Bacteriophages are viruses that target bacteria, playing a crucial role in microbial ecology. Phage proteins are important in understanding phage biology, such as virus infection, replication, and evolution. Although a large number of new phages have been identified via metagenomic sequencing, many of them have limited protein function annotation. Accurate function annotation of phage proteins presents several challenges, including their inherent diversity and the scarcity of annotated ones. Existing tools have yet to fully leverage the unique properties of phages in annotating protein functions. In this work, we propose a new protein function annotation tool for phages by leveraging the modular genomic structure of phage genomes. By employing embeddings from the latest protein foundation models and Transformer to capture contextual information between proteins in phage genomes, GOPhage surpasses state-of-the-art methods in annotating diverged proteins and proteins with uncommon functions by 6.78% and 13.05% improvement, respectively. GOPhage can annotate proteins lacking homology search results, which is critical for characterizing the rapidly accumulating phage genomes. We demonstrate the utility of GOPhage by identifying 688 potential holins in phages, which exhibit high structural conservation with known holins. The results show the potential of GOPhage to extend our understanding of newly discovered phages.

Abstract Receptor binding proteins (RBPs) are critical for bacteriophage infection, mediating the initial recognition and attachment to specific bacterial surface receptors, thereby determining host range and infection specificity. Despite their functional importance, RBPs exhibit high sequence diversity, vary significantly in length (200–2000+ amino acids), and remain poorly annotated in current databases. This low conservation limits the effectiveness of homology-based search methods, which often fail to detect distantly related RBPs. Deep learning models offer a more flexible alternative by learning complex sequence patterns, but face challenges in training due to extreme class imbalance—RBPs are vastly outnumbered by non-RBP phage proteins, leading to inefficient learning and biased predictions. To address this, we propose a deep learning framework enhanced with a reinforcement learning-based sample selection strategy that dynamically prioritizes the most informative negative samples during training. Evaluated on a curated phage protein dataset, our method achieves a precision of 0.9455 and an F1-score of 0.7309, outperforming existing homology-based and deep learning approaches. Our results demonstrate that integrating strategic sample selection significantly improves RBP detection in diverse phage genomes.

Accurate prediction of virus-host interactions is critical for understanding viral ecology and developing applications like phage therapy. However, the growing number of computational tools has created a complex landscape, making direct performance comparison challenging due to inconsistent benchmarks and varying usability. Here, we provide a systematic review and a rigorous benchmark of 27 virus-host prediction tools. We formulate the host prediction task into two primary frameworks-link prediction and multi-class classification-and construct two benchmark datasets to evaluate tool performance in distinct scenarios: a database-centric dataset (RefSeq-VHDB) and a metagenomic discovery dataset (MetaHiC-VHDB). Our results reveal that no single tool is universally optimal. Performance is highly context-dependent, with tools like CHERRY and iPHoP demonstrating robust, broad applicability, while others, such as RaFAH and PHIST, excel in specific contexts. We further identify a critical trade-off between predictive accuracy, prediction rate, and computational cost. This work serves as a practical guide for researchers and establishes a standardized benchmark to drive future innovation in deciphering complex virus-host interactions.

MOTIVATION: Nucleocytoplasmic large DNA viruses (NCLDVs) are notable for their large genomes and extensive gene repertoires, which contribute to their widespread environmental presence and critical roles in processes such as host metabolic reprogramming and nutrient cycling. Metagenomic sequencing has emerged as a powerful tool for uncovering novel NCLDVs in environmental samples. However, identifying NCLDV sequences in metagenomic data remains challenging due to their high genomic diversity, limited reference genomes, and shared regions with other microbes. Existing alignment-based and machine learning methods struggle with achieving optimal trade-offs between sensitivity and precision. RESULTS: In this work, we present GiantHunter, a reinforcement learning-based tool for identifying NCLDVs from metagenomic data. By employing a Monte Carlo tree search strategy, GiantHunter dynamically selects representative non-NCLDV sequences as the negative training data, enabling the model to establish a robust decision boundary. Benchmarking on rigorously designed experiments shows that GiantHunter achieves high precision while maintaining competitive sensitivity, improving the F1-score by 10% and reducing computational cost by 90% compared to the second-best method. To demonstrate its real-world utility, we applied GiantHunter to 60 metagenomic datasets collected from six cities along the Yangtze River, located both upstream and downstream of the Three Gorges Dam. The results reveal significant differences in NCLDV diversity correlated with proximity to the dam, likely influenced by reduced flow velocity caused by the dam. These findings highlight GiantHunter's potential to advance our understanding of NCLDVs and their ecological roles in diverse environments. AVAILABILITY AND IMPLEMENTATION: The source code of GiantHunter is available via: https://github.com/FuchuanQu/GiantHunter.

