Biology subjects

Chen, M.

Publications and source records attributed to Chen, M..

At least 199 records · Page 11Linked to original sources

bioRxiv2025

EPSD 2.0: An Updated Database of Protein Phosphorylation Sites across Eukaryotic Species

As one of the most crucial post-translational modifications (PTMs), protein phosphorylation regulates a broad range of biological processes in eukaryotes. Biocuration, integration and annotation of reported phosphorylation events will deliver a valuable resource for the community. Here, we present an updated database, the eukaryotic phosphorylation site database 2.0 (EPSD 2.0), which includes 2,769,163 experimentally identified phosphorylation sites (p-sites) in 362,707 phosphoproteins from 223 eukaryotes. From the literature, 873,718 new p-sites identified through high-throughput phosphoproteomic research were first collected, and 1,078,888 original phosphopeptides together with primary references were reserved. Then, this dataset was merged into EPSD 1.0, comprising 1,616,804 p-sites within 209,326 proteins across 68 eukaryotic organisms [1]. We also integrated 362,190 additional known p-sites from 10 public databases. After redundancy clearance, we manually re-checked each p-site and annotated 88,074 functional events for 32,762 p-sites, covering 58 types of downstream effects on phosphoproteins, and regulatory impacts on 107 biological processes. In addition, phosphoproteins and p-sites in 8 model organisms were meticulously annotated utilizing information supplied by 100 external platforms encompassing 15 areas. These areas included kinase/phosphatase, transcription regulators, three-dimensional structures, physicochemical characteristics, genomic variations, functional descriptions, protein domains, molecular interactions, drug-target associations, disease-related data, orthologs, transcript expression levels, proteomics, subcellular localization, and regulatory pathways. We expect that EPSD 2.0 will become a useful database supporting comprehensive studies on phosphorylation in eukaryotes. The EPSD 2.0 database is freely accessible online at https://epsd.biocuckoo.cn/.

bioinformatics↗

bioRxiv2025

AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities

Currently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone. In this system, the adenovirus helper genes (E2A, E4orf6, and VA RNA), packaging genes (rep and cap), and the vector transgene cassette are consolidated into a single compact plasmid with a 13-kb backbone. The AAVone system achieves a two- to four-fold increase in yields compared to the traditional triple-plasmid system. Furthermore, the AAVone system exhibits low batch-to-batch variation and eliminates the need for fine-tuning the ratios of the three plasmids, simplifying the production process. In terms of vector quality, AAVs generated by the AAVone system show similar in vitro and in vivo transduction efficiency, but a substantial reduction in sequences attributed to plasmid backbones and a marked reduction in non-functional snap-back genomes. In Summary, the AAVone platform is a straightforward, cost-effective, and highly consistent AAV production system - making it particularly suitable for GMP-grade AAV vectors.

bioengineering↗

bioRxiv2024

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery

Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how they are influenced by the lipid composition and mixing method. Here we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multi-angle light scattering, and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering. We show that LNPs have intrinsic polydispersity in size, RNA loading, and shape, which depends on both the formulation technique and lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.

bioengineering↗

bioRxiv2024

High potential contribution of intercropping to soybean and maize self-sufficiency in Europe

This study examines whether maize-soybean intercropping could improve the European Unions soybean self-sufficiency while minimizing trade-offs with maize and other crops production. Assuming a four-year return, intercropping on 6.3, 13.1, and 20.1 Mha could produce 9.1 (28.8), 18.2 (63.7), and 27.2 (99.0) Mt of soybean (maize), achieving 25% (34%), 50% (75%), and 75% (116%) self-sufficiency. Reaching similar co-productions with sole crops require an additional 11.1, 24.7, and 36.5 Mha, implying 20-21% land savings with intercrops. Full soybean self-sufficiency could require more than 25 Mha of intercrops (25% of European cropland) annually. These estimates held under a large range of nitrogen inputs and temporal overlaps between crop species, provided 1 hectare of intercropping produced more soybean than 0.5 hectare of sole soybean. Geographic variation in sole crop yields should therefore be considered when assessing intercrops land-use efficiency. Our results highlight intercropping as a strategy to simultaneously improve European soybean production, satisfy maize needs, and save lands.

