Ductal carcinoma in situ (DCIS) is the most common form of early-stage breast cancer. Patients with high-grade DCIS who undergo only surgical treatment face a high recurrence rate (up to 35%). However, the genomic relationships between primary DCIS and recurrence, and the genomic events associated with progression, are poorly understood. A major technical hurdle is that most samples are preserved as formalin-fixed paraffin-embedded (FFPE) blocks, which, although ideal for histopathological analysis, creates challenges for genomic analysis due to extensive DNA damage caused by formalin fixation. To address this, we developed archival nanowell sequencing (Arc-well), a high-throughput single-cell DNA sequencing method tailored to the analysis of archival FFPE tissues. Arc-well applies optimized tagmentation-based chemistry to efficiently label and amplify the degraded DNA fragments in FFPE tissues. By integrating this chemistry with a nanowell dispensing and imaging platform, Arc-well achieves high cellular throughput (up to 2,600 cells), while substantially reducing reagent costs. Arc-well enables the processing of thousands of cells in parallel within a two-day timeframe, considerably shortening experimental time by weeks compared with the degenerate oligonucleotide-primed PCR method. Those features allow Arc-well to profile large numbers of samples at single-cell resolution.

Using Arc-well, we profiled 29,434 single cells of 27 FFPE samples from breast, lung and prostate cancers. Arc-well showed reliable technical performance in these archival samples with the median age of FFPE blocks being about 20 years (the oldest being 31 years).