Photoselective sequencing: microscopically-guided genomic measurements with subcellular resolution

Abstract

In biological systems, spatial organization is interconnected with genome function and regulation. However, methods that couple high-throughput genomic and epigenomic profiling with spatial information are lacking. Here, we developed Photoselective Sequencing, a spatially-informed DNA sequencing method to assay collections of cells or subcellular regions that share a unifying morphological trait. In Photoselective Sequencing, we prepare a blocked fragment library within a fixed biological specimen. Guided by fluorescence imaging, we remove the block in specific regions of interest using targeted illumination with near-UV light, ultimately allowing high-throughput sequencing of the selected fragments. To validate Photoselective Sequencing, we profile chromatin openness in fluorescently-labeled cell types within the mouse brain and demonstrate strong agreement with published single-cell ATAC-seq data. Using Photoselective Sequencing, we characterize the accessibility profiles of oligodendrocyte-lineage cells within the cortex and corpus-callosum regions of the brain. We develop a new computational strategy for decomposing bulk accessibility profiles by individual cell types, and report a relative enrichment of oligodendrocyte-progenitor-like cells in the cortex. Finally, we leverage Photoselective Sequencing for unbiased profiling of DNA at the nuclear periphery, a key chromatin organizing region. We compare and contrast the Photoselective Sequencing profile with lamin ChIP-seq data, and identify features beyond lamin interaction that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that Photoselective Sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.

OpenTn5: Open-Source Resource for Robust and Scalable Tn5 Transposase Purification and Characterization

“…Tagmentation has been leveraged for streamlined preparation of high-throughput DNA sequencing libraries from sample DNA using custom sequencing adapter sequences. The efficiency and versatility of Tn5 tagmentation has given rise to a variety of genomics technologies for investigating chromatin organization, transcription and cell identity, with innovations continuing to be made in novel Tn5 protein fusions as well as custom MEDS adapter designs [10][11][12][13][14][15][16][17][18][19]. Despite the accomplishments of tagmentation-based methods to date, in-house production of high-quality Tn5 enzyme remains a persistent challenge [10] which hinders both ongoing efforts in the development of novel tagmentation applications and the equitable, broad adoption of existing methods.…”

Enablers and challenges of spatial omics, a melting pot of technologies

“…Examples include the studies by Vickovic et al (2022), Liu et al (2023) and Ben‐Chetrit et al (2023). A subset of these ex situ strategies have been combined with in situ transposition to incorporate sequencing adapters into fixed genomic DNA, using Tn5 transposase similarly to ATAC‐Seq (Buenrostro et al , 2013) so that native spatial positions of accessible genomic DNA are preserved, enabling studies of spatial (epi)genomics (Chen et al , 2016b; Payne et al , 2020; Deng et al , 2022; Mangiameli et al , 2023).…”