Abstract
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.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. Raw and processed RNA-seq data are available from GEO (GSE190219). The source image data for this study are available via Zenodo at https://zenodo.org/records/10837475 (ref. 56). Publicly available datasets used in the study include GSE113576 and GSE74672. The data of conventional in situ hybridization used in this study were obtained from the Allen Institute of Brain Science (https://mouse.brain-map.org/search/index). Other cited data are all indicated with corresponding references in the paper.
Code availability
The custom written package and scripts used in this study are available in GitHub at https://github.com/GangCaoLab/InnerEye.
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Acknowledgements
We thank F. Yang (Huazhong Agricultural University) for helping with tissue processing; Z. Hu, Y. Li and C. Shi in the Core Facility at HZAU for technical support in imaging; Z. Zhang (Spatial FISH Co., Ltd) for technical support in FISH; M. Liang for helping with early experimental design; X. Li and J. Li for helping with RCA; Xiaojian Cao for helping with statistical analysis on differential gene expression in pathogen infection assay; R Tyagi for proofreading; J. He, H. Zhang and M. Jin (State Key Laboratory of Neuroscience, Institute of Neuroscience, Centre for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences) for providing zebrafish brain and related help; X. Li (Hubei Cancer Hospital) for providing human diffuse large B cell lymphoma samples; Z. Huang (Huazhong University of Science and Technology) for helping with SRS imaging; Wuhan Keqian Biology Company for providing PCV2 and CSFV virus. This work was supported by the National Natural Science Foundation of China (Grant No. 32221005,32171022, U21A20259, 31872470 and 81827901).
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Authors and Affiliations
Contributions
G.C., J.D. and X.W. conceived and designed the project. X.W. performed probe-library design and preparation, cell and tissue experiment processing and sample preparation, in situ sequencing experiments, imaging, and data analysis. W.X. performed image-processing-pipeline development, in situ image processing, and analysis of the spatial patterns of cells and genes. W.X., C.W. and Y.Z. performed probe-design-software development, spatial-gene-decoding-pipeline design, and analysis. L.D. performed the spatial trajectory and spatial Delaunay network analysis. Y.L. performed C6 glioma-model establishment, Raman imaging analysis and the vascular-imaging experiment. Z.W. performed gene in situ verification in brain tissue, the parental orthogonal experiment, methylation in situ detection and in situ hybridization of the Raman tissue slice experiment. L.S. and G.D. performed conjugation of nucleic acid to antibody and in situ detection of proteins. A.G. performed MiP-seq and HCR3.0 efficiency comparison experiments. H.W. performed MiP-seq in situ DNA detection. X.Y. performed in vivo calcium imaging and analysis. K.Y. performed RNA-seq library sequencing for C6 cells and offspring brain tissue for the parental orthogonal experiment. L.Z. and L.Y. performed neuron culture and efficiency verification. Z.L. and D.L. performed bioinformatic analysis. J.Y. performed pathological diagnosis of DLBCL samples. G.C., J.D., Y.H., Z.F., P.W., X.W., W.X. and L.D. wrote the paper. All authors read and approved the final paper.
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Extended data
Extended Data Fig. 1 In situ detection of gene expression in brain slices via MiP-seq.
a, b, The expression patterns of Cck, Penk, Crh, Oxt, Avp, Nov, Gad1, Npy, Cux2, Rorb, and Sst genes detected by MiP-seq were comparable to that by conventional in situ hybridization (from Allen Institute of Brain Science). c, In situ co-detection of ten genes (Kif5a, Snhg11, Tau, Gja1, Nr4a1, Rbfox3, Dpf1, Map2, Thy1, and Eef2) in mice brain slices based on dual barcode sequencing. Different pseudo-colours indicate the corresponding gene transcripts of ten genes, which display distinct subcellular localization (Kif5a in cytoplasm; Snhg11 in nucleus). The subcellular localization of Kif5a, Snhg11, Tau, and Eef2 genes transcripts are presented in the lower panels respectively. The detection of these genes’ mRNA in a-c was replicated at least three times. Scale bars, 50 µm in left two panels of a, 1000 µm in right panel of a and middle panel of b, 200 µm in left two panels and right two panels of b, 10 µm in c.
