Research Article

In situ genome sequencing resolves DNA sequence and structure in intact biological samples

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Science  26 Feb 2021:
Vol. 371, Issue 6532, eaay3446
DOI: 10.1126/science.aay3446

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Visualizing the 3D genome in situ

The conformation of the genome within the cell changes depending on cell state, such that being able to visualize genome structure can identify cis and trans interactions among regulatory genetic elements. Payne et al. have developed an unbiased genome-sequencing technique in single cells in situ that can infer the chromatin structure by imaging. They were able to identify sequences at subnuclei locations to analyze the proximity relationships among genetic elements within and across chromosomes in single cells. Using this technique, they could detect chromosome territories and distinctions between different types of repetitive sequences and chromosomal features. This method can map and image genomic coordinates with submicrometer resolution in intact single cells.

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Structured Abstract


Genomes are spatially organized across length scales from single base pairs to whole chromosomes. This organization is thought to regulate gene expression and control cellular function and varies across cells within organisms. Current methods based on DNA sequencing achieve genome-wide coverage with base-pair resolution but lack spatial context. Alternatively, current methods based on imaging capture spatial context but are targeted and lack base-pair resolution. Thus, a method bridging sequencing and imaging modalities for mapping genome structure is lacking.


Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. Sequencing enables parental alleles and repetitive elements to be distinguished and included in genomic analyses. Further, imaging enables genome-wide study of spatial relationships within cells, such as association of genomic loci with nuclear structures, and between cells, such as structural similarities within cell lineages.


We applied IGS to cultured human fibroblasts and intact early mouse embryos at the pronuclear stage 4 zygote, late two-cell, and early four-cell stages of development, spatially localizing hundreds to thousands of DNA sequences in individual cells. In embryos, we integrated genotype information and immunostaining to identify and characterize parent-specific changes in genome structure between embryonic stages, including parental genome mixing, chromosome polarization, and nuclear lamina association. We further uncovered and characterized single-cell domain structures with lamina-distal boundaries and lamina-proximal interiors in paternal zygotic pronuclei. Finally, we demonstrated epigenetic memory of global chromosome positioning within clonal cell lineages of individual embryos.


IGS unifies sequencing and imaging of genomes, offering a method that connects DNA sequences to their native spatial context within and between the cells of intact biological samples. The single-cell domain structures that we observed in zygotes open opportunities for further investigation, including addressing questions about how nuclear structures such as the lamina may modulate the epigenetic or intrinsic domain-forming behaviors of chromatin. Additionally, our observation of epigenetic memory of chromosome positioning highlights how genome organization during mitosis may influence genome structure at later stages of development. We anticipate that further development of IGS and integration with existing in situ molecular profiling technologies will provide increased resolution and enable multiomic measurements, creating new opportunities to study the structure and function of genomes across length scales and organisms.

IGS unifies sequencing and imaging of genomes.

In situ sequencing of spatial barcodes within intact samples is followed by high-throughput paired-end sequencing. Data from the two modalities are computationally integrated, yielding spatially localized paired-end genomic reads. IGS in early mouse embryos enables the identification of chromosome territories that are assigned to parent-of-origin using base-pair–resolved genotype information. By preserving spatial organization in multicellular samples, IGS allows intercellular comparison of genome structure within individual embryos.


Understanding genome organization requires integration of DNA sequence and three-dimensional spatial context; however, existing genome-wide methods lack either base pair sequence resolution or direct spatial localization. Here, we describe in situ genome sequencing (IGS), a method for simultaneously sequencing and imaging genomes within intact biological samples. We applied IGS to human fibroblasts and early mouse embryos, spatially localizing thousands of genomic loci in individual nuclei. Using these data, we characterized parent-specific changes in genome structure across embryonic stages, revealed single-cell chromatin domains in zygotes, and uncovered epigenetic memory of global chromosome positioning within individual embryos. These results demonstrate how IGS can directly connect sequence and structure across length scales from single base pairs to whole organisms.

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