The major interest domains of single-cell RNA sequential analysis are identification of existing and novel types of cells, depiction of cells, cell fate prediction, classification of several types of tumor, and investigation of heterogeneity in different cells. Single-cell clustering plays an important role to solve the aforementioned questions of interest. Cluster identification in high dimensional single-cell sequencing data faces some challenges due to its nature. Dimensionality reduction models can solve the problem. Here, we introduce a potential cluster specified frequent biomarkers discovery framework using dimensionality reduction and hierarchical agglomerative clustering Louvain for single-cell RNA sequencing data analysis. First, we pre-filtered the features with fewer number of cells and the cells with fewer number of features. Then we created a Seurat object to store data and analysis together and used quality control metrics to discard low quality or dying cells. Afterwards we applied global-scaling normalization method “LogNormalize” for data normalization. Next, we computed cell-to-cell highly variable features from our dataset. Then, we applied a linear transformation and linear dimensionality reduction technique, Principal Component Analysis (PCA) to project high dimensional data to an optimal low-dimensional space. After identifying fifty “significant”principal components (PCs) based on strong enrichment of low p-value features, we implemented a graph-based clustering algorithm Louvain for the cell clustering of 10 top significant PCs. We applied our model to a single-cell RNA sequential dataset for a rare intestinal cell type in mice (NCBI accession ID:GSE62270, 23,630 features and 1872 samples (cells)). We obtained 10 cell clusters with a maximum modularity of 0.885 1. After detecting the cell clusters, we found 3871 cluster-specific biomarkers using an expression feature extraction statistical tool for single-cell sequencing data, Model-based Analysis of Single-cell Transcriptomics (MAST) with a log ₂FC threshold of 0.25 and a minimum feature detection of 25%. From these cluster-specific biomarkers, we found 1892 most frequent markers, i.e., overlapping biomarkers. We performed degree hub gene network analysis using Cytoscape and reported the five highest degree genes (Rps4x, Rps18, Rpl13a, Rps12 and Rpl18a). Subsequently, we performed KEGG pathway and Gene Ontology enrichment analysis of cluster markers using David 6.8 software tool. In summary, our proposed framework that integrated dimensionality reduction and agglomerative hierarchical clustering provides a robust approach to efficiently discover cluster-specific frequent biomarkers, i.e., overlapping biomarkers from single-cell RNA sequencing data.

DNA methylation change has been useful for cancer biomarker discovery, classification, and potential treatment development. So far, existing methods use either differentially methylated CpG sites or combined CpG sites, namely differentially methylated regions, that can be mapped to genes. However, such methylation signal mapping has limitations. To address these limitations, in this study, we introduced a combinatorial framework using linear regression, differential expression, deep learning method for accurate biological interpretation of DNA methylation through integrating DNA methylation data and corresponding TCGA gene expression data. We demonstrated it for uterine cervical cancer. First, we pre-filtered outliers from the data set and then determined the predicted gene expression value from the pre-filtered methylation data through linear regression. We identified differentially expressed genes (DEGs) by Empirical Bayes test using $\mathit{Limma}$. Then we applied a deep learning method, “nnet” to classify the cervical cancer label of those DEGs to determine all classification metrics including accuracy and area under curve (AUC) through 10-fold cross validation. We applied our approach to uterine cervical cancer DNA methylation dataset (NCBI accession ID: GSE30760, 27,578 features covering 63 tumor and 152 matched normal samples). After linear regression and differential expression analysis, we obtained 6287 DEGs with false discovery rate (FDR) $<0.001$. After performing deep learning analysis, we obtained average classification accuracy $90.69\%$ ($\pm 1.97\%$) of the uterine cervical cancerous labels. This performance is better than that of other peer methods. We performed in-degree and out-degree hub gene network analysis using $\mathit{Cytoscape}$. We reported five top in-degree genes ($\mathit{PAIP}\mathit{2}$, $\mathit{GRWD}\mathit{1}$, $\mathit{VPS}\mathit{4}\mathit{B}$, $\mathit{CRADD}$ and $\mathit{LLPH}$) and five top out-degree genes ($\mathit{MRPL}\mathit{35}$, $\mathit{FAM}\mathit{177}\mathit{A}\mathit{1}$, $\mathit{STAT}\mathit{4}$, $\mathit{ASPSCR}\mathit{1}$ and $\mathit{FABP}\mathit{7}$). After that, we performed KEGG pathway and Gene Ontology enrichment analysis of DEGs using tool WebGestalt(WEB-based Gene SeT AnaLysis Toolkit). In summary, our proposed framework that integrated linear regression, differential expression, deep learning provides a robust approach to better interpret DNA methylation analysis and gene expression data in disease study.

