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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, PhaGO surpasses state-of-the-art methods in annotating diverged proteins and proteins with uncommon functions by 6.78% and 13.05% improvement, respectively. PhaGO can annotate proteins lacking homology search results, which is critical for characterizing the rapidly accumulating phage genomes. We demonstrate the utility of PhaGO by identifying 688 potential holins in phages, which exhibit high structural conservation with known holins. The results show the potential of PhaGO to extend our understanding of newly discovered phages., Comment: 17 pages,6 figures

The smoothing issue leads to indistinguishable node representations, which poses a significant challenge in the field of graph learning. However, this issue also presents an opportunity to reveal underlying properties behind different types of nodes, which have been overlooked in previous studies. Through empirical and theoretical analysis of real-world node anomaly detection (NAD) datasets, we observe that anomalous and normal nodes show different patterns in the smoothing process, which can be leveraged to enhance NAD tasks. Motivated by these findings, in this paper, we propose a novel unsupervised NAD framework. Specifically, according to our theoretical analysis, we design a Smoothing Learning Component. Subsequently, we introduce a Smoothing-aware Spectral Graph Neural Network, which establishes the connection between the spectral space of graphs and the smoothing process. Additionally, we demonstrate that the Dirichlet Energy, which reflects the smoothness of a graph, can serve as coefficients for node representations across different dimensions of the spectral space. Building upon these observations and analyses, we devise a novel anomaly measure for the NAD task. Extensive experiments on 9 real-world datasets show that SmoothGNN outperforms the best rival by an average of 14.66% in AUC and 7.28% in Precision, with 75x running time speed-up, which validates the effectiveness and efficiency of our framework.

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 almost the same 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., Comment: 3 pages; 1 figure

Motivation: There is accumulating evidence showing the important roles of bacteriophages (phages) in regulating the structure and functions of the microbiome. However, lacking an easy-to-use and integrated phage analysis software hampers microbiome-related research from incorporating phages in the analysis. Results: In this work, we developed a web server, PhaBOX, which can comprehensively identify and analyze phage contigs in metagenomic data. It supports integrated phage analysis, including phage contig identification from the metagenomic assembly, lifestyle prediction, taxonomic classification, and host prediction. Instead of treating the algorithms as a black box, PhaBOX also supports visualization of the essential features for making predictions. The web server is designed with a user-friendly graphical interface that enables both informatics-trained and non-specialist users to analyze phages in microbiome data with ease. Availability: The web server of PhaBOX is available via: https://phage.ee.cityu.edu.hk. The source code of PhaBOX is available at: https://github.com/KennthShang/PhaBOX Contact: yannisun@cityu.edu.hk, Comment: 7 pages, 3 figures, 1 table

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 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 gaming 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 show the benefit of using classified proteins rather than all proteins., Comment: 15 pages, 13 figures

Bacteriophages, which are viruses infecting bacteria, are the most ubiquitous and diverse entities in the biosphere. There is accumulating evidence revealing their important roles in shaping the structure of various microbiomes. Thanks to (viral) metagenomic sequencing, a large number of new bacteriophages have been discovered. However, lacking a standard and automatic virus classification pipeline, the taxonomic characterization of new viruses seriously lag behind the sequencing efforts. In particular, according to the latest version of ICTV, several large phage families in the previous classification system are removed. Therefore, a comprehensive review and comparison of taxonomic classification tools under the new standard are needed to establish the state-of-the-art. In this work, we retrained and tested four recently published tools on newly labeled databases. We demonstrated their utilities and tested them on multiple datasets, including the RefSeq, short contigs, simulated metagenomic datasets, and low-similarity datasets. This study provides a comprehensive review of phage family classification in different scenarios and a practical guidance for choosing appropriate taxonomic classification pipelines. To our best knowledge, this is the first review conducted under the new ICTV classification framework. The results show that the new family classification framework overall leads to better-conserved groups and thus makes family-level classification more feasible., Comment: 13 pages, 6 figures, 6 tables

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., Comment: 16 pages, 11 figures

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\%., Comment: 15 phages, 11 figures

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., Comment: 20 pages, 14 figures

Background: Prokaryotic viruses, which infect bacteria and archaea, are the most abundant and diverse biological entities in the biosphere. To understand their regulatory roles in various ecosystems and to harness the potential of bacteriophages for use in therapy, more knowledge of viral-host relationships is required. High-throughput sequencing and its application to the microbiome have offered new opportunities for computational approaches for predicting which hosts particular viruses can infect. However, there are two main challenges for computational host prediction. First, the empirically known virus-host relationships are very limited. Second, although sequence similarity between viruses and their prokaryote hosts have been used as a major feature for host prediction, the alignment is either missing or ambiguous in many cases. Thus, there is still a need to improve the accuracy of host prediction. Results: In this work, we present a semi-supervised learning model, named HostG, to conduct host prediction for novel viruses. We construct a knowledge graph by utilizing both virus-virus protein similarity and virus-host DNA sequence similarity. Then graph convolutional network (GCN) is adopted to exploit viruses with or without known hosts in training to enhance the learning ability. During the GCN training, we minimize the expected calibrated error (ECE) to ensure the confidence of the predictions. We tested HostG on both simulated and real sequencing data and compared its performance with other state-of-the-art methods specifcally designed for virus host classification (VHM-net, WIsH, PHP, HoPhage, RaFAH, vHULK, and VPF-Class). Conclusion: HostG outperforms other popular methods, demonstrating the efficacy of using a GCN-based semi-supervised learning approach. A particular advantage of HostG is its ability to predict hosts from new taxa., Comment: 16 pages, 14 figures

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 (CNN) and protein sequence similarity gained from gene-sharing network. Then we apply graph convolutional network (GCN) 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., Comment: 15 pages, 10 figures

Motivation: With the development of third-generation sequencing technologies, people are able to obtain DNA sequences with lengths from 10s to 100s of kb. These long reads allow protein domain annotation without assembly, thus can produce important insights into the biological functions of the underlying data. However, the high error rate in third-generation sequencing data raises a new challenge to established domain analysis pipelines. The state-of-the-art methods are not optimized for noisy reads and have shown unsatisfactory accuracy of domain classification in third-generation sequencing data. New computational methods are still needed to improve the performance of domain prediction in long noisy reads. Results: In this work, we introduce ProDOMA, a deep learning model that conducts domain classification for third-generation sequencing reads. It uses deep neural networks with 3-frame translation encoding to learn conserved features from partially correct translations. In addition, we formulate our problem as an open-set problem and thus our model can reject unrelated DNA reads such as those from noncoding regions. In the experiments on simulated reads of protein coding sequences and real reads from the human genome, our model outperforms HMMER and DeepFam on protein domain classification. In summary, ProDOMA is a useful end-to-end protein domain analysis tool for long noisy reads without relying on error correction. Availability: The source code and the trained model are freely available at https://github.com/strideradu/ProDOMA. Contact: yannisun@cityu.edu.hk, Comment: 13 pages, 8 figures (three of them are gourped in Figre. 6)