Extended Data Fig. 2 Parallel comparison of in situ detection for 77 genes expression in preoptic area of mice between MiP-seq and MERFISH.
a, Detection of 77 genes in preoptic area (POA) by MiP-seq, which is for parallel comparison to their expression in coronal section of POA via MERFISH (data from the work of Moffitt et al., 2018, Science). The grey signal for visualizing the entire cell morphology were the whole cell mRNA signal detected by oligo-dT-Cy3. The DAPI image for the coronal section of POA were illustrated in the right-lower panel, and the white boxed area indicated the area for MiP-seq image. b, Comparison of 77 genes expression in POA detected by MiP-seq (n= 913 cells) with MERFISH (n= 841 cells). The Y-axis represents the values of the expression normalized by respective standards for each of the two methods. The detection of 77 genes expression in preoptic area of mice was replicated at least three times. Scale bars, 50 µm in upper image and 100 µm in lower image of a.
Extended Data Fig. 3 In situ detection of gene transcripts with single-base variance via MiP-seq.
a, Detection of single-base variance in human and mouse β-actin gene transcripts in cocultured HBMEC (human origin) cells and Bend3 (mouse origin) cells. Red and green dot signals indicate human β-actin gene (ACTB) and mouse β-actin gene (Actb) mRNA sequence, respectively. The detection of ACTB and Actb mRNA was replicated at least three times. b, In situ sequencing for allele-specific gene expression of Fam171b, Rpl15, Hba-a2, and Rps11 in hippocampus of the offspring mice from C57BL/6 female mice crossed with BALB/c male mice. Cell morphology stained with Oligo-dT(grey) were overlaid with corresponding gene expression profiles. The expression level of Fam171b, Rps11, Hba-a2, and Rpl15 alleles for each cell (RCPs per cell) in hippocampus were presented in the upper panels. RH, right hippocampus; LH, left hippocampus. Numbers indicate different mice, and the presented data were from three different mice. n=5412 cells in 1-RH detection, n=5979 cells in 2-LH detection, n=4538 cells in 3-LH detection. c, In situ sequencing for allele-specific gene expression of Fam171b, Rpl15, Hba-a2, and Rps11 in PVN of the offspring mice from C57BL/6 female mice crossed with BALB/c male mice. Cell morphology stained with Oligo-dT(grey) were overlaid with corresponding gene expression profiles. The quantification of Fam171b, Rps11, Hba-a2, and Rpl115 alleles expression for each cell (RCPs per cell) in PVN were presented in the upper panels. Numbers indicate different mice, and the data from three different mice were presented here. n=2471 cells in 1-PVN detection, n=2510 cells in 3-PVN detection, n=2418 cells in 4-PVN detection. d, The correlation of alleles expression in hippocampus and PVN from different mice. Numbers indicate different mice. RH, right hippocampus; LH, left hippocampus. e, In situ co-detection of mutations for multiple genes (Eno1, Nptxr, Spp1, Tnc, and Tuba1a) in glioma area of rat brain from C6 glioma model, and the box plot for the expression levels of wild-type and mutant variances was presented. The detection of allele-specific gene expression and the detection of Eno1, Nptxr, Spp1, Tnc, and Tuba1a mRNA was replicated at least three times. Scale bars, 10 µm in a, 50 µm in b and c, 30 µm in e.
