Abstract
BACKGROUND: The rapid increase in nucleotide sequence data generated by next-generation sequencing (NGS) technologies demands efficient computational tools for sequence comparison. Alignment-free (AF) methods offer a scalable alternative to traditional alignment-based approaches such as BLAST. This study evaluates alignment-free methods as scalable and rapid alternatives for viral sequence classification, focusing on identifying techniques that maintain high accuracy and efficiency when applied to extremely large datasets. RESULTS: We employed six established AF techniques to extract feature vectors from viral genomes, which were subsequently used to train Random Forest classifiers. Our primary dataset comprises 297,186 SARS-CoV- 2 nucleotide sequences, categorized into 3502 distinct lineages. Furthermore, we validated our models using dengue and HIV sequences to demonstrate robustness across different viral datasets. Our AF classifiers achieved 97.8% accuracy on the SARS-CoV- 2 test set, and 99.8% and 89.1% accuracy on dengue and HIV test sets, respectively. CONCLUSION: Despite the high-class dimensionality, we show that word-based AF methods effectively represent viral sequences. Our study highlights the practical advantages of AF techniques, including significantly faster processing compared to alignment-based methods and the ability to classify sequences using modest computational resources.