ClusterV-Web: a user-friendly tool for profiling HIV quasispecies and generating drug resistance reports from nanopore long-read data.

Summary: Third-generation long-read sequencing is an increasingly utilized technique for profiling human immunodeficiency virus (HIV) quasispecies and detecting drug resistance mutations due to its ability to cover the entire viral genome in individual reads. Recently, the ClusterV tool has demonstrated accurate detection of HIV quasispecies from Nanopore long-read sequencing data. However, the need for scripting skills and a computational environment may act as a barrier for many potential users. To address this issue, we have introduced ClusterV-Web, a user-friendly web-based application that enables easy configuration and execution of ClusterV, both remotely and locally. Our tool provides interactive tables and data visualizations to aid in the interpretation of results. This development is expected to democratize access to long-read sequencing data analysis, enabling a wider range of researchers and clinicians to efficiently profile HIV quasispecies and detect drug resistance mutations. Availability and implementation: ClusterV-Web is freely available and open source, with detailed documentation accessible at http://www.bio8.cs.hku.hk/ClusterVW/. The standalone Docker image and source code are also available at https://github.com/HKU-BAL/ClusterV-Web.

Integrated modeling framework reveals co-regulation of transcription factors, miRNAs and lncRNAs on cardiac developmental dynamics.

AIMS: Dissecting complex interactions among transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are central for understanding heart development and function. Although computational approaches and platforms have been described to infer relationships among regulatory factors and genes, current approaches do not adequately account for how highly diverse, interacting regulators that include noncoding RNAs (ncRNAs) control cardiac gene expression dynamics over time. METHODS: To overcome this limitation, we devised an integrated framework, cardiac gene regulatory modeling (CGRM) that integrates LogicTRN and regulatory component analysis bioinformatics modeling platforms to infer complex regulatory mechanisms. We then used CGRM to identify and compare the TF-ncRNA gene regulatory networks that govern early- and late-stage cardiomyocytes (CMs) generated by in vitro differentiation of human pluripotent stem cells (hPSC) and ventricular and atrial CMs isolated during in vivo human cardiac development. RESULTS: Comparisons of in vitro versus in vivo derived CMs revealed conserved regulatory networks among TFs and ncRNAs in early cells that significantly diverged in late staged cells. We report that cardiac genes ("heart targets") expressed in early-stage hPSC-CMs are primarily regulated by MESP1, miR-1, miR-23, lncRNAs NEAT1 and MALAT1, while GATA6, HAND2, miR-200c, NEAT1 and MALAT1 are critical for late hPSC-CMs. The inferred TF-miRNA-lncRNA networks regulating heart development and contraction were similar among early-stage CMs, among individual hPSC-CM datasets and between in vitro and in vivo samples. However, genes related to apoptosis, cell cycle and proliferation, and transmembrane transport showed a high degree of divergence between in vitro and in vivo derived late-stage CMs. Overall, late-, but not early-stage CMs diverged greatly in the expression of "heart target" transcripts and their regulatory mechanisms. CONCLUSIONS: In conclusion, we find that hPSC-CMs are regulated in a cell autonomous manner during early development that diverges significantly as a function of time when compared to in vivo derived CMs. These findings demonstrate the feasibility of using CGRM to reveal dynamic and complex transcriptional and posttranscriptional regulatory interactions that underlie cell directed versus environment-dependent CM development. These results with in vitro versus in vivo derived CMs thus establish this approach for detailed analyses of heart disease and for the analysis of cell regulatory systems in other biomedical fields.

Shape Dependence of Silver-Nanoparticle-Mediated Synthesis of Gold Nanoclusters with Small Molecules as Capping Ligands.

