Single-cell ribonucleic acid sequencing (scRNA-seq) technology can be used to perform high-resolution analysis of the transcriptomes of individual cells. Therefore, its application has gained popularity for accurately analyzing the ever-increasing content of heterogeneous single-cell datasets. Central to interpreting scRNA-seq data is the clustering of cells to decipher transcriptomic diversity and infer cell behavior patterns. However, its complexity necessitates the application of advanced methodologies capable of resolving the inherent heterogeneity and limited gene expression characteristics of single-cell data. Herein, we introduce a novel deep learning-based algorithm for single-cell clustering, designated scDFN, which can significantly enhance the clustering of scRNA-seq data through a fusion network strategy. The scDFN algorithm applies a dual mechanism involving an autoencoder to extract attribute information and an improved graph autoencoder to capture topological nuances, integrated via a cross-network information fusion mechanism complemented by a triple self-supervision strategy. This fusion is optimized through a holistic consideration of four distinct loss functions. A comparative analysis with five leading scRNA-seq clustering methodologies across multiple datasets revealed the superiority of scDFN, as determined by better the Normalized Mutual Information (NMI) and the Adjusted Rand Index (ARI) metrics. Additionally, scDFN demonstrated robust multi-cluster dataset performance and exceptional resilience to batch effects. Ablation studies highlighted the key roles of the autoencoder and the improved graph autoencoder components, along with the critical contribution of the four joint loss functions to the overall efficacy of the algorithm. Through these advancements, scDFN set a new benchmark in single-cell clustering and can be used as an effective tool for the nuanced analysis of single-cell transcriptomics.

Liquid-liquid phase separation (LLPS) is one of the mechanisms mediating the compartmentalization of macromolecules (proteins and nucleic acids) in cells, forming biomolecular condensates or membraneless organelles. Consequently, the systematic identification of potential LLPS proteins is crucial for understanding the phase separation process and its biological mechanisms. A two-task predictor, Opt_PredLLPS, was developed to discover potential phase separation proteins and further evaluate their mechanism. The first task model of Opt_PredLLPS combines a convolutional neural network (CNN) and bidirectional long short-term memory (BiLSTM) through a fully connected layer, where the CNN utilizes evolutionary information features as input, and BiLSTM utilizes multimodal features as input. If a protein is predicted to be an LLPS protein, it is input into the second task model to predict whether this protein needs to interact with its partners to undergo LLPS. The second task model employs the XGBoost classification algorithm and 37 physicochemical properties following a three-step feature selection. The effectiveness of the model was validated on multiple benchmark datasets, and in silico saturation mutagenesis was used to identify regions that play a key role in phase separation. These findings may assist future research on the LLPS mechanism and the discovery of potential phase separation proteins.

Circular RNAs (circRNAs) exist in vivo and are a class of noncoding RNA molecules. They have a single-stranded, closed, annular structure. Many studies have shown that circRNAs and diseases are linked. Therefore, it is critical to build a reliable and accurate predictor to find the circRNA-disease association. In this paper, we presented a meta-learning model named MAMLCDA to identify the circRNA-disease association, which is based on model-agnostic meta-learning (MAML) combined with CNN classification. Specifically, similarities between diseases and circRNAs are extracted and integrated to characterize their relationships, and k-means is used to cluster majority samples and select a certain number of samples from each cluster to obtain the same number of negative samples as the positive samples. To further reduce the dimension of the features and save operation time, we applied probabilistic principal component analysis (PPCA) to compact the integrated circRNA and disease similarity network feature vectors. The feature vectors are converted into images. At this time, the prediction problem is transformed into the 2-way 1-shot problem of the image and input into the model with MAML as the meta-learner and CNN as the base-learner. Comparison results of five-fold cross-validation on two benchmark datasets illustrate that MAMLCDA outperforms several state-of-the-art approaches with the best accuracies of 95.33% and 98%. Therefore, MAMLCDA can help to understand the pathogenesis of complex diseases at the circRNA level.