plant biology↗

bioRxiv2024

GraphVelo allows inference of multi-modal single cell velocities and molecular mechanisms

RNA velocities and generalizations emerge as powerful approaches for extracting time-resolved information from high-throughput snapshot single-cell data. Yet, several inherent limitations restrict applying the approaches to genes not suitable for RNA velocity inference due to complex transcriptional dynamics, low expression, or lacking splicing dynamics, or data of non-transcriptomic modality. Here, we present GraphVelo, a graph-based machine learning procedure that uses as input the RNA velocities inferred from existing methods and infers velocity vectors lying in the tangent space of the low-dimensional manifold formed by the single cell data. GraphVelo preserves vector magnitude and direction information during transformations across different data representations. Tests on synthetic and experimental single-cell data including viral-host interactome, multi-omics, and spatial genomics datasets demonstrate that GraphVelo, together with downstream generalized dynamo analyses, extends RNA velocities to multi-modal data and reveals quantitative nonlinear regulation relations between genes, virus and host cells, and different layers of gene regulation.

bioinformatics↗

bioRxiv2024

A CRISPR/SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus

As members of the -proteobacteria group, Caulobacter crescentus and its relatives are known for their asymmetric life cycle and comprehensive applications in gene delivery, agricultural biotechnology, and the production of high-value compounds. However, genetic manipulations of these bacteria are often time-consuming and labor-intensive due to the lack of efficient genome editing tools. Here, we report a practical CRISPR/SpCas9M-reporting system that overcomes the limitations of SpCas9 expression, enabling efficient, markerless, and rapid genome editing in C. crescentus. As a demonstration, we successfully knocked out two genes encoding the scaffold proteins, achieving apparent editing efficiencies up to 80%. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized to enhance the editing efficiency or decrease the cell lethality. A nearly zero off-target ratio was observed after the curing of the editor plasmid in editing strains. Furthermore, we applied the CRISPR/SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and reliable editing strategy. We anticipate that this system could be applied to other hard-to-edit organisms, accelerating both basic and applied research in -proteobacteria.

bioengineering↗

bioRxiv2024

TurboID-based proximity labeling discovers ABCF2 as an adhesion receptor for the zoonotic pathogen Pasteurella multocida

Pasteurella multocida is a zoonotic pathogen that can cause fatal infections in both animals and humans. A significant number of putative adhesive factors have been predicted to contribute to the pathogenesis of P. multocida, but their interactive proteins on host cells remain unclear. In this study, we experimentally verified the roles of three previously proposed proteins (PlpE, PtfA, Hsf-2) in the adherence of P. multocida. Through turboID-based proximity labeling screening, we identified ATP-binding cassette sub-family F member 2 (ABCF2) as a host interactive protein for PlpE/PtfA/Hsf-2. Crucial amino acid residues in PlpE, PtfA, and/or Hsf-2 that are essential for interacting with ABCF2 included Asp-123 (PlpE), Lys-88 (PtfA), Asp-136 (PtfA), Ala-464 (Hsf-2), Glu-473 (Hsf-2), and Arg-489 (Hsf-2). Knocking down or knocking out ABCF2 significantly reduced the adherence and invasion of P. multocida to host cells, while overexpression of ABCF2 markedly increased these effects. However, ABCF2 did not contribute to the adherence of other bacterial species such as Klebsiella pneumoniae and Bordetella bronchiseptica. Additionally, we demonstrated that P. multocida infection upregulated the expression of host ABCF2 by activating the p38 MAPK and NF-{kappa}B signaling pathways. Furthermore, we showed that ABCF2 was involved in the P. multocida-induced p53-dependent apoptotic signaling pathway. To the best of our knowledge, this is the first identification of ABCF2 as a host factor contributing to the adherence of P. multocida and only the second report of ABCF2s involvement in bacterial pathogenesis. ImportanceP. multocida can cause fatal infections in both animals and humans, yet the mechanisms related to its pathogenesis remain to be fully explored. In this study, we identified ABCF2 as a crucial host interactive protein for three adhesive proteins encoded by P. multocida and experimentally verified its role in the adherence and invasion of P. multocida. Furthermore, we elucidated how P. multocida modulates ABCF2 during its infection and revealed p53-dependent apoptosis as a downstream effect of ABCF2 during P. multocida infection. Given the absence of reports on ABCF2 contributing to the pathogenesis of P. multocida and only one previous report on the involvement of ABCF2 in bacterial pathogenesis prior to this study, this research could be valuable for comprehending the interactions between bacteria and hosts during bacterial infections.

