BACKGROUND: The immune response to SARS-CoV-2 varies greatly among individuals yielding highly varying severity levels among the patients. While there are various methods to spot severity associated biomarkers in COVID-19 patients, we investigated highly mutated regions, or mutation hotspots, within the SARS-CoV-2 genome that correlate with patient severity levels. SARS-CoV-2 mutation hotspots were searched in the GISAID database using a density based clustering algorithm, Mutclust, that searches for loci with high mutation density and diversity. RESULTS: Using Mutclust, 477 mutation hotspots were searched in the SARS-CoV-2 genome, of which 28 showed significant association with severity levels in a multi-omics COVID-19 cohort comprised of 387 infected patients. The patients were further stratified into moderate and severe patient groups based on the 28 severity related mutation hotspots that showed distinctive cytokine and gene expression levels in both cytokine profile and single-cell RNA-seq samples. The effect of the SARS-CoV-2 mutation hotspots on human genes was further investigated by network propagation analysis, where two mutation hotspots specific to the severe group showed association with NK cell activity. One of them showed to decrease the affinity between the viral epitope of the hotspot region and its binding HLA when compared to the non-mutated epitope. CONCLUSION: Genes related to the immunological function of NK cells, especially the NK cell receptor and co-activating receptor genes, were significantly dysregulated in the severe patient group in both cytokine and single-cell levels. Collectively, mutation hotspots associated with severity and their related NK cell associated gene expression regulation were identified.

BACKGROUND: Rapid advancements in high-throughput sequencing technologies allow for detailed and accurate measurement of omics features within their biological context. The integration of different omics types creates heterogeneous datasets, presenting challenges in analysis due to variations in measurement units, sample numbers, and features. Currently, there is a lack of generalized guidelines for making decisions in multi-omics study design (MOSD), such as selecting an appropriate number of samples and features, type of preprocessing and integration for robust analysis results. We propose a suggestive guideline for MOSD, involving nine important factors: sample size, feature selection, preprocessing strategy, noise characterization, class balance, number of classes, cancer subtype combination, omics combination, and clinical features. RESULTS: To assess the effectiveness of our proposed MOSD guidelines, we designed and conducted seven benchmark tests using 10 clustering methods on various TCGA cancer datasets with an objective of clustering cancer subtypes. The results indicated robust performance in terms of cancer subtype discrimination when adhering to the following criteria: 26 or more samples per class, selecting less than 10% of omics features, maintaining a sample balance under a 3:1 ratio, and keeping the noise level below 30%. Feature selection was particularly important, improving clustering performance by 34%. CONCLUSION: These findings provide evidence-based recommendations for MOSD, enabling researchers to optimize analytical approaches and enhance the reliability of results across cancer datasets. The proposed MOSD framework offers a suggestive guideline addressing both computational and biological factors for multi-omics data integration.