The formation of biomolecular condensates by liquid-liquid phase separation (LLPS) has become a universal mechanism for spatiotemporal coordination of biological activities in cells and has been widely observed to directly regulate the key cellular processes involved in cancer cell pathology. However, the complexity of protein sequences and the diversity of conformations are inherently disordered, which poses great challenges for LLPS protein calculations and experimental research. Herein, we proposed a novel predictor named PredLLPS_PSSM for LLPS protein identification based only on sequence evolution information. Because finding real and reliable samples is the cornerstone of building predictors, we collected anew and collated the LLPS proteins from the latest versions of three databases. By comparing the performance of the position-specific score matrix (PSSM) and word embedding, PredLLPS_PSSM combined PSSM-based information and two deep learning frameworks. Independent tests using three existing independent test datasets and two newly constructed independent test datasets demonstrated the superiority of PredLLPS_PSSM compared with state-of-the-art methods. Furthermore, we tested PredLLPS_PSSM on nine experimentally identified LLPS proteins from three insects that were not included in any of the databases. In addition, the powerful Shapley Additive exPlanation algorithm and heatmap were applied to find the most critical amino acids relevant to LLPS.

BACKGROUND: Promoters are DNA regions that initiate the transcription of specific genes near the transcription start sites. In bacteria, promoters are recognized by RNA polymerases and associated sigma factors. Effective promoter recognition is essential for synthesizing the gene-encoded products by bacteria to grow and adapt to different environmental conditions. A variety of machine learning-based predictors for bacterial promoters have been developed; however, most of them were designed specifically for a particular species. To date, only a few predictors are available for identifying general bacterial promoters with limited predictive performance. RESULTS: In this study, we developed TIMER, a Siamese neural network-based approach for identifying both general and species-specific bacterial promoters. Specifically, TIMER uses DNA sequences as the input and employs three Siamese neural networks with the attention layers to train and optimize the models for a total of 13 species-specific and general bacterial promoters. Extensive 10-fold cross-validation and independent tests demonstrated that TIMER achieves a competitive performance and outperforms several existing methods on both general and species-specific promoter prediction. As an implementation of the proposed method, the web server of TIMER is publicly accessible at http://web.unimelb-bioinfortools.cloud.edu.au/TIMER/.

Subcellular localization of messenger RNAs (mRNAs) plays a key role in the spatial regulation of gene activity. The functions of mRNAs have been shown to be closely linked with their localizations. As such, understanding of the subcellular localizations of mRNAs can help elucidate gene regulatory networks. Despite several computational methods that have been developed to predict mRNA localizations within cells, there is still much room for improvement in predictive performance, especially for the multiple-location prediction. In this study, we proposed a novel multi-label multi-class predictor, termed Clarion, for mRNA subcellular localization prediction. Clarion was developed based on a manually curated benchmark dataset and leveraged the weighted series method for multi-label transformation. Extensive benchmarking tests demonstrated Clarion achieved competitive predictive performance and the weighted series method plays a crucial role in securing superior performance of Clarion. In addition, the independent test results indicate that Clarion outperformed the state-of-the-art methods and can secure accuracy of 81.47, 91.29, 79.77, 92.10, 89.15, 83.74, 80.74, 79.23 and 84.74% for chromatin, cytoplasm, cytosol, exosome, membrane, nucleolus, nucleoplasm, nucleus and ribosome, respectively. The webserver and local stand-alone tool of Clarion is freely available at http://monash.bioweb.cloud.edu.au/Clarion/.