In this study, differently shaped silver nanoparticles used for the synthesis of gold nanoclusters with small capping ligands were demonstrated. Silver nanoparticles provide a reaction platform that plays dual roles in the formation of Au NCs. One is to reduce gold ions and the other is to attract capping ligands to the surface of nanoparticles. The binding of capping ligands to the AgNP surface creates a restricted space on the surface while gold ions are being reduced by the particles. Four different shapes of AgNPs were prepared and used to examine whether or not this approach is dependent on the morphology of AgNPs. Quasi-spherical AgNPs and silver nanoplates showed excellent results when they were used to synthesize Au NCs. Spherical AgNPs and triangular nanoplates exhibited limited synthesis of Au NCs. TEM images demonstrated that Au NCs were transiently assembled on the surface of silver nanoparticles in the method. The formation of Au NCs was observed on the whole surface of the QS-AgNPs if the synthesis of Au NCs was mediated by QS-AgNPs. In contrast, formation of Au NCs was only observed on the edges and corners of AgNPts if the synthesis of Au NCs was mediated by AgNPts. All of the synthesized Au NCs emitted bright red fluorescence under UV-box irradiation. The synthesized Au NCs displayed similar fluorescent properties, including quantum yields and excitation and emission wavelengths.

Boosting variant-calling performance with multi-platform sequencing data using Clair3-MP.

BACKGROUND: With the continuous advances in third-generation sequencing technology and the increasing affordability of next-generation sequencing technology, sequencing data from different sequencing technology platforms is becoming more common. While numerous benchmarking studies have been conducted to compare variant-calling performance across different platforms and approaches, little attention has been paid to the potential of leveraging the strengths of different platforms to optimize overall performance, especially integrating Oxford Nanopore and Illumina sequencing data. RESULTS: We investigated the impact of multi-platform data on the performance of variant calling through carefully designed experiments with a deep learning-based variant caller named Clair3-MP (Multi-Platform). Through our research, we not only demonstrated the capability of ONT-Illumina data for improved variant calling, but also identified the optimal scenarios for utilizing ONT-Illumina data. In addition, we revealed that the improvement in variant calling using ONT-Illumina data comes from an improvement in difficult genomic regions, such as the large low-complexity regions and segmental and collapse duplication regions. Moreover, Clair3-MP can incorporate reference genome stratification information to achieve a small but measurable improvement in variant calling. Clair3-MP is accessible as an open-source project at: https://github.com/HKU-BAL/Clair3-MP . CONCLUSIONS: These insights have important implications for researchers and practitioners alike, providing valuable guidance for improving the reliability and efficiency of genomic analysis in diverse applications.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

BACKGROUND: HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. METHODS: A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. RESULTS: In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/µL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. CONCLUSIONS: Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

Evolution of aniline degradation and nitrogen removal performance in electro-enhanced sequence batch reactor under salinity stress: Sludge characteristics and microbial diversity.

To explore the influence mechanism of different concentrations of salinity on the electro-enhanced aniline biodegradation system, a control group and experimental groups (0%-NaCl, 0.5%-NaCl, 1.5%-NaCl, 2.5%-NaCl, 3.5%-NaCl) were established. The experimental results showed that the electric field strengthened the denitrification performance, while salinity had little effect on the degradation efficiency of aniline and chemical oxygen demand (COD). The removal rate of TN reached 79.6% and 74.9% in 0.5%-NaCl and 1.5%-NaCl, respectively, which were superior than 0%-NaCl. As salinity increased, the nitrogen removal effect was negatively affected. Microbial diversity analysis indicated that the microbial community structure was uniform in the control group, 0%-NaCl, and 0.5%-NaCl, with the dominant genus OLB8 ensuring the nitrogen removal performance. In contrast, in the 2.5%-NaCl and 3.5%-NaCl experimental groups, the organic degrading bacteria were still active, while nitrifiers and denitrifiers were severely damaged. In conclusion, this study suggested that low concentrations of salinity can improve the decontamination performance of the electro-enhanced aniline biodegradation system, while high concentrations of salinity could lead to the collapse of the decontamination mechanism.

Understanding the impacts of intermittent electro field on the bioelectrochemical aniline degradation system: Performance, microbial community and functional enzyme.