microbiology↗

bioRxiv2024

Structure and mechanism of the RalGAP tumor suppressor complex

The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract (oncogenic) Ras signaling. However, no structural information on the architecture of this tumor suppressor complex is available hampering a mechanistic understanding of its functionality. Here, we present a cryo-EM structure of RalGAP that reveals an extended 58 nm tetrameric architecture comprising two heterodimers of the RalGAP and RalGAP{beta} subunits. We show that the catalytic domain of RalGAP requires stabilization by a unique domain of RalGAP{beta}, providing the molecular basis for why RalGAP complexes are obligatory heterodimers. Formation of RalGAP tetramers is not required for activity in vitro, but essential for function of the complex in vivo. Structural analysis of RalGAP subunit variants reported in cancer patients suggests effects on complex formation and thus functional relevance in tumor development, emphasizing the significance of the obtained structural information for medical research.

biochemistry↗

bioRxiv2024

scPrediXcan integrates advances in deep learning and single-cell data into a powerful cell-type-specific transcriptome-wide association study framework

Transcriptome-wide association studies (TWAS) help identify disease causing genes, but often fail to pinpoint disease mechanisms at the cellular level because of the limited sample sizes and sparsity of cell-type-specific expression data. Here we propose scPrediXcan which integrates state-of-the-art deep learning approaches that predict epigenetic features from DNA sequences with the canonical TWAS framework. Our prediction approach, ctPred, predicts cell-type-specific expression with high accuracy and captures complex gene regulatory grammar that linear models overlook. Applied to type 2 diabetes and systemic lupus erythematosus, scPrediXcan outperformed the canonical TWAS framework by identifying more candidate causal genes, explaining more genome-wide association studies (GWAS) loci, and providing insights into the cellular specificity of TWAS hits. Overall, our results demonstrate that scPrediXcan represents a significant advance, promising to deepen our understanding of the cellular mechanisms underlying complex diseases.

genetics↗

bioRxiv2024

Ophiostomatoid fungi associated with bark beetles infesting Pinus armandii, with characterizations of seven new species from western China

Ophiostomatoid fungi mainly refer to the order Ophiostomatales (Ascomycota), which are known to be symbiotic with bark beetles infesting pine trees. These fungi are primarily associated with Dendroctonus armandi and other bark beetles, significantly impacting the local pines Pinus armandii in western China. However, this fungal diversity must be further explored. This study aims to identify Ophiostomatoid fungi associated with P. armandii in Ningshaan, Shaanxi Province. A total of 225 Ophiostomatalean isolates were obtained from 46 gallery samples, accounting for 77.6 % of the total number of isolated strains. Twenty species were identified based on the phylogenetic analyses of ITS, LSU, {beta}-tubulin, and EF-1, as well as morphological characteristics. These include seven new taxa, Ceratocystiopsis rugosa, Graphilbum prolificum, G. ramosus, Masuyamyces huoditangensis, Ophiostoma flavescens, O. lenta, O. mediotumida, and thirteen known species. This study laid a theoretical foundation for understanding the relationship between the beetles and the ophiostomatalean fungi in the Ningshan region, China.

microbiology↗

bioRxiv2024

YY1 Enhances the Stability of HIF-1α Protein by Interacting with NUSAP1 in Macrophages within the Prostate Cancer Microenvironment

