Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.

空间多组学技术有助于发现细胞功能和疾病机制的新见解。在这里，我们报告了多组学原位成对测序 (MiP-seq) 的发展和应用，这是一种以亚细胞分辨率同时检测 DNA、RNA、蛋白质和生物分子的方法。与其他原位测序方法相比，MiP-seq增强了解码能力，降低了测序和成像成本，同时保持了基因突变、等位基因特异性表达和RNA修饰检测的功效。 MiP-seq 可以与体内钙成像和拉曼成像集成，这使我们能够生成小鼠脑组织的空间多组学图谱，并将基因表达与神经元活动和细胞生化指纹相关联。我们还报告了一种连续稀释策略，用于在原位测序过程中解决光学拥挤信号。高通量原位成对测序可以促进组织分子和功能图谱的多维分析。