In this era, feature clustering is a prominent technique in data mining. Feature clustering has also huge applications in biomedical research for multiple purposes including grouping, feature reduction, and many more. The Internet of Medical Things (IoMT) is a promising and emerging field of research that is having a major impact on knowledge retrieval and networking. IoMT also has significant application in biomedical research regarding remote monitoring and remote healthcare services. In this COVID-19 pandemic situation, psychological effects and human reactions have become a major concern of further research. A dataset can be reduced in size by using feature selection techniques. To facilitate subsequent processing, this will make the data easier to manage. Feature selection is also used to clean, reduce, and reduce dimensions of data. The clustering method has proven to be a powerful tool for finding patterns and structures in both labeled and unlabeled datasets. Our study basically provides various state-of-the-art methods regarding medical IoMT for remote healthcare, feature clustering for information retrieval regarding biomedical science. In this study, we have studied five different types of feature selection algorithms such as minimum redundancy maximum relevance (mRMR), random forest, normalized mutual information feature selection (NMIFS), F-test, and chi-square and five different types of clustering algorithms like hierarchical clustering, density-based spatial clustering of applications with noise (DBSCAN) clustering, K-means clustering, shrinkage clustering, and fuzzy C-means clustering. Finally, this study is very useful to understand and apply the appropriate IoMT, feature clustering, and catharsis on the various biomedical applications for the benevolence of society. Received: 31 March 2023 | Revised: 22 June 2023 | Accepted: 30 June 2023 Conflicts of Interest The authors declare that they have no conflicts of interest to this work. Data Availability Statement The data that support the findings of this study are openly available in UCSC Xena at https://xenabrowser.net/datapages. The data that support the findings of this study are openly available in National Cancer Institute at https://www.cancer.gov/ccg/access-data. The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov/geo/. Author Contribution Statement Atikul Islam: Conceptualization, Methodology, Software, Validation, Formal analysis, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration. Soumita Seth: Methodology, Software, Validation, Formal analysis, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration. Tapas Bhadra: Methodology, Software, Validation, Formal analysis, Resources, Data curation, Writing - review & editing, Visualization, Supervision. Saurav Mallik: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration. Arup Roy: Software, Validation, Formal analysis, Investigation, Resources, Data curation. Aimin Li: Resources, Data curation. Manash Sarkar: Formal analysis, Investigation, Resources, Data curation.

In this current era, the identification of both known and novel cell types, the representation of cells, predicting cell fates, classifying various tumor types, and studying heterogeneity in various cells are the key areas of interest in the analysis of single-cell RNA sequencing (scRNA-seq) data. Due to the nature of the data, cluster identification in single-cell sequencing data with high dimensions presents several difficulties. In this paper, we introduce a new framework that combines various strategies such as imputed matrix, minimum redundancy maximum relevance (MRMR) feature selection, and shrinkage clustering to discover gene signatures from scRNA-seq data. Firstly, we conducted the pre-filtering of the “drop-out” value in the data focusing solely on imputing the identified “drop-out” values. Next, we applied the MRMR feature selection method to the imputed data and obtained the top 100 features based on the MRMR feature selection optimization scores for further downstream analysis. Thereafter, we employed shrinkage clustering on the selected feature matrix to identify the cell clusters using a global optimization approach. Finally, we applied the Limma-Voom R tool employing voom normalization and an empirical Bayes test to detect differentially expressed features with a false discovery rate (FDR) < 0.001. In addition, we performed the KEGG pathway and gene ontology enrichment analysis of the identified biomarkers using David 6.8 software. Furthermore, we conducted miRNA target detection for the top gene markers and performed miRNA target gene interaction network analysis using the Cytoscape online tool. Subsequently, we compared our detected 100 markers with our previously detected top 100 cluster-specified markers ranked by FDR of the latest published article and discovered three common markers; namely, $Cyp2b10$, $Mt1$, $Alpi$, along with 97 novel markers. In addition, the Gene Set Enrichment Analysis (GSEA) of both marker sets also yields similar outcomes. Apart from this, we performed another comparative study with another published method, demonstrating that our model detects more significant markers than that model. To assess the efficiency of our framework, we apply it to another dataset and identify 20 strongly significant up-regulated markers. Additionally, we perform a comparative study of different imputation methods and include an ablation study to prove that every key phase of our framework is essential and strongly recommended. In summary, our proposed integrated framework efficiently discovers differentially expressed stronger gene signatures as well as up-regulated markers in single-cell RNA sequencing data.