MOTIVATION: Viruses, with their ubiquitous presence and high diversity, play pivotal roles in ecological systems and public health. Accurate identification of viruses in various ecosystems is essential for comprehending their variety and assessing their ecological influence. Metagenomic sequencing has become a major strategy to survey the viruses in various ecosystems. However, accurate and comprehensive virus detection in metagenomic data remains difficult. Limited reference sequences prevent alignment-based methods from identifying novel viruses. Machine learning-based tools are more promising in novel virus detection but often miss short viral contigs, which are abundant in typical metagenomic data. The inconsistency in virus search results produced by available tools further highlights the urgent need for a more robust tool for virus identification. RESULTS: In this work, we develop ViraLM for identifying novel viral contigs in metagenomic data. By using the latest genome foundation model as the backbone and training on a rigorously constructed dataset, the model is able to distinguish viruses from other organisms based on the learned genomic characteristics. We thoroughly tested ViraLM on multiple datasets and the experimental results show that ViraLM outperforms available tools in different scenarios. In particular, ViraLM improves the F1-score on short contigs by 22%. AVAILABILITY AND IMPLEMENTATION: The source code of ViraLM is available via: https://github.com/ChengPENG-wolf/ViraLM.

MOTIVATION: Protein embedding, which represents proteins as numerical vectors, is a crucial step in various learning-based protein annotation/classification problems, including gene ontology prediction, protein-protein interaction prediction, and protein structure prediction. However, existing protein embedding methods are often computationally expensive due to their large number of parameters, which can reach millions or even billions. The growing availability of large-scale protein datasets and the need for efficient analysis tools have created a pressing demand for efficient protein embedding methods. RESULTS: We propose a novel protein embedding approach based on multi-teacher distillation learning, which leverages the knowledge of multiple pre-trained protein embedding models to learn a compact and informative representation of proteins. Our method achieves comparable performance to state-of-the-art methods while significantly reducing computational costs and resource requirements. Specifically, our approach reduces computational time by ∼70% and maintains ±1.5% accuracy as the original large models. This makes our method well-suited for large-scale protein analysis and enables the bioinformatics community to perform protein embedding tasks more efficiently. AVAILABILITY AND IMPLEMENTATION: The source code of MTDP is available via https://github.com/KennthShang/MTDP.

MOTIVATION: Bacteriophages (phages for short), which prey on and replicate within bacterial cells, have a significant role in modulating microbial communities and hold potential applications in treating antibiotic resistance. The advancement of high-throughput sequencing technology contributes to the discovery of phages tremendously. However, the taxonomic classification of assembled phage contigs still faces several challenges, including high genetic diversity, lack of a stable taxonomy system and limited knowledge of phage annotations. Despite extensive efforts, existing tools have not yet achieved an optimal balance between prediction rate and accuracy. RESULTS: In this work, we develop a learning-based model named PhaGenus, which conducts genus-level taxonomic classification for phage contigs. PhaGenus utilizes a powerful Transformer model to learn the association between protein clusters and support the classification of up to 508 genera. We tested PhaGenus on four datasets in different scenarios. The experimental results show that PhaGenus outperforms state-of-the-art methods in predicting low-similarity datasets, achieving an improvement of at least 13.7%. Additionally, PhaGenus is highly effective at identifying previously uncharacterized genera that are not represented in reference databases, with an improvement of 8.52%. The analysis of the infants' gut and GOV2.0 dataset demonstrates that PhaGenus can be used to classify more contigs with higher accuracy.

Viruses are the most ubiquitous and diverse entities in the biome. Due to the rapid growth of newly identified viruses, there is an urgent need for accurate and comprehensive virus classification, particularly for novel viruses. Here, we present PhaGCN2, which can rapidly classify the taxonomy of viral sequences at the family level and supports the visualization of the associations of all families. We evaluate the performance of PhaGCN2 and compare it with the state-of-the-art virus classification tools, such as vConTACT2, CAT and VPF-Class, using the widely accepted metrics. The results show that PhaGCN2 largely improves the precision and recall of virus classification, increases the number of classifiable virus sequences in the Global Ocean Virome dataset (v2.0) by four times and classifies more than 90% of the Gut Phage Database. PhaGCN2 makes it possible to conduct high-throughput and automatic expansion of the database of the International Committee on Taxonomy of Viruses. The source code is freely available at https://github.com/KennthShang/PhaGCN2.0.