On account of the lack of a sustainable electron donor source and the inhibitory effect of aniline on denitrogenation make it tough to achieve simultaneous removal of aniline and nitrogen. Herein, the strategy of adjusting electric field mode was applied to the electro-enhanced sequential batch reactors (E-SBRs: R1 (continuous ON), R2 (2 h-ON/2 h-OFF), R3 (12 h-ON/12 h-OFF), R4 (in the aerobic phase ON), R5 (in the anoxic phase ON)) to treat aniline wastewater. Aniline removal rate reached approximately 99% in the five systems. Decreasing electrical stimulation interval from 12 to 2 h significantly improved the electron utilization efficiency for aniline degradation and nitrogen metabolism. The total nitrogen removal was achieved from 70.31% to 75.63%. Meanwhile, the hydrogenotrophic denitrifiers of Hydrogenophaga, Thauera, and Rhodospirillales, enriched in reactors of minor electrical stimulation interval. Accordingly, the expression of functional enzyme related to electron transport was incremental with the proper electrical stimulation frequency.

Unraveling the nexus of Cr (VI), aniline, and microbial ecology on aniline-degrading biosystem: Removal efficiency, sludge type, microbial ecology.

In order to explore the stress principle of Cr (Ⅵ) on aniline biodegradation system, a control group and experimental groups with the concentration of Cr (Ⅵ) at 2, 5, 8 mg/L were set up. The results demonstrated that Cr (Ⅵ) had minimal effects on the degradation efficiency of aniline but significantly inhibited nitrogen removal function. When Cr (Ⅵ) concentration was below 5 mg/L, the nitrification performance recovered spontaneously, while denitrification performance was severely impaired. Furthermore, the secretion of extracellular polymeric substances (EPS) and its fluorescence substance concentration were strongly inhibited with increasing Cr (Ⅵ) concentration. High-throughput sequencing revealed that the experimental groups were enriched with Leucobacter and Cr (Ⅵ)-reducing bacteria, but the abundance of nitrifiers and denitrifiers was significantly decreased compared to the control group. Overall, the effects of Cr (Ⅵ) stress at different concentrations on nitrogen removal performance were more significant than those on aniline degradation.

Integrated fixed-film activated sludge systems in continuous-flow and batch mode acclimated from low to high aniline concentrations: Performance, mechanism and metabolic pathways.

Integrated fixed-film activated sludge (IFAS) system has considerable advantages in treating aniline wastewater economically and efficiently. However, the response mechanism of IFAS to aniline needs further study. Herein, IFAS in continuous-flow (CF-IFAS) and batch mode (B-IFAS) were set up to investigate it. The removal efficiency of aniline exceeded 99% under different stress intensities. At low stress intensity (aniline ≈ 200 mg/L), the total nitrogen removal efficiency of B-IFAS was approximately 37.76% higher than CF-IFAS. When the stress intensity increased (aniline ≥ 400 mg/L), both were over 82%. CF-IFAS was restrained by denitrification while nitrification in B-IFAS. The legacy effect of perturbation of B-IFAS made microflora quickly reach new stability. The closer interspecific relationship in B-IFAS and more key species: Leucobacter, Rhodococcus, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Ellin6067 and norank_f_NS9_marine_group. Metabolic and Cell growth and death were the most abundant metabolic pathways, resulting both systems the excellent pollutant removal and stability under high stress intensity.

Evaluation of Mycobacterium tuberculosis enrichment in metagenomic samples using ONT adaptive sequencing and amplicon sequencing for identification and variant calling.

Sensitive detection of Mycobacterium tuberculosis (TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford nanopore technologies (ONT) MinION sequencing allows rapid and simple sample preparation for sequencing. Its recently developed adaptive sequencing selects reads from targets while allowing real-time base-calling to achieve sequence enrichment or depletion during sequencing. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods using ONT MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human (95%) and other microbial DNA. Adaptive sequencing with readfish and UNCALLDE achieved a 3.9-fold and 2.2-fold enrichment compared to the control run. We provide a simple automatic analysis framework to support the detection of TB for clinical use, openly available at https://github.com/HKU-BAL/ONT-TB-NF . Depending on the patient's medical condition and sample type, we recommend users evaluate and optimize their workflow for different clinical specimens to improve the detection limit.