Extended Data Fig. 4 Spatial profiling of multiple genes in mice brain at a large-field of view and 3D reconstruction of the spatial profiling of specific marker genes in the whole cerebellum.
a, Expression of Plp1, Neurod1, Gad1, and Calb1 in mice brain detected by MiP-seq is consistent with conventional in situ hybridization results from Allen Institute of Brain Science. b, The Left panel is the enlarged view of rectangle region in Fig. 2a for ten genes codetection in hippocampus. The gene expression patterns in hippocampus detected by MiP-seq are consistent with the results from Allen Institute of Brain Science (middle and right panels). c, 3D view of Calb1, Gad1, Neurod1, and Plp1 gene expression in the whole cerebellum are illustrated, respectively. The detection of Calb1, Gad1, Neurod1, and Plp1 gene expression in cerebellum was replicated at least three times. Scale bars, 1000 µm in a, 300 µm in b, 500 µm in c.
Extended Data Fig. 5 Image processing pipeline for spatial transcriptome analysis by MiP-seq.
a, Diagram of image processing pipeline for spatial transcriptome analysis by MiP-seq. The raw images from multiple rounds sequencing are first matched to the first-round image by affine transformations that allow rotation, translation, scaling, and shear transformations. The candidate points are selected according to the aligned images, and are decoded according to the intensity of each channel in each round at its location. The cell contour is calculated by cellpose based on the merged images from three sequencing rounds after alignment. Finally, the decoded gene points are allocated to neighbouring cells according to the principle of proximity, and the expression profile corresponding to each cell is obtained. b, The image (i) before registration. Three rounds of the channel merged images are represented in cyan, yellow, and magenta, respectively. c, The image (ii) after registration. d, Image and candidate spots in a local area. (i) Gray scale image of the combined channel images after alignment. (ii) Candidate spots are marked as white points on the image. (iii) Decoded spots are marked as points with different colours. The 'general channel' of each round is calculated by averaging the images from different signal channels. e, The cell mask is calculated from the averaged general channel image from three sequencing rounds by cellpose. Different cells are indicated by different colours. f, The results of gene assignment in this field of view. Different coloured dots on the graph represent different genes. The red connecting line represents the gene to the corresponding cell. Scale bars, 30 µm in left image of b and c, 10 µm in right image of b and c, 10 µm in d, 30 µm in e and f.
Extended Data Fig. 6 Spatial transcriptomic analysis of 100 genes in HDB by MiP-seq and gene co-expression between cell clusters.
a, 3D projection image for in situ detection of 100 genes mRNAs in the first sequencing round by MiP-seq in HDB. b, The images of three round sequencing by MiP-seq. The signals lined by the rectangle box and circle are further illustrated in the middle and lower panel respectively. c, The spatial distribution of neurotransmitter-related and peptide hormone genes (blue) and their receptor genes (yellow). When the counts of the corresponding gene in each cell is greater than 10, the whole cell is marked by the colour of the corresponding gene. d, Spatial expression analysis of Hap1 and Pdgfra genes in HDB. The unit for the X and Y axes is pixel. e, The spatial distance of Hap1 (green curve) and Pdgfra (red curve) gene to the top 50 expression genes. The 'mean min distance' refers to the average minimal distance (measured by mean pixels) from the signal points of Hap1 or Pdgfra gene transcripts to the nearest signal points for each of top 50 expressed genes. The unit for the Y axis is pixel. f, Cells of HDB are clustered into 6 groups (0–5) based on the expression profile of single cells detected by MiP-seq. g, UMAP of featured genes expression in certain cell clusters and the corresponding spatial expression pattern in HDB. n=521 cells for data analysis of d-g. Scale bars, 50 µm in a, 10 µm in b.
Extended Data Fig. 7 Spatial transcriptomic alteration of PVN upon Mycobacterium tuberculosis (M.tb) infection.
a, Detection of 217 genes including cell markers, hormones, receptors, as well as immune related genes in mice PVN upon Mycobacterium tuberculosis (M.tb) infection and saline control treatment by MiP-seq. b, Expression of Ly6h and Gpr165 were up-regulated while C1qb and Pgm2l1 were down-regulated in response to M.tb infection. c, Heatmap of the expression of cell marker genes. d, Differentially expressed genes in endothelial cells, astrocytes, microglia, and neurons. Genes with significantly changes (P value ≤0.05 and log(fold change) ≥0.3) were labelled in green. n= 491 cells in saline control, n= 533 cells in M.tb infection group. The spatial transcriptomic detection in PVN upon M.tb infection via MiP-seq was replicated at least three times. Statistical significance was measured by Student’s t-test (Two-tailed). The raw data of d can be found in Supplementary Table 3. Scale bars, 100 µm in DAPI images of a, 10 µm in multiplexed genes detection image of a and in b.