Bacteriophages (or phages), which infect bacteria, have two distinct lifestyles: virulent and temperate. Predicting the lifestyle of phages helps decipher their interactions with their bacterial hosts, aiding phages' applications in fields such as phage therapy. Because experimental methods for annotating the lifestyle of phages cannot keep pace with the fast accumulation of sequenced phages, computational method for predicting phages' lifestyles has become an attractive alternative. Despite some promising results, computational lifestyle prediction remains difficult because of the limited known annotations and the sheer amount of sequenced phage contigs assembled from metagenomic data. In particular, most of the existing tools cannot precisely predict phages' lifestyles for short contigs. In this work, we develop PhaTYP (Phage TYPe prediction tool) to improve the accuracy of lifestyle prediction on short contigs. We design two different training tasks, self-supervised and fine-tuning tasks, to overcome lifestyle prediction difficulties. We rigorously tested and compared PhaTYP with four state-of-the-art methods: DeePhage, PHACTS, PhagePred and BACPHLIP. The experimental results show that PhaTYP outperforms all these methods and achieves more stable performance on short contigs. In addition, we demonstrated the utility of PhaTYP for analyzing the phage lifestyle on human neonates' gut data. This application shows that PhaTYP is a useful means for studying phages in metagenomic data and helps extend our understanding of microbial communities.

MOTIVATION: As prevalent extrachromosomal replicons in many bacteria, plasmids play an essential role in their hosts' evolution and adaptation. The host range of a plasmid refers to the taxonomic range of bacteria in which it can replicate and thrive. Understanding host ranges of plasmids sheds light on studying the roles of plasmids in bacterial evolution and adaptation. Metagenomic sequencing has become a major means to obtain new plasmids and derive their hosts. However, host prediction for assembled plasmid contigs still needs to tackle several challenges: different sequence compositions and copy numbers between plasmids and the hosts, high diversity in plasmids, and limited plasmid annotations. Existing tools have not yet achieved an ideal tradeoff between sensitivity and precision on metagenomic assembled contigs. RESULTS: In this work, we construct a hierarchical classification tool named HOTSPOT, whose backbone is a phylogenetic tree of the bacterial hosts from phylum to species. By incorporating the state-of-the-art language model, Transformer, in each node's taxon classifier, the top-down tree search achieves an accurate host taxonomy prediction for the input plasmid contigs. We rigorously tested HOTSPOT on multiple datasets, including RefSeq complete plasmids, artificial contigs, simulated metagenomic data, mock metagenomic data, the Hi-C dataset, and the CAMI2 marine dataset. All experiments show that HOTSPOT outperforms other popular methods. AVAILABILITY AND IMPLEMENTATION: The source code of HOTSPOT is available via: https://github.com/Orin-beep/HOTSPOT.

MOTIVATION: With advances in metagenomic sequencing technologies, there are accumulating studies revealing the associations between the human gut microbiome and some human diseases. These associations shed light on using gut microbiome data to distinguish case and control samples of a specific disease, which is also called host disease status classification. Importantly, using learning-based models to distinguish the disease and control samples is expected to identify important biomarkers more accurately than abundance-based statistical analysis. However, available tools have not fully addressed two challenges associated with this task: limited labeled microbiome data and decreased accuracy in cross-studies. The confounding factors, such as the diet, technical biases in sample collection/sequencing across different studies/cohorts often jeopardize the generalization of the learning model. RESULTS: To address these challenges, we develop a new tool GDmicro, which combines semi-supervised learning and domain adaptation to achieve a more generalized model using limited labeled samples. We evaluated GDmicro on human gut microbiome data from 11 cohorts covering 5 different diseases. The results show that GDmicro has better performance and robustness than state-of-the-art tools. In particular, it improves the AUC from 0.783 to 0.949 in identifying inflammatory bowel disease. Furthermore, GDmicro can identify potential biomarkers with greater accuracy than abundance-based statistical analysis methods. It also reveals the contribution of these biomarkers to the host's disease status. AVAILABILITY AND IMPLEMENTATION: https://github.com/liaoherui/GDmicro.

