Multiplex generation and single cell analysis of structural variants in a mammalian genome

Abstract

AbstractThe functional consequences of structural variants (SVs) in mammalian genomes are challenging to study. This is due to several factors, including: 1) their numerical paucity relative to other forms of standing genetic variation such as single nucleotide variants (SNVs) and short insertions or deletions (indels); 2) the fact that a single SV can involve and potentially impact the function of more than one gene and/orcisregulatory element; and 3) the relative immaturity of methods to generate and map SVs, either randomly or in targeted fashion, inin vitroorin vivomodel systems. Towards addressing these challenges, we developedGenome-Shuffle-seq, a straightforward method that enables the multiplex generation and mapping of several major forms of SVs (deletions, inversions, translocations) throughout a mammalian genome.Genome-Shuffle-seqis based on the integration of “shuffle cassettes’’ to the genome, wherein each shuffle cassette contains components that facilitate its site-specific recombination (SSR) with other integrated shuffle cassettes (via Cre-loxP), its mapping to a specific genomic location (via T7-mediatedin vitrotranscription or IVT), and its identification in single-cell RNA-seq (scRNA-seq) data (via T7-mediatedin situtranscription or IST). In this proof-of-concept, we applyGenome-Shuffle-seqto induce and map thousands of genomic SVs in mouse embryonic stem cells (mESCs) in a single experiment. Induced SVs are rapidly depleted from the cellular population over time, possibly due to Cre-mediated toxicity and/or negative selection on the rearrangements themselves. Leveraging T7 IST of barcodes whose positions are already mapped, we further demonstrate that we can efficiently genotype which SVs are present in association with each of many single cell transcriptomes in scRNA-seq data. Finally, preliminary evidence suggests our method may be a powerful means of generating extrachromosomal circular DNAs (ecDNAs). Looking forward, we anticipate thatGenome-Shuffle-seqmay be broadly useful for the systematic exploration of the functional consequences of SVs on gene expression, the chromatin landscape, and 3D nuclear architecture. We further anticipate potential uses forin vitromodeling of ecDNAs, as well as in paving the path to a minimal mammalian genome.