Conventional supervised binary classification algorithms have been widely applied to address significant research questions using biological and biomedical data. This classification scheme requires two fully labeled classes of data (e.g. positive and negative samples) to train a classification model. However, in many bioinformatics applications, labeling data is laborious, and the negative samples might be potentially mislabeled due to the limited sensitivity of the experimental equipment. The positive unlabeled (PU) learning scheme was therefore proposed to enable the classifier to learn directly from limited positive samples and a large number of unlabeled samples (i.e. a mixture of positive or negative samples). To date, several PU learning algorithms have been developed to address various biological questions, such as sequence identification, functional site characterization and interaction prediction. In this paper, we revisit a collection of 29 state-of-the-art PU learning bioinformatic applications to address various biological questions. Various important aspects are extensively discussed, including PU learning methodology, biological application, classifier design and evaluation strategy. We also comment on the existing issues of PU learning and offer our perspectives for the future development of PU learning applications. We anticipate that our work serves as an instrumental guideline for a better understanding of the PU learning framework in bioinformatics and further developing next-generation PU learning frameworks for critical biological applications.

Promoters are crucial regulatory DNA regions for gene transcriptional activation. Rapid advances in next-generation sequencing technologies have accelerated the accumulation of genome sequences, providing increased training data to inform computational approaches for both prokaryotic and eukaryotic promoter prediction. However, it remains a significant challenge to accurately identify species-specific promoter sequences using computational approaches. To advance computational support for promoter prediction, in this study, we curated 58 comprehensive, up-to-date, benchmark datasets for 7 different species (i.e. Escherichia coli, Bacillus subtilis, Homo sapiens, Mus musculus, Arabidopsis thaliana, Zea mays and Drosophila melanogaster) to assist the research community to assess the relative functionality of alternative approaches and support future research on both prokaryotic and eukaryotic promoters. We revisited 106 predictors published since 2000 for promoter identification (40 for prokaryotic promoter, 61 for eukaryotic promoter, and 5 for both). We systematically evaluated their training datasets, computational methodologies, calculated features, performance and software usability. On the basis of these benchmark datasets, we benchmarked 19 predictors with functioning webservers/local tools and assessed their prediction performance. We found that deep learning and traditional machine learning-based approaches generally outperformed scoring function-based approaches. Taken together, the curated benchmark dataset repository and the benchmarking analysis in this study serve to inform the design and implementation of computational approaches for promoter prediction and facilitate more rigorous comparison of new techniques in the future.

DNA N4-methylcytosine (4mC) is an important epigenetic modification that plays a vital role in regulating DNA replication and expression. However, it is challenging to detect 4mC sites through experimental methods, which are time-consuming and costly. Thus, computational tools that can identify 4mC sites would be very useful for understanding the mechanism of this important type of DNA modification. Several machine learning-based 4mC predictors have been proposed in the past 3 years, although their performance is unsatisfactory. Deep learning is a promising technique for the development of more accurate 4mC site predictions. In this work, we propose a deep learning-based approach, called DeepTorrent, for improved prediction of 4mC sites from DNA sequences. It combines four different feature encoding schemes to encode raw DNA sequences and employs multi-layer convolutional neural networks with an inception module integrated with bidirectional long short-term memory to effectively learn the higher-order feature representations. Dimension reduction and concatenated feature maps from the filters of different sizes are then applied to the inception module. In addition, an attention mechanism and transfer learning techniques are also employed to train the robust predictor. Extensive benchmarking experiments demonstrate that DeepTorrent significantly improves the performance of 4mC site prediction compared with several state-of-the-art methods.

The development of deep sequencing technologies has led to the discovery of novel transcripts. Many in silico methods have been developed to assess the coding potential of these transcripts to further investigate their functions. Existing methods perform well on distinguishing majority long noncoding RNAs (lncRNAs) and coding RNAs (mRNAs) but poorly on RNAs with small open reading frames (sORFs). Here, we present DeepCPP (deep neural network for coding potential prediction), a deep learning method for RNA coding potential prediction. Extensive evaluations on four previous datasets and six new datasets constructed in different species show that DeepCPP outperforms other state-of-the-art methods, especially on sORF type data, which overcomes the bottleneck of sORF mRNA identification by improving more than 4.31, 37.24 and 5.89% on its accuracy for newly discovered human, vertebrate and insect data, respectively. Additionally, we also revealed that discontinuous k-mer, and our newly proposed nucleotide bias and minimal distribution similarity feature selection method play crucial roles in this classification problem. Taken together, DeepCPP is an effective method for RNA coding potential prediction.

