UCLA researchers in the Department of Human Genetics have developed a scalable, high-precision in situ spatial barcoding method, enabling spatially-resolved single-cell and subcellular genomic profiling, including transcriptomics and epigenomics, with targeted cell selection guided by immunofluorescence.
BACKGROUND: Recent advancements in spatial transcriptomics have enabled the study of gene expression patterns in tissues with varying spatial resolutions and throughputs. These approaches include in situ RNA hybridization (ISH), highly multiplexed single-molecule RNA in situ methods such as MERFISH and seqFISH(+), and sequencing-based in situ transcriptomic methods. Despite these advancements, challenges persist in achieving single-cell resolution. Spatial transcriptomics methods based on microarray or microbead platforms have limited resolution and often fail to distinguish adjacent cells. While ISH methods achieve subcellular and single-molecule resolution, their low imaging throughput limits their scalability to whole-transcriptome and large-tissue studies. Additionally, the extension of spatial genomics to epigenomics, such as DNA methylation, chromatin accessibility, and histone modifications, is underdeveloped with no existing method capable of generating spatially resolved, single-cell epigenomic data.
INNOVATION: UCLA researchers have developed photonic-indexing sequencing (pi-seq), a novel approach for in situ spatial barcoding with single-cell and subcellular resolution. Pi-seq achieves spatial barcoding by writing DNA barcodes directly into tissue slices using highly parallel ligation reactions controlled by high-resolution patterned UV light. The process involves iterative cycles of photonic cleavage and subsequent ligation, with distinct illumination patterns used in each cycle to effectively generate millions of unique barcodes within the tissue, thus providing exceptional spatial specificity and high barcoding accuracy. In addition, the pi-seq strategy is versatile and compatible with various molecular assays. Researchers have combined pi-seq with established single-cell techniques, including the chromatin accessibility profiling method ATAC-seq and the methylome profiling method snmC-seq2, to develop spatially resolved approaches for profiling of chromatin accessibility (pi-ATAC-seq) and methylcytosine (pi-mC-seq).
POTENTIAL APPLICATIONS:
- Spatially resolved genomic profiles for studying single-cell transcriptomics and epigenomics within three-dimensional tissue structures. Spatial epigenomic profiling includes chromatin accessibility and methylcytosine analysis.
ADVANTAGES:
- Barcoding single cells for genomic profiling with exceptional spatial specificity and high accuracy
- Can be scaled to large tissue areas and allows the targeted selection of specific cells of interest using immunofluorescent labeling as a guide
- The sequencing library generated by pi-seq is fully compatible with commercial short-read sequencing platforms
- The pi-seq spatial barcoding strategy can be applied to multiple molecular assays (e.g. RNA-seq, ATAC-seq or snmC-seq2)
DEVELOPMENT-TO-DATE: Researchers have successfully conducted in situ regional and single-cell pi-ATAC-seq and pi-mC-seq indexing experiments in mouse brain slices to explore the spatial diversity and organization of brain cell types.
KEYWORDS: Spatially resolved genomics, in situ sequencing, photonic-indexing sequencing, single-cell resolution, single-molecule resolution, spatial barcoding, DNA barcoding, immunofluorescent labeling, high-throughput, spatial transcriptomics, spatial epigenomics, chromatin accessibility, methylcytosine, pi-seq, RNA-seq, ATAC-seq, snmC-seq2