Full-range genomic analysis at single-cell resolution reveals genetic, epigenetic, and parallel evolution of melanoma subclones

Tumor evolution is driven by various mutational processes, ranging from single nucleotide variants (SNVs) to large structural variants (SVs) to dynamic shifts in DNA methylation. Current short-read sequencing methods struggle to accurately capture the full spectrum of these genomic and epigenomic alterations, as well as their relations, due to inherent technical limitations. Here we used Nanopore long-read sequencing to profile 23 subclones, each derived from a single cell of a mouse melanoma cell line, for precise detection and evolutionary ordering of SNVs, SVs, copy number alterations (CNAs), and DNA methylation changes at subclonal level. Through phylogenetic analysis of these subclones, we reconstruct the timing of mutational processes and their contributions to diverse clonal phenotypes. The analysis reveals recurrent amplifications of putative driver genes, generated by independent SVs across different lineages, suggesting parallel evolution. Additionally, we described lineage-specific methylation changes associated with aggressive tumor subclones, highlighting epigenetic trajectories linked to tumor progression. Overall, we demonstrate that our long-read approach enables a uniquely comprehensive view of melanoma progression, highlighting that SVs and methylation played an important role in initiation, clonal diversification, and development of therapeutic resistance in this tumor, in consistence with recent clinical findings. We will release the sequencing data and curated variant calls to encourage developments of new computational methods.