Long noncoding RNAs (lncRNAs) play significant roles in various physiological and pathological processes via their interactions with biomolecules like DNA, RNA and protein. The existing in silico methods used for predicting the functions of lncRNA mainly rely on calculating the similarity of lncRNA or investigating whether an lncRNA can interact with a specific biomolecule or disease. In this work, we explored the functions of lncRNA from a different perspective: we presented a tool for predicting the interaction biomolecule type for a given lncRNA. For this purpose, we first investigated the main molecular mechanisms of the interactions of lncRNA-RNA, lncRNA-protein and lncRNA-DNA. Then, we developed an ensemble deep learning model: lncIBTP (lncRNA Interaction Biomolecule Type Prediction). This model predicted the interactions between lncRNA and different types of biomolecules. On the 5-fold cross-validation, the lncIBTP achieves average values of 0.7042 in accuracy, 0.7903 and 0.6421 in macro-average area under receiver operating characteristic curve and precision-recall curve, respectively, which illustrates the model effectiveness. Besides, based on the analysis of the collected published data and prediction results, we hypothesized that the characteristics of lncRNAs that interacted with DNA may be different from those that interacted with only RNA.

A promoter is a region in the DNA sequence that defines where the transcription of a gene by RNA polymerase initiates, which is typically located proximal to the transcription start site (TSS). How to correctly identify the gene TSS and the core promoter is essential for our understanding of the transcriptional regulation of genes. As a complement to conventional experimental methods, computational techniques with easy-to-use platforms as essential bioinformatics tools can be effectively applied to annotate the functions and physiological roles of promoters. In this work, we propose a deep learning-based method termed Depicter (Deep learning for predicting promoter), for identifying three specific types of promoters, i.e. promoter sequences with the TATA-box (TATA model), promoter sequences without the TATA-box (non-TATA model), and indistinguishable promoters (TATA and non-TATA model). Depicter is developed based on an up-to-date, species-specific dataset which includes Homo sapiens, Mus musculus, Drosophila melanogaster and Arabidopsis thaliana promoters. A convolutional neural network coupled with capsule layers is proposed to train and optimize the prediction model of Depicter. Extensive benchmarking and independent tests demonstrate that Depicter achieves an improved predictive performance compared with several state-of-the-art methods. The webserver of Depicter is implemented and freely accessible at https://depicter.erc.monash.edu/.

Lysine formylation is a reversible type of protein post-translational modification and has been found to be involved in a myriad of biological processes, including modulation of chromatin conformation and gene expression in histones and other nuclear proteins. Accurate identification of lysine formylation sites is essential for elucidating the underlying molecular mechanisms of formylation. Traditional experimental methods are time-consuming and expensive. As such, it is desirable and necessary to develop computational methods for accurate prediction of formylation sites. In this study, we propose a novel predictor, termed Formator, for identifying lysine formylation sites from sequences information. Formator is developed using the ensemble learning (EL) strategy based on four individual support vector machine classifiers via a voting system. Moreover, the most distant undersampling and Safe-Level-SMOTE oversampling techniques were integrated to deal with the data imbalance problem of the training dataset. Four effective feature extraction methods, namely bi-profile Bayes (BPB), k-nearest neighbor (KNN), amino acid physicochemical properties (AAindex), and composition and transition (CTD) were employed to encode the surrounding sequence features of potential formylation sites. Extensive empirical studies show that Formator achieved the accuracy of 87.24 and 74.96 percent on jackknife test and the independent test, respectively. Performance comparison results on the independent test indicate that Formator outperforms current existing prediction tool, LFPred, suggesting that it has a great potential to serve as a useful tool in identifying novel lysine formylation sites and facilitating hypothesis-driven experimental efforts.

