High-throughput linear amplification for single-cell template strand sequencing with sci-L3-Strand-seq

Despite the many advances in single cell genomics, detecting structural rearrangements in single cells, particularly error-free sister-chromatid exchanges, remains challenging. Here we describe sci-L3-Strand-seq, a combinatorial indexing method with linear amplification for DNA template strand sequencing that cost-effectively scales to millions of single cells, as a platform for mapping mitotic crossover (CO) and resulting genome instability events. We provide a computational framework to fully leverage the throughput, as well as the relatively sparse but multifaceted genotype information within each cell that includes strandedness, digital counting of copy numbers, and haplotype-aware chromosome segmentation, to systematically distinguish seven possible types of mitotic CO outcomes. We showcase the power of sci-L3-Strand-seq by quantifying the rates of error-free and mutational COs in thousands of cells, enabling us to explore enrichment patterns of genomic and epigenomic features. The throughput of sci-L3-Strand-seq also gave us the ability to measure subtle phenotypes, opening the door for future large mutational screens. Furthermore, mapping clonal lineages provided insights into the temporal order of certain genome instability events, showcasing the potential to dissect cancer evolution. Altogether, we show the wide applicability of sci-L3-Strand-seq to the study of DNA repair and structural variations. Overall design: We performed replication template strand sequencing of single cells using sci-L3-Strand-seq for mouse diploid Patski, human diploid BJ-5ta, human haploid HAP1, and HAP1 LIG4- knockout cells.