Extended Data Fig. 8 In situ detection of multiple genomic loci via MiP-seq.
a, The remaining data for co-detection of Rbfox3 and Nr4a1 genes in mice tissue by MiP-seq. Different cells are numbered. The data for #1 cell are shown in Fig. 4b. b, The intranuclear location of Rbfox3 and Nr4a1 loci for 20 cells in mice brain. c, The histogram of Euclidean distance between two Rbfox3 loci, two Nr4a1 loci, and the two loci to nuclear edge, respectively. n= 20 cells in each group. d, More images for in situ sequencing of Nptxr point mutation located in different chromosomes in C6 cell. Different cells are numbered. The data for #1 cell are shown in Fig. 4c. e, The intranuclear location of wildtype and mutant Nptxr loci for 20 C6 cells. f, The histogram of Euclidean distance between wildtype and mutant Nptxr loci, and between their locus to nuclear edge. n= 20 cells in each group. Data was presented as mean ± SD in c and f. The raw data can be found in Supplementary Table 3. The detection of these genomic loci was replicated at least three times. Scale bars, 2 µm in a, b and d, e.
Extended Data Fig. 9 In situ detection of neuropeptide and multiple proteins via MiP-seq.
a, Nearly identical signals for neuropeptide orexin detected by MiP-seq (Cy3 red fluorescence) and classic immunostaining (Alexa fluor 488 green fluorescence) were observed in lateral hypothalamus (LH) region of mice brain. For orexin detection by MiP-seq, the targeting oligonucleotides was conjugated with secondary antibody. b, In situ co-detection of four proteins (E2 of CSFV, Cap of PCV2, flag for IFNG, and Pol II) conjugated with distinct DNA oligonucleotides via MiP-seq. The comparison experiment between MiP-seq and classic immunostaining in Orexin detection and the codetection of four proteins (E2 of CSFV, Cap of PCV2, flag for IFNG, and Pol II) by MiP-seq was replicated at least three times. Scale bar, 50 µm in a, 20 µm in b.
Extended Data Fig. 10 Co-detection of proteins and mRNAs in tumour samples via MiP-seq.
a, Co-detection of 30 immune related proteins with 6 diagnostic marker genes mRNAs in human diffuse large B cell lymphoma (DLBCL) sections. The images for DAPI staining and oligo-dT-CY3 hybridization were illustrated in the right lower panel which are used for cell segmentation. b, UMAP for 16 cell clusters in DLBCL based on the analysis of expression profile of single cells (n=2597 cells) in a. c, The spatial map of 16 cell clusters. d, The heatmap of spatial interaction frequency among 16 cell clusters. e, The spatial map of three cell clusters showing high interaction frequency. f, The normalized spatial expression heatmap of detected proteins and mRNAs in DLBCL. g, The spatial expression of CD27, CD45RA, CCR7 and CXCR6 in DLBCL sections. The codetection of 30 proteins and 6 mRNAs in human DLBCL was replicated at least three times. Scale bar, 50 μm in a.
Supplementary information
Main Supplementary Information
Supplementary figures.
Supplementary Video
3D gene-expression architecture for the whole cerebellum.
Supplemantary Data
Probe sequences.
Supplemantary Data 2
Raw data for efficiency comparison between different methods.
Supplemantary Data 3
Raw data for statistical analysis, and cell cluster genes list.
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Wu, X., Xu, W., Deng, L. et al. Spatial multi-omics at subcellular resolution via high-throughput in situ pairwise sequencing. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01205-7
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DOI: https://doi.org/10.1038/s41551-024-01205-7