Adaptive Multi-ROI Agricultural Robot Navigation Line Extraction Based on Image Semantic Segmentation.

Automated robots are an important part of realizing sustainable food production in smart agriculture. Agricultural robots require a powerful and precise navigation system to be able to perform tasks in the field. Aiming at the problems of complex image background, as well as weed and light interference factors of the visual navigation system in field and greenhouse environments, a Faster-U-net model that retains the advantages of the U-net model feature jump connection is proposed. Based on the U-net model, pruning and optimization were carried out to predict crop ridges. Firstly, a corn dataset was trained to obtain the weight of the corn dataset. Then, the training weight of the obtained corn dataset was used as the pretraining weight for the cucumber, wheat, and tomato datasets, respectively. The three datasets were trained separately. Finally, the navigation line between ridges and the yaw angle of the robot were generated by B-spline curve fitting. The experimental results showed that the parameters of the improved path segmentation model were reduced by 65.86%, and the mPA was 97.39%. The recognition accuracy MIoU of the Faster-U-net model for maize, tomatoes, cucumbers, and wheat was 93.86%, 94.01%, 93.14%, and 89.10%, respectively. The processing speed of the single-core CPU was 22.32 fps/s. The proposed method had strong robustness in predicting rows of different crops. The average angle difference of the navigation line under a ridge environment such as that for corn, tomatoes, cucumbers, or wheat was 0.624°, 0.556°, 0.526°, and 0.999°, respectively. This research can provide technical support and reference for the research and development of intelligent agricultural robot navigation equipment in the field.

The changing C/N of aggressive aniline: Metagenomic analysis of pollutant removal, metabolic pathways and functional genes.

In order to optimize the degradation of high-concentration aniline wastewater, the operation of sequencing batch bioaugmentation reactors with different aniline concentrations (200 mg/L, 600 mg/L, 1000 mg/L) was studied. The results showed that the removal rates of aniline and COD in the three reactors could reach 100%. When the aniline increased to 600 mg/L, the nitrogen removal efficiency reached the peak (51.85%). The increase of aniline inhibited the nitrification, while denitrification was enhanced due to the increase of C/N ratio. But this change was reversed by the toxicity of high concentrations of aniline. The metagenomic analysis showed that when the aniline concentration was 600 mg/L, the abundance distribution of microbial samples was more uniform. The improved of aniline concentration had led to the increase of aromatic compounds degradation metabolic pathways. In addition, the abundance of aniline degradation and nitrogen metabolism genes (dmpB, xylE, norB) was also promoted.

Clair3-trio: high-performance Nanopore long-read variant calling in family trios with trio-to-trio deep neural networks.

Accurate identification of genetic variants from family child-mother-father trio sequencing data is important in genomics. However, state-of-the-art approaches treat variant calling from trios as three independent tasks, which limits their calling accuracy for Nanopore long-read sequencing data. For better trio variant calling, we introduce Clair3-Trio, the first variant caller tailored for family trio data from Nanopore long-reads. Clair3-Trio employs a Trio-to-Trio deep neural network model, which allows it to input the trio sequencing information and output all of the trio's predicted variants within a single model to improve variant calling. We also present MCVLoss, a novel loss function tailor-made for variant calling in trios, leveraging the explicit encoding of the Mendelian inheritance. Clair3-Trio showed comprehensive improvement in experiments. It predicted far fewer Mendelian inheritance violation variations than current state-of-the-art methods. We also demonstrated that our Trio-to-Trio model is more accurate than competing architectures. Clair3-Trio is accessible as a free, open-source project at https://github.com/HKU-BAL/Clair3-Trio.

Exploring the impact of intensity and duration of Cu (II) depression on aniline-degrading biosystem: Performance, sludge activity and microbial diversity.