Immune checkpoint therapy for prostate cancer (PCa) has failed in clinical trials; however, the precise underlying mechanisms involved remain elusive. PCa, a classic "immune-cold" tumor, is characterized by an immunosuppressive tumor microenvironment. Within this milieu, macrophages, the predominant immune cell population, have a propensity to infiltrate the hypoxic zones of tumors. In a previous study, we showed that Yin Yang 1 (YY1) is highly expressed in macrophages in PCa tissues. Here, through multiplexed imaging mass cytometry (IMC) of a PCa tissue microarray, we further demonstrate that YY1+ macrophages aggregate in hypoxic areas of tumors and that hypoxia promotes the phase separation of YY1 in the nucleus by increasing YY1 tyrosine phosphorylation in macrophages. Furthermore, YY1 binds to NUSAP1 and promotes the SUMOylation of HIF-1, which promotes phase separation and stabilization of the HIF-1 protein. We also demonstrated that either treatment with a small molecule inhibitor (tenapanor) to decrease the YY1-NUSAP1-HIF-1 interaction or myeloid-specific YY1 gene knockout impairs subcutaneous PCa tumor formation. Furthermore, we present a first-generation tetrahedral DNA nanostructure (TDN) based on the proteolysis targeting chimera (PROTAC) technique, named YY1-DcTAC, which targets and degrades YY1 in tumor-associated macrophages. In a PCa mouse model, YY1-DcTAC exhibited prolonged drug efficacy, robust macrophage-specific responsiveness, potent antitumor effects, and increased CD8+ T cell tumor infiltration. In summary, our findings underscore the pivotal role of YY1 within the hypoxia/HIF-1 pathway in tumor-associated macrophages and affirm the therapeutic potential of targeting YY1 for treating PCa.

cancer biology↗

bioRxiv2024

Ophiostomatalean fungi associated with bark beetles infesting Pinus koraiensis, including the description of Ceratocystiopsis fushunensis sp. nov. in China

Fungi of Ophiostomatales (Sordariomycetes, Ascomycota) always have a symbiotic relationship with bark beetles (Coleoptera: Scolytinae). In this study, we investigated the fungi associated with Cryphalus piceae and Hylastes sp in Fushun. Based on the four gene fragments of TUB, ITS, LSU, and TEF and morphological characteristics, 206 strains of Ophiostomatales were identified to be distributed in 21 taxa; these included an undescribed taxon. Here, we described it as Ceratocystiopsis fushunensis sp. nov. The results of this study provided valuable information on the relationship between fungi and bark beetles.

microbiology↗

bioRxiv2024

Shared Transcriptomic Signatures Reveal Synaptic Pruning as a Link Between Alzheimer's Disease and Epilepsy

Alzheimers disease (AD) and epilepsy (EP) share a complex bidirectional relationship, yet the molecular mechanisms underlying their comorbidity remain insufficiently explored. To identify potential transcriptional programs across animal models and human patients with AD and EP, we conducted a comprehensive genome-wide transcriptomic analysis. Our investigation included mouse models of temporal lobe epilepsy (pilocarpine- and kainic acid-induced; n = 280), AD transgenic models (7 transgenic models expressing human tau or amyloid pathology; n = 257), and performed cross-species validation in human cohorts (EP: n = 182; AD: n = 301). We identified a highly conserved immune-related module across all models and patient cohorts. The hub consensus signatures of this module were centered around a microglial synaptic pruning pathway involving TYROBP, TREM2, and C1Q complement components. Gene regulatory network analysis identified TYROBP as the key upstream hub signature. These signatures showed consistent up-regulation in both conditions and strong diagnostic potential. Differential expression analyses revealed their predominant expression in specific microglial subpopulations associated with complement-mediated synaptic pruning and immune activation. Neural circuit modeling further demonstrates the asymmetric sensitivity of synaptic pruning to network dynamics. Loss of inhibitory synapses has a disproportionately significant impact on neural network excitation/inhibition balance and synchronization. Our findings support microglial complement-mediated synaptic pruning as a conserved central pathway linking neurodegeneration to epileptogenesis, suggesting a promising therapeutic target for AD and EP comorbidity. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/618752v12_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@179aa28org.highwire.dtl.DTLVardef@922dfaorg.highwire.dtl.DTLVardef@108a99eorg.highwire.dtl.DTLVardef@1b7d2f6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGenome-wide transcriptomic analysis across epilepsy (EP) and Alzheimers disease (AD) models and patients identified a conserved immune module. C_LIO_LIThe TYROBP-TREM2-C1Q microglial synaptic pruning pathway was identified as a central consensus signature across model and patient cohorts. C_LIO_LIConsensus signatures possess potential diagnostic utility in AD and EP patients, with predominant expression in specific microglial subpopulations C_LIO_LINeural circuit modeling demonstrates the asymmetric effects of synaptic pruning, whereby loss of inhibitory synapses disproportionately disrupts the E/I balance, leading to increased network synchronization. C_LI

