Scalable spatial DNA sequencing from archival tissue maps copy number subclones

Spatially resolved DNA sequencing holds promise due to its potential utility in understanding cancer intra-tumour heterogeneity and tumour evolution in relation to tissue architecture. However, it has so far been used to a limited extent due to technical challenges and high cost of existing methods. Hence we aimed to develop a high throughput spatial genomic assay to obtain copy number alteration (CNA) information at user-defined spatial resolution. We derived CNA profiles from ultra-low coverage whole genome sequencing at sub-millimetre resolution from archival samples using a novel method called Adaptive Resolution Multiscale Spatial DNA sequencing (ARMS DNAseq). We used it to profile CNAs from more than 766 regions (tiles) from 3 patients, covering a total area of over 300 mm2, with 1.2-2.6 million mapped reads per tile and tile sizes of 0.1-0.99mm2. Using ARMS DNAseq, we delineate tumour evolution in a spatial context, and identify more tumour subclones that were obscured or incompletely represented in bulk multi-region whole genome sequencing. Next, we show associations between tumour subclones and morphology, and prediction of subclone identity from deep learning-derived image representations. Finally, we demonstrate multi-omic integration by alignment with spatial transcriptomic data, showing subclone-specific immune cell co-occurrence as well as transcriptional programmes cutting across subclone boundaries. ARMS DNAseq converts low-throughput, region-by-region profiling into a scalable and adaptable workflow for direct spatial copy number profiling from archival tissue sections.