To evaluate the ecological risk of aniline wastewater biodegradation, the aniline wastewater (200 mg/L) was treated in this work under the stress of Cu (II) at 3, 6 and 10 mg/L, respectively. The slight fluctuation of aniline-degrading performance and the significant inhibition of nitrogen removal was caused by the Cu (II) stress at below 6 mg/L. Meanwhile, the tolerance of nitrifying performance to Cu (II) was higher than denitrifying. The collapse of biosystem was caused by the Cu (II) stress at 10 mg/L and the decontamination function was disabled within 8 days. The activity and stability of sludge declined under the increase of Cu (II) content. Microbial diversity results demonstrated that the genera with heavy-metal tolerance represented by Zoogloea and Azospira significantly dominated under the continuously Cu (II) stress. Whereas, the biosystem with these dominant genera did not achieve the comparable aniline and nitrogen removal performance as the control group.

Understanding the effect of residual aluminum salt coagulant on activated sludge in sequencing batch reactor: Performance response, activity restoration and microbial community evolution.

To investigate the effect of residual coagulant after coagulation pretreatment on activated sludge system of wastewater treatment plants (WWTPs), comparative evaluation of lab-scale sequencing batch reactors under different poly-aluminum chloride (PAC) concentrations (20 and 55 mg/L), presenting the performance differences of reactors. Results showed that the PAC concentration of 20 mg/L slightly enhanced the average removal efficiencies of chemical oxygen demand (COD) and total nitrogen (TN), up to 93.43% and 72.52%. Whereas, an inhibition effect was exerted at the PAC concentration of 55 mg/L, the average removal efficiencies decreased to 88.56% and 57.80% respectively. Similarly, the residual aluminum salts showed a concentration effect of low promotion and high inhibition on sludge activity index. The content of specific oxygen utilization rate (SOUR) and dehydrogenase (DHA) sharply decreased by 30.17% and 53.56% under the high PAC concentration of 55 mg/L. Activity recovery phase showed that the suppression of aluminum salt coagulant on biological system was reversible. High-throughput sequencing presented that the relative abundance of microbes showed obvious variations at different PAC concentrations, and certain bacteria in Chloroflexi and Bacteroidota exhibited better adaptability to the high PAC concentration environment. Nevertheless, the antagonism action between denitrifying genera and other genera as well as the downregulation of functional enzymes regarding nitrogen metabolism gave rise to the deterioration of denitrification under the high PAC concentration of 55 mg/L. This study revealed the influence mechanism of residual aluminum salt coagulant on activated sludge system, providing strategies for efficient decontamination and long-term stable operation of biological system in wastewater treatment plant under the condition of adding PAC.

Transfer of functional microorganism: Regulation of N-acyl-homoserine lactones on the microbial community in aniline-degrading sequencing batch biofilm reactor.

Due to the inhibition of nitrification from aniline toxicity, exogenous N-acyl-homoserine lactones (AHLs) addition was attempted to enhance nitrogen removal in this work. Two sequencing batch biofilm reactors (SBBRs): S1 (the control) and S2 (C6-HSL and 3-oxo-C8-HSL dosing) were used to treat aniline wastewater. The NH4+-N and TN removal rates of S2 were 42.50% and 26.99% higher than S1 in the aerobic phase, respectively. It revealed the nitrogen removal performance of S2 much better than S1. High-throughput sequencing results indicated that many nitrifiers and denitrifiers of S2, such as Nitrosomonas and Thauera, transferred from sludge to biofilm significantly and built closer relationships each other. Overall, main nitrogen removal was contributed by biofilm rather than sludge with the regulation of AHLs. A mild and collaborative environment of biofilms for microorganisms enhanced nitrogen removal. The work provided a new idea for reconciling the contradiction between nitrification and denitrification in aniline wastewater treatment.

Preventive effects of traditional Chinese medicine formula Huoxiangzhengqi against lipopolysaccharide-induced inflammatory response.