bioinformatics↗

bioRxiv2024

Organic opto-nanobiointerface enables multiscale biomodulation

Virtually all organic material on Earth has been produced converting solar energy through photosynthesis in chloroplasts, a sack-like, double membrane organelle in plants and algae, where transmembrane electron transfer occurs from lumen to stroma. Although animals hardly harness the power of photosynthesis, their bioelectrical signals extensively regulate complex electrophysiological behaviors, rendering it a superior target for biomedical innovation. Here, a crude structural mimicry of chloroplast has led us to discover that hollow sphere graphitic carbon nitride nanoparticles (hg-C3N4 NPs) endowed non-genetic, subcellular and intercellular photo-modulation of various excitable and non-excitable cells, accumulatively achieving modulation at tissue/organ function level. The homogeneous hg-C3N4 NPs showed responsiveness to light via both photoelectrochemical and photothermal mechanisms. The hg-C3N4 NPs can be spontaneously internalized with excellent cytocompatibility. Using a focusing laser, the hg-C3N4 NPs enable intracellular optical stimulation with subcellular resolution, inducing calcium transient release in multiple cells and propagation in primary cardiomyocytes and cardiac fibroblasts. At multicellular scale, optical pacing and synchronization of cardiomyocyte beating is readily achieved by LED. Further, we demonstrate that hg-C3N4 nanoparticles can be safely delivered into the mouse eye and elicit measurable cortical and behavioral light responses in a subset of animals in a model of advanced retinal degeneration. Finally, application of hg-C3N4 NPs to porcine retinal tissue ex vivo confirmed their modulation capability to directly activate RGCs activity under LED photostimulation. Taken together, these nanostructured biomimetic photocatalytic NPs offer high resolution, leadless optical probing, non-invasive delivery and great biocompatibility, serving as a versatile tool for addressing a range of complex biomedical challenges through subcellular, intercellular and tissue-level photo-modulation across a broad spectrum of scales.

bioengineering↗

bioRxiv2024

Wnt specifically induces FZD5/8 endocytosis and degradation and the involvement of RSPO-ZNRF3/RNF43 and DVL

Frizzled (FZD) proteins are the principal receptors of the Wnt signaling pathway. However, whether Wnt ligands induce FZD endocytosis and degradation remains elusive. The transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 were reported to promote the endocytosis and degradation of FZD receptors to inhibit Wnt signaling, and their function is antagonized by R-spondin (RSPO) proteins. However, the dependency of RSPO-ZNRF3/RNF43-mediated FZD endocytosis and degradation on Wnt stimulation, as well as the specificity of this degradation for different FZD, remains unclear. Here, we demonstrated that Wnt induces FZD5/8 endocytosis and degradation in a ZNRF3/RNF43-dependent manner. ZNRF3/RNF43 selectively targets FZD5/8 for degradation upon Wnt stimulation. RSPO1 enhances Wnt signaling by specifically stabilizing FZD5/8. Wnt promotes the interaction between FZD5 and RNF43. We further demonstrated that DVL proteins promote ligand-independent endocytosis of FZD but are dispensable for Wnt-induced FZD5/8 endocytosis and degradation. Our results reveal a novel negative regulatory mechanism of Wnt signaling at the receptor level and illuminate the mechanism by which RSPO-ZNRF3/RNF43 regulates Wnt signaling, which may provide new insights into regenerative medicine and cancer therapy.