Post-translational modifications (PTMs) play very important roles in various cell signaling pathways and biological process. Due to PTMs' extremely important roles, many major PTMs have been studied, while the functional and mechanical characterization of major PTMs is well documented in several databases. However, most currently available databases mainly focus on protein sequences, while the real 3D structures of PTMs have been largely ignored. Therefore, studies of PTMs 3D structural signatures have been severely limited by the deficiency of the data. Here, we develop PRISMOID, a novel publicly available and free 3D structure database for a wide range of PTMs. PRISMOID represents an up-to-date and interactive online knowledge base with specific focus on 3D structural contexts of PTMs sites and mutations that occur on PTMs and in the close proximity of PTM sites with functional impact. The first version of PRISMOID encompasses 17 145 non-redundant modification sites on 3919 related protein 3D structure entries pertaining to 37 different types of PTMs. Our entry web page is organized in a comprehensive manner, including detailed PTM annotation on the 3D structure and biological information in terms of mutations affecting PTMs, secondary structure features and per-residue solvent accessibility features of PTM sites, domain context, predicted natively disordered regions and sequence alignments. In addition, high-definition JavaScript packages are employed to enhance information visualization in PRISMOID. PRISMOID equips a variety of interactive and customizable search options and data browsing functions; these capabilities allow users to access data via keyword, ID and advanced options combination search in an efficient and user-friendly way. A download page is also provided to enable users to download the SQL file, computational structural features and PTM sites' data. We anticipate PRISMOID will swiftly become an invaluable online resource, assisting both biologists and bioinformaticians to conduct experiments and develop applications supporting discovery efforts in the sequence-structural-functional relationship of PTMs and providing important insight into mutations and PTM sites interaction mechanisms. The PRISMOID database is freely accessible at http://prismoid.erc.monash.edu/. The database and web interface are implemented in MySQL, JSP, JavaScript and HTML with all major browsers supported.

