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

Payne, Andrew; Chiang, Zachary; Reginato, Paul; Mangiameli, Sarah; Murray, Evan; Yao, Chun-Chen; Markoulaki, Styliani; Earl, Andrew; Labade, Ajay S.; Jaenisch, Rudolf; Church, George M.; Boyden, Edward S.; Buenrostro, Jason D.; Chen, Fei

doi:10.1126/science.aay3446

Cited by 154 publications

(122 citation statements)

References 99 publications

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“…We also note the recent reports of multiple highly variant chromatin domain structures at the single-cell level observed with different advanced technologies, e.g . nucleosome clutches 8 , chromatin nanodomains (CNDs) 20 , packing domains (PDs) 48 , and larger single-cell domains (SCDs) that are multi-megabase in size 49 . The relationship between these structures and the TAD-like single-cell domains identified by chromatin tracing also requires further investigation.…”

Section: Discussionmentioning

confidence: 99%

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Cheng

Liu

et al. 2021

Preprint

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Background: Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs was shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing led to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains - highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Several recent computational modeling studies suggest that single-cell variations of epigenetic profiles may underlie the formation of the single-cell domains. Results: Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components did not significantly affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains. Conclusions: In sum, this study suggests that single-cell domains are genome architecture building blocks independent of variations in major epigenetic landscapes.

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Section: Discussionmentioning

confidence: 99%

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Cheng

Liu

et al. 2021

Preprint

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“…Complementary to the genomics approaches, imaging-based approaches such as DNA fluorescence in situ hybridization (FISH) (20) can directly obtain 3D chromosome structures from measured loci in single cells without computational reconstructions. Recent multiplexed imaging-based methods (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32), such as sequential DNA FISH (22) and in situ genome sequencing (IGS) (31), have directly characterized the variabilities of chromosome structures in 3D, even between homologous chromosomes in a cell, and reported TAD-like domain structures in single cells (23), which when averaged over populations of cells are consistent with sequencing-based bulk measurements. Furthermore, super-resolution imaging studies (23,33,34) have shown that architectural proteins such as CTCF and cohesin can play important roles in the single-cell domain structures.…”

Section: Main Textmentioning

confidence: 73%

“…Lastly, we examined the spatial distance between pairs of intra-chromosomal loci to understand cell-type specific chromosomal scaling in the nucleus. Although previous imaging studies in cell culture and embryos showed differences in the chromosomal scaling of spatial distance as a function of genomic distance (22,31,32), it remains unclear how the scaling principles operate across different cell types within the same tissues. We found that the relationship between spatial versus genomic distance scaling was distinct in different cell types, in both 1-Mb and 25-kb resolution data (Fig.…”

Section: Distinct Nuclear Features Across Cell Types In the Brainmentioning

confidence: 98%

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Integrated spatial genomics in tissues reveals invariant and cell type dependent nuclear architecture

Takei

Zheng

Yun

et al. 2021

Preprint

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Nuclear architecture in tissues can arise from cell-type specific organization of nuclear bodies, chromatin states and chromosome structures. However, the lack of genome-wide measurements to interrelate such modalities within single cells limits our overall understanding of nuclear architecture. Here, we demonstrate integrated spatial genomics in the mouse brain cortex, imaging thousands of genomic loci along with RNAs and subnuclear markers simultaneously in individual cells. We revealed chromatin fixed points, combined with cell-type specific organization of nuclear bodies, arrange the interchromosomal organization and radial positioning of chromosomes in diverse cell types. At the sub-megabase level, we uncovered a collection of single-cell chromosome domain structures, including those for the active and inactive X chromosomes. These results advance our understanding of single-cell nuclear architecture in complex tissues.

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“…In imaging, a cell’s position within a microscopic field of view is the unique piece of identifying information used to map phenotype to perturbation. Some platforms use in situ sequencing 27,28 to generate sequencing results that contain positional data to a specific cell 24,29 . However, a platform that can be integrated into existing next-generation sequencing (NGS) pipelines would be more accessible and robust.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Yenkin

Kremitzki

et al. 2021

Preprint

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Most human genetic variation is classified as VUS - variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a new method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a new way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen.

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

Cited by 154 publications

References 99 publications

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations

Integrated spatial genomics in tissues reveals invariant and cell type dependent nuclear architecture

Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

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