cell biology↗

bioRxiv2024

The Taiwan Precision Medicine Initiative: A Cohort for Large-Scale Studies

The Taiwan Precision Medicine Initiative (TPMI), a project initiated by the Academia Sinica in collaboration with 16 major medical centers around Taiwan, has recruited 565,390 participants who consented to provide DNA samples for genetic profiling and grant access to their electronic medical records (EMR) for studies to develop precision medicine. Access to the EMR is both retrospective and prospective, allowing researchers to conduct prospective studies over time. Genetic profiling is done with population-optimized SNP arrays for the Han Chinese populations that enable genetic analyses such as genome-wide association, phenome-wide association, and polygenic risk score studies to evaluate common disease risk and pharmacogenetic response. Furthermore, the TPMI participants agree to be contacted for future research opportunities related to their genetic risks and receive personalized genetic risk profiles with health management recommendations. TPMI has established the TPMI Data Access Platform (TDAP), a central database and analysis platform that both safeguards the security of the data and facilitates academic research. The TPMI is the largest non-European cohort that merges genetic profiles with EMR in the world. With a cohort that can be followed over time, it can be utilized to validate genetic risk prediction models, conduct clinical trials to show the efficacy of risk-based health management, and optimize health policies based on genetic risks. In this report, we describe the TPMI study design, the population and genetic characteristics of the TPMI cohort, and the power it provides to conduct crucial studies in developing precision medicine on a population and personal level. As Han Chinese represent almost 20% of the worlds population, the results of TPMI studies will benefit >1.4 billion people around the world and serve as a model for developing population-based precision medicine.

genetics↗

bioRxiv2024

DEMINERS enables clinical metagenomics and comparative transcriptomic analysis by increasing throughput and accuracy of nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) advances RNA biology but is limited by relatively low basecalling accuracy, low throughput, yet high RNA input and costs. Here we introduce a novel DRS toolkit, DEMINERS, which integrates an RNA multiplexing experimental workflow, a machine-learning barcode classifier based on Random Forest and a novel basecaller built on an optimized convolutional neural network providing an additional species-specific training module. With the increased accuracy in barcode classification and basecalling, DEMINERS can demultiplex up to 24 samples and the required RNA input and running time are both substantially reduced. We demonstrated the applications of DEMINERS in clinical metagenomics, cancer transcriptomics and parallel comparison of transcriptomic features in different biological conditions, revealing altered airway microbial diversity in COVID-19 and a potential role of m6A in increasing transcriptomic diversity in glioma and the mature blood-stage of malaria parasites. Overall, DEMINERS is a simple, robust, high-throughput DRS method for accurately estimating transcript levels, poly(A) lengths, and mutation and RNA modification heterogeneity at single-read level, with minimal sequencing biases.

bioinformatics↗

bioRxiv2024

Benchmarking alternative polyadenylation detection in single-cell and spatial transcriptomes

Background3-tag-based sequencing methods have become the predominant approach for single-cell and spatial transcriptomics, with some protocols proven effective in detecting alternative polyadenylation (APA). While numerous computational tools have been developed for APA detection from these sequencing data, the absence of comprehensive benchmarks and the diversity of sequencing protocols and tools make it challenging to select appropriate methods for APA analysis in these contexts. ResultsWe systematically compared seven 3-tag-based sequencing protocols and identified key peak features affecting APA detection performance. We developed a simulation pipeline that generates realistic datasets preserving protocol-specific characteristics. Using simulated and real data, we comprehensively assessed six computational tools for their ability to identify polyA sites, quantify polyA site expression, detect differentially expressed (DE) APA genes, filter sequencing artifacts, and their computational efficiency. We also investigated factors influencing APA detection. Our evaluation revealed that SCAPE and scAPAtrap generally outperformed other tools across various performance metrics and protocols. ConclusionOur systematic evaluation provides guidance for tool selection, experiment design, and future tool development in APA analysis for singlecell and spatial transcriptomics, paving the way for investigating APA in these contexts.

bioinformatics↗