Multiplex generation and single cell analysis of structural variants in mammalian genomes [IVT-seq]

Studying the functional consequences of structural variants (SVs) in mammalian genomes is challenging because: 1) SVs arise much less commonly than single nucleotide variants or small indels; and 2) methods to generate, map and characterize SVs in model systems are underdeveloped. To address these challenges, we developed Genome-Shuffle-seq, a method that enables the multiplex generation and mapping of thousands of SVs (deletions, inversions, translocations, extrachromosomal circles) throughout mammalian genomes. We also demonstrate the co-capture of SV identity with single-cell transcriptomes, facilitating the measurement of SVs' impact on gene expression. We anticipate Genome-Shuffle-seq will be broadly useful for the systematic exploration of the functional consequences of SVs on gene expression, chromatin landscape, and 3D nuclear architecture, while also initiating a path towards a minimal mammalian genome. Overall design: IVT-seq libraries were constructed with T7 In vitro transcription (IVT) derived RNA from genomic DNA extracted from mammalian cell lines (mESC, K562) in which shuffle cassettes were integrated. IVT-seq was largely performed to map integration sites of shuffle cassettes in parental populations but also to validate rearrangements induced by the recombinase.