Cell-type-specific consequences of mosaic structural variants in hematopoietic stem and progenitor cells.

The functional impact and cellular context of mosaic structural variants (mSVs) in normal tissues is understudied. Utilizing Strand-seq, we sequenced 1,133 single-cell genomes from 19 human donors of increasing age, and discovered the heterogeneous mSV landscapes of hematopoietic stem and progenitor cells. While mSVs are continuously acquired throughout life, expanded subclones in our cohort are confined to individuals >60. Cells already harboring mSVs are more likely to acquire additional somatic structural variants, including megabase-scale segmental aneuploidies. Capitalizing on comprehensive single-cell micrococcal nuclease digestion with sequencing reference data, we conducted high-resolution cell-typing for eight hematopoietic stem and progenitor cells. Clonally expanded mSVs disrupt normal cellular function by dysregulating diverse cellular pathways, and enriching for myeloid progenitors. Our findings underscore the contribution of mSVs to the cellular and molecular phenotypes associated with the aging hematopoietic system, and establish a foundation for deciphering the molecular links between mSVs, aging and disease susceptibility in normal tissues.

Functional analysis of structural variants in single cells using Strand-seq.

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations.