Multiplex generation and single cell analysis of structural variants in mammalian genomes [scRNA-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. scRNA-seq libraries were constructed using the 10X Genomics 3' Gene expression with feature barcoding HT kit. Cells were fixed with methanol, subject to in situ transcription (IST) with T7 polymerase and then loaded on to the 10X Genomics chip. Gene expression libraries were constructed according to the manufacturer's protocol. A custom library protocol was employed to sequence T7 derived transcripts corresponding to integrated shuffle-cassettes.