MOTIVATION: As viruses that mainly infect bacteria, phages are key players across a wide range of ecosystems. Analyzing phage proteins is indispensable for understanding phages' functions and roles in microbiomes. High-throughput sequencing enables us to obtain phages in different microbiomes with low cost. However, compared to the fast accumulation of newly identified phages, phage protein classification remains difficult. In particular, a fundamental need is to annotate virion proteins, the structural proteins, such as major tail, baseplate, etc. Although there are experimental methods for virion protein identification, they are too expensive or time-consuming, leaving a large number of proteins unclassified. Thus, there is a great demand to develop a computational method for fast and accurate phage virion protein (PVP) classification. RESULTS: In this work, we adapted the state-of-the-art image classification model, Vision Transformer, to conduct virion protein classification. By encoding protein sequences into unique images using chaos game representation, we can leverage Vision Transformer to learn both local and global features from sequence "images". Our method, PhaVIP, has two main functions: classifying PVP and non-PVP sequences and annotating the types of PVP, such as capsid and tail. We tested PhaVIP on several datasets with increasing difficulty and benchmarked it against alternative tools. The experimental results show that PhaVIP has superior performance. After validating the performance of PhaVIP, we investigated two applications that can use the output of PhaVIP: phage taxonomy classification and phage host prediction. The results showed the benefit of using classified proteins over all proteins. AVAILABILITY AND IMPLEMENTATION: The web server of PhaVIP is available via: https://phage.ee.cityu.edu.hk/phavip. The source code of PhaVIP is available via: https://github.com/KennthShang/PhaVIP.

Prokaryotic viruses, which infect bacteria and archaea, are key players in microbial communities. Predicting the hosts of prokaryotic viruses helps decipher the dynamic relationship between microbes. Experimental methods for host prediction cannot keep pace with the fast accumulation of sequenced phages. Thus, there is a need for computational host prediction. Despite some promising results, computational host prediction remains a challenge because of the limited known interactions and the sheer amount of sequenced phages by high-throughput sequencing technologies. The state-of-the-art methods can only achieve 43% accuracy at the species level. In this work, we formulate host prediction as link prediction in a knowledge graph that integrates multiple protein and DNA-based sequence features. Our implementation named CHERRY can be applied to predict hosts for newly discovered viruses and to identify viruses infecting targeted bacteria. We demonstrated the utility of CHERRY for both applications and compared its performance with 11 popular host prediction methods. To our best knowledge, CHERRY has the highest accuracy in identifying virus-prokaryote interactions. It outperforms all the existing methods at the species level with an accuracy increase of 37%. In addition, CHERRY's performance on short contigs is more stable than other tools.

MOTIVATION: Bacteriophages are viruses infecting bacteria. Being key players in microbial communities, they can regulate the composition/function of microbiome by infecting their bacterial hosts and mediating gene transfer. Recently, metagenomic sequencing, which can sequence all genetic materials from various microbiome, has become a popular means for new phage discovery. However, accurate and comprehensive detection of phages from the metagenomic data remains difficult. High diversity/abundance, and limited reference genomes pose major challenges for recruiting phage fragments from metagenomic data. Existing alignment-based or learning-based models have either low recall or precision on metagenomic data. RESULTS: In this work, we adopt the state-of-the-art language model, Transformer, to conduct contextual embedding for phage contigs. By constructing a protein-cluster vocabulary, we can feed both the protein composition and the proteins' positions from each contig into the Transformer. The Transformer can learn the protein organization and associations using the self-attention mechanism and predicts the label for test contigs. We rigorously tested our developed tool named PhaMer on multiple datasets with increasing difficulty, including quality RefSeq genomes, short contigs, simulated metagenomic data, mock metagenomic data and the public IMG/VR dataset. All the experimental results show that PhaMer outperforms the state-of-the-art tools. In the real metagenomic data experiment, PhaMer improves the F1-score of phage detection by 27%.

With advances in library construction protocols and next-generation sequencing technologies, viral metagenomic sequencing has become the major source for novel virus discovery. Conducting taxonomic classification for metagenomic data is an important means to characterize the viral composition in the underlying samples. However, RNA viruses are abundant and highly diverse, jeopardizing the sensitivity of comparison-based classification methods. To improve the sensitivity of read-level taxonomic classification, we developed an RNA-dependent RNA polymerase (RdRp) gene-based read classification tool RdRpBin. It combines alignment-based strategy with machine learning models in order to fully exploit the sequence properties of RdRp. We tested our method and compared its performance with the state-of-the-art tools on the simulated and real sequencing data. RdRpBin competes favorably with all. In particular, when the query RNA viruses share low sequence similarity with the known viruses ($\sim 0.4$), our tool can still maintain a higher F-score than the state-of-the-art tools. The experimental results on real data also showed that RdRpBin can classify more RNA viral reads with a relatively low false-positive rate. Thus, RdRpBin can be utilized to classify novel and diverged RNA viruses.

