppression in an orthotopic mouse model of IDH-mutant glioma. Strikingly, loss of VRAC causes intracellular accumulation of D-2HG, which paradoxically limits tumor cell proliferation by driving epigenetic remodeling and mitochondrial metabolic stress. Pharmacological inhibition of VRAC with dicumarol suppresses IDH-mutant glioma growth, enhances intratumoral T cell activation, synergizes with immune checkpoint blockade, and prolongs survival in tumor-bearing mice. In human IDH-mutant lower-grade gliomas, elevated LRRC8C expression correlates with DNA hypermethylation and an immunosuppressive tumor microenvironment (TME), and predicts poor overall survival. Together, these findings establish VRAC-mediated D-2HG export as a central mechanism regulating both immune evasion and tumor cell fitness, uncovering new therapeutic opportunities across IDH-mutant cancers.

cancer biology↗

bioRxiv2026

Genes controlling polysaccharide synthesis, protein folding and basic metabolism ensure intra-host cell adaptation and replication of Yersinia enterocolitica

Pathogenesis of yersiniosis comprises cell-invasive events in which the bacteria subvert the endocytic-autophagolysosomal pathway of infected host cells to create an intracellular niche for bacterial replication. To determine potential virulence factors that enable intracellular proliferation of Yersinia enterocolitica, we performed a high-throughput transposon-directed insertion site sequencing approach (TraDIS) with infected epithelial HeLa cells. In this screen, we identified seven particular genes that have either metabolic activities in disulfide bond formation (dsbA), glutamine synthesis (glnA), and energy generation (acnB), or dictate synthesis of enterobacterial common antigen ECA (webB/C/F) and LPS (wzx). Individual knockout of the identified genes impaired intracellular replication of Yersinia. Furthermore, these mutants differentially affected host cell autophagy and the acidification of Yersinia-containing vacuoles (YCVs). The results indicate that glnA, acnB, dsbA, wecB, and wecC support autophagy-promoted, intracellular replication of Yersinia. Folding of yet to be defined proteins dependent on dsbA seems to be most critically required for mediating these events. A wzx-controlled LPS synthesis pathway appears implicated in preventing YCV acidification. These results suggest that autophagy induction and adjustment of Yersinia to the intravacuolar environment result from a multifactorial adaptation process that involves formation of a protective carbohydrate envelope and basic metabolic enzymes needed for the refinement of productive intracellular replication.

microbiology↗

bioRxiv2026

An ancestral mitochondrial DNA insertion disrupts RNAi and enables persistence of a novel mycovirus in Cryptococcus neoformans

RNA interference (RNAi) is a widely conserved genome-defense mechanism that protects eukaryotes against viruses and transposable elements. We previously showed that RNAi loss can lead to hypermutation and antifungal drug resistance in the human fungal pathogen Cryptococcus neoformans, illuminating the potential clinical relevance of this pathway. In this study, we identified another function of RNAi in C. neoformans: mycovirus restriction. By screening known RNAi-deficient C. neoformans isolates, we identified a novel dsRNA mycovirus of the Orthototiviridae family, which we named CnTV1. We subsequently detected CnTV1 in three additional RNAi-deficient isolates. All CnTV1-positive isolates shared an ancestral nuclear mitochondrial DNA segment (NUMT) insertion that disrupts the gene encoding Argonaute (Ago1). Restoration of RNAi in sexually produced zygotes efficiently eliminated CnTV1. RNAi rescue by CRISPR-Cas9-mediated allele exchange eliminated CnTV1 during vegetative growth, further demonstrating RNAi is sufficient for mycoviral control. Loss of the RNA helicase Ski2 or the exoribonuclease Xrn1 increased CnTV1 abundance, revealing RNAi-independent restriction of the virus. Restoration of RNAi cleared the virus even in the absence of Ski2 or Xrn1, indicating a dominant role for RNAi in antiviral defense. To investigate the biological implications of CnTV1 infection, we developed a cytoplasmic-mixing approach and generated isogenic strain pairs in an RNAi-deficient background that differ only in viral infection status. Leveraging these strains, we show that CnTV1 infection leads to coordinated, low-magnitude transcriptomic changes and that strains lacking the mycovirus were moderately less virulent in a murine infection model. Our findings reveal that RNAi serves as a dominant antiviral defense system in C. neoformans and suggest that naturally occurring RNAi deficiency may be more prevalent than previously appreciated. This work highlights mycovirus persistence as an important consequence of RNAi loss in this WHO-designated critical priority fungal pathogen.

genetics↗

bioRxiv2026

A T2T Benchmark Reveals How Reference Choice Shapes Human Genome Interpretation

The completion of telomere-to-telomere (T2T) human genomes has expanded the accessible landscape of human genetic variation, yet benchmark resources remain limited to conventional high-confidence regions defined by existing reference frameworks. Here, we generated a near-perfect diploid T2T genome (T2T-LIN) from a Chinese individual and established assembly-based truth sets by comparison with T2T-YAO, an ancestry-matched near-perfect T2T reference genome. The benchmark showed a heterozygous/homozygous SNV ratio of ~2, consistent with expectations under Hardy-Weinberg equilibrium, and enabled genome-wide evaluation of reference-dependent biases. We found that reference choice substantially influences genome interpretation: ancestry-matched linear T2T references provided the most faithful representation of individual genomic variation and enabled more accurate genome reconstruction than unmatched linear, diploid and graph-based references. Benchmarking previously inaccessible repetitive and structurally complex regions revealed substantial limitations of current variant callers that were masked by conventional metrics. The T2T-LIN and YAO-LIN benchmarks establish a T2T-era framework for evaluating reference-dependent genome interpretation and variant discovery across nearly the complete human genome.

genomics↗