BACKGROUND: Huoxiangzhengqi oral liquid (HX), a pharmaceutical product made from traditional Chinese medicine formulas, has been commonly used in household medication for gastrointestinal disorders, but the mode of action remains largely unclear. PURPOSE: This study aims to investigate whether pretreatment with HX prevents lipopolysaccharide (LPS)-induced adverse effects and the potential mechanisms involved. METHODS: Seven-week-old male C57BL/6J mice were orally administered low (1.3 ml/kg) and high doses (2.6 ml/kg) of HX for 7 days, and subsequently subjected to a single dose of LPS at 6 mg/kg. Dexamethasone served as the positive control. Each group had ten animals. RESULTS: The data demonstrated that either a low or high dose of HX significantly reduced the levels of inflammation induced by LPS in both small intestinal and cortical tissues. LPS profoundly decreased the richness and evenness of the microbiota and disrupted the composition of the intestinal microbial community, but pretreatment with HX did not successfully prevent dysbiosis. No significant improvements in HX against LPS were observed in intestinal local immunity or the secretion of partial gut-brain peptides. In addition, pretreatment with HX prevented the alterations in the expression levels of proteins related to the NF-κB pathway, including phospho-p38, p38, phospho-p44/42, p44/42, p50 and p65 induced by LPS. CONCLUSION: Herein, we demonstrated for the first time that the preventive effects of HX against LPS mainly occur through the inhibition of inflammation. These findings provide novel evidence that HX may serve as a new agent for the prevention of gastrointestinal inflammation-related disorders.

An attempt to stimulate aniline degrading bioreactor by exogenous auto-inducer: Decontamination performance, sludge characteristics, and microbial community structure response.

To break the contradiction between aniline and nitrogen metabolism in activated sludge reactor by influencing microbial interspecific communication, Auto-inducer C6-HSL and 3-oxo-C8-HSL were selected in this study to interfere with aniline degradation system. The two Auto-inducers enhanced the aniline degradation rate and ammonia removal efficiency of the systems, especially C6-HSL. Meanwhile, the main ammonia removal way was assimilation. Exogenous Auto-inducer effectively stabilized the sludge structure and activity from the destruction of aniline, and promoted EPS secretion. Microbial diversity analysis showed that most of functional microflora of seed sludge gradually deactivated with the operation of the reactor, while Rhodococcus, Leucobacter, g_norank_f_Saprospiraceae proliferated wildly under the action of Auto-inducer. Additionally, the interspecific relationship also demonstrated a different trend. Exogenous Auto-inducer was proved to exert positive effects on aniline degradation system to a certain extent, providing new insights in the field of aniline wastewater bio-degradation.

Control of aeration time in the aniline degrading-bioreactor with the analysis of metagenomic: Aniline degradation and nitrogen metabolism.

The strategy of adjusting aeration time (5 h/6 h/7 h) was applied to the sequential batch reactors to optimize the treatment of aniline wastewater (600 mg/L) conveniently and economically. Three reactors degraded aniline effectively. The nitrogen removal ability of system with 6 h aeration time was better, performing the similar denitrification property as 5 h and nitrification performance as 7 h. Meanwhile, longer aeration time potentially damaged the sludge structure. The metagenomic analysis explained the micro-mechanism for the better performance of the system with 6 h aeration time. Appropriate aeration time was conducive to the enrichment of synergistic microflora, including aniline degrading-bacteria, heterotrophic nitrifiers and denitrifiers. Then, the tilt of environmental resources to these floras in the system was beneficial to the maximum value utilization of living substrates. Accordingly, these bacteria were more closely related to genes, resulting in higher expression of functional genes in the system.

Symphonizing pileup and full-alignment for deep learning-based long-read variant calling.

Deep learning-based variant callers are becoming the standard and have achieved superior single nucleotide polymorphisms calling performance using long reads. Here we present Clair3, which leverages two major method categories: pileup calling handles most variant candidates with speed, and full-alignment tackles complicated candidates to maximize precision and recall. Clair3 runs faster than any of the other state-of-the-art variant callers and demonstrates improved performance, especially at lower coverage.