MOTIVATION: Different from traditional linear RNAs (containing 5' and 3' ends), circular RNAs (circRNAs) are a special type of RNAs that have a closed ring structure. Accumulating evidence has indicated that circRNAs can directly bind proteins and participate in a myriad of different biological processes. RESULTS: For identifying the interaction of circRNAs with 37 different types of circRNA-binding proteins (RBPs), we develop an ensemble neural network, termed PASSION, which is based on the concatenated artificial neural network (ANN) and hybrid deep neural network frameworks. Specifically, the input of the ANN is the optimal feature subset for each RBP, which has been selected from six types of feature encoding schemes through incremental feature selection and application of the XGBoost algorithm. In turn, the input of the hybrid deep neural network is a stacked codon-based scheme. Benchmarking experiments indicate that the ensemble neural network reaches the average best area under the curve (AUC) of 0.883 across the 37 circRNA datasets when compared with XGBoost, k-nearest neighbor, support vector machine, random forest, logistic regression and Naive Bayes. Moreover, each of the 37 RBP models is extensively tested by performing independent tests, with the varying sequence similarity thresholds of 0.8, 0.7, 0.6 and 0.5, respectively. The corresponding average AUC obtained are 0.883, 0.876, 0.868 and 0.883, respectively, highlighting the effectiveness and robustness of PASSION. Extensive benchmarking experiments demonstrate that PASSION achieves a competitive performance for identifying binding sites between circRNA and RBPs, when compared with several state-of-the-art methods. AVAILABILITY AND IMPLEMENTATION: A user-friendly web server of PASSION is publicly accessible at http://flagship.erc.monash.edu/PASSION/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: N4-methylcytosine (4mC), an important epigenetic modification formed by the action of specific methyltransferases, plays an essential role in DNA repair, expression and replication. The accurate identification of 4mC sites aids in-depth research to biological functions and mechanisms. Because, experimental identification of 4mC sites is time-consuming and costly, especially given the rapid accumulation of gene sequences. Supplementation with efficient computational methods is urgently needed. RESULTS: In this study, we developed a new tool, 4mCPred, for predicting 4mC sites in Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Escherichia coli, Geoalkalibacter subterraneus and Geobacter pickeringii. 4mCPred consists of two independent models, 4mCPred_I and 4mCPred_II, for each species. The predictive results of independent and cross-species tests demonstrated that the performance of 4mCPred_I is a useful tool. To identify position-specific trinucleotide propensity (PSTNP) and electron-ion interaction potential features, we used the F-score method to construct predictive models and to compare their PSTNP features. Compared with other existing predictors, 4mCPred achieved much higher accuracies in rigorous jackknife and independent tests. We also analyzed the importance of different features in detail. AVAILABILITY AND IMPLEMENTATION: The web-server 4mCPred is accessible at http://server.malab.cn/4mCPred/index.jsp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Promoters are short DNA consensus sequences that are localized proximal to the transcription start sites of genes, allowing transcription initiation of particular genes. However, the precise prediction of promoters remains a challenging task because individual promoters often differ from the consensus at one or more positions. RESULTS: In this study, we present a new multi-layer computational approach, called MULTiPly, for recognizing promoters and their specific types. MULTiPly took into account the sequences themselves, including both local information such as k-tuple nucleotide composition, dinucleotide-based auto covariance and global information of the entire samples based on bi-profile Bayes and k-nearest neighbour feature encodings. Specifically, the F-score feature selection method was applied to identify the best unique type of feature prediction results, in combination with other types of features that were subsequently added to further improve the prediction performance of MULTiPly. Benchmarking experiments on the benchmark dataset and comparisons with five state-of-the-art tools show that MULTiPly can achieve a better prediction performance on 5-fold cross-validation and jackknife tests. Moreover, the superiority of MULTiPly was also validated on a newly constructed independent test dataset. MULTiPly is expected to be used as a useful tool that will facilitate the discovery of both general and specific types of promoters in the post-genomic era. AVAILABILITY AND IMPLEMENTATION: The MULTiPly webserver and curated datasets are freely available at http://flagshipnt.erc.monash.edu/MULTiPly/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Motivation: Protein O-GlcNAcylation (O-GlcNAc) is an important post-translational modification of serine (S)/threonine (T) residues that involves multiple molecular and cellular processes. Recent studies have suggested that abnormal O-G1cNAcylation causes many diseases, such as cancer and various neurodegenerative diseases. With the available protein O-G1cNAcylation sites experimentally verified, it is highly desired to develop automated methods to rapidly and effectively identify O-GlcNAcylation sites. Although some computational methods have been proposed, their performance has been unsatisfactory, particularly in terms of prediction sensitivity. Results: In this study, we developed an ensemble model O-GlcNAcPRED-II to identify potential O-GlcNAcylation sites. A K-means principal component analysis oversampling technique (KPCA) and fuzzy undersampling method (FUS) were first proposed and incorporated to reduce the proportion of the original positive and negative training samples. Then, rotation forest, a type of classifier-integrated system, was adopted to divide the eight types of feature space into several subsets using four sub-classifiers: random forest, k-nearest neighbour, naive Bayesian and support vector machine. We observed that O-GlcNAcPRED-II achieved a sensitivity of 81.05%, specificity of 95.91%, accuracy of 91.43% and Matthew's correlation coefficient of 0.7928 for five-fold cross-validation run 10 times. Additionally, the results obtained by O-GlcNAcPRED-II on two independent datasets also indicated that the proposed predictor outperformed five published prediction tools. Availability and implementation: http://121.42.167.206/OGlcPred/. Supplementary information: Supplementary data are available at Bioinformatics online.