Scalable Methods for In Situ Genomics

Abstract

The maxim that biological structure determines function was inspired by the discovery of the DNA double helix, yet mapping the structure of genomic DNA within a cell remains challenging, and accordingly the role of genome structure and organization in determining cellular function is an open question. Mapping cellular genome organization is difficult because it requires joint measurement of linear DNA sequence and 3D spatial context, however, existing genome-scale methods lack either base-pair sequence information or direct spatial localization. To overcome these limitations, we invented In Situ Genome Sequencing (IGS), a set of scalable methods for simultaneously sequencing and imaging cellular genomes within intact biological samples. We first report technological developments enabling IGS, including new chemistries for in situ sequencing library construction, workflows for multimodal sequencing of libraries, and strategies for computational integration of spatial and genetic information. Next, we use IGS to map spatial genome organization in cultured human fibroblasts, validating and benchmarking our results against key genomic features such as chromosome positioning, chromosome folding, and repetitive sequence localization. Finally, we apply IGS to map genome organization in intact mouse early embryos, extending known features and uncovering new features of embryonic genome architecture. We characterize parent-specific changes in genome structure across embryonic stages, reveal single-cell chromatin domains in zygotes, and uncover epigenetic memory of global chromosome positioning within individual embryos. We conclude with a discussion of IGS scaling properties, by which we can anticipate many-fold future improvements in yield and resolution. We anticipate IGS and related scalable in situ methods will be instrumental in unifying genomics and microscopy, enabling scientists to map genome organization from single base pairs to whole organisms and ultimately to connect genome structure and function.