MOTIVATION: Lacking strict proofreading mechanisms, many RNA viruses can generate progeny with slightly changed genomes. Being able to characterize highly similar genomes (i.e. haplotypes) in one virus population helps study the viruses' evolution and their interactions with the host/other microbes. High-throughput sequencing data has become the major source for characterizing viral populations. However, the inherent limitation on read length by next-generation sequencing makes complete haplotype reconstruction difficult. RESULTS: In this work, we present a new tool named HaploDMF that can construct complete haplotypes using third-generation sequencing (TGS) data. HaploDMF utilizes a deep matrix factorization model with an adapted loss function to learn latent features from aligned reads automatically. The latent features are then used to cluster reads of the same haplotype. Unlike existing tools whose performance can be affected by the overlap size between reads, HaploDMF is able to achieve highly robust performance on data with different coverage, haplotype number and error rates. In particular, it can generate more complete haplotypes even when the sequencing coverage drops in the middle. We benchmark HaploDMF against the state-of-the-art tools on simulated and real sequencing TGS data on different viruses. The results show that HaploDMF competes favorably against all others. AVAILABILITY AND IMPLEMENTATION: The source code and the documentation of HaploDMF are available at https://github.com/dhcai21/HaploDMF. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Bacteriophages (aka phages), which mainly infect bacteria, play key roles in the biology of microbes. As the most abundant biological entities on the planet, the number of discovered phages is only the tip of the iceberg. Recently, many new phages have been revealed using high-throughput sequencing, particularly metagenomic sequencing. Compared to the fast accumulation of phage-like sequences, there is a serious lag in taxonomic classification of phages. High diversity, abundance and limited known phages pose great challenges for taxonomic analysis. In particular, alignment-based tools have difficulty in classifying fast accumulating contigs assembled from metagenomic data. RESULTS: In this work, we present a novel semi-supervised learning model, named PhaGCN, to conduct taxonomic classification for phage contigs. In this learning model, we construct a knowledge graph by combining the DNA sequence features learned by convolutional neural network and protein sequence similarity gained from gene-sharing network. Then we apply graph convolutional network to utilize both the labeled and unlabeled samples in training to enhance the learning ability. We tested PhaGCN on both simulated and real sequencing data. The results clearly show that our method competes favorably against available phage classification tools. AVAILABILITY AND IMPLEMENTATION: The source code of PhaGCN is available via: https://github.com/KennthShang/PhaGCN.

BACKGROUND: Infections by RNA viruses such as Influenza, HIV still pose a serious threat to human health despite extensive research on viral diseases. One challenge for producing effective prevention and treatment strategies is high intra-species genetic diversity. As different strains may have different biological properties, characterizing the genetic diversity is thus important to vaccine and drug design. Next-generation sequencing technology enables comprehensive characterization of both known and novel strains and has been widely adopted for sequencing viral populations. However, genome-scale reconstruction of haplotypes is still a challenging problem. In particular, haplotype assembly programs often produce contigs rather than full genomes. As a mutation in one gene can mask the phenotypic effects of a mutation at another locus, clustering these contigs into genome-scale haplotypes is still needed. RESULTS: We developed a contig binning tool, VirBin, which clusters contigs into different groups so that each group represents a haplotype. Commonly used features based on sequence composition and contig coverage cannot effectively distinguish viral haplotypes because of their high sequence similarity and heterogeneous sequencing coverage for RNA viruses. VirBin applied prototype-based clustering to cluster regions that are more likely to contain mutations specific to a haplotype. The tool was tested on multiple simulated sequencing data with different haplotype abundance distributions and contig sizes, and also on mock quasispecies sequencing data. The benchmark results with other contig binning tools demonstrated the superior sensitivity and precision of VirBin in contig binning for viral haplotype reconstruction. CONCLUSIONS: In this work, we presented VirBin, a new contig binning tool for distinguishing contigs from different viral haplotypes with high sequence similarity. It competes favorably with other tools on viral contig binning. The source codes are available at: https://github.com/chjiao/VirBin .

Quick response (QR) code, one of the two‐dimensional barcodes, is now being widely used in all fields. The effectiveness of decoding, however, needs to be improved in real‐time application. In most cases, the decoding procedure is time consuming, in which the detection of QR code plays an essential part. Therefore, this study proposes a fast detection method of QR code based on run‐length coding: firstly, a novel approach is proposed to detect the minimum region containing position detection pattern (PDP) in QR code. Second, coordinates of central PDP in QR code are calculated by using run‐length coding. The highlight in this step is the calculation, which utilises modified Knuth–Morris–Pratt algorithm. By this means, the computational complexity can be reduced tremendously. Finally, QR code can be detected successfully with the coordinates. The experimental results show that the proposed method is time saving and suitable for real‐time application.