Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

Xinbin Tang; Jiayu Chen; Xinya Zhang; Xuzhu Liu; Zhaoxiang Xie; Kaipeng Wei; Jianlong Qiu; Weiyan Ma; Chen Lin; Rongqin Ke

doi:10.1016/j.jgg.2023.02.004

Volume 50 Issue 9

Sep. 2023

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Article Navigation > Journal of Genetics and Genomics > 2023 > 50(9): 652-660

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Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections

doi: 10.1016/j.jgg.2023.02.004 cstr: 32370.14.j.jgg.2023.02.004

a. School of Medicine and School of Biomedical Sciences, Huaqiao University, Quanzhou, Fujian 362021, China;
b. Department of Pathology, The 910 Hospital, Quanzhou, Fujian 362000, China

Funds:

This study was supported by the Natural Science Foundation of Fujian Province (2022J06022), the Quanzhou Science and Technology Plan Project (2021C040R), and the Scientific Research Funds of Huaqiao University.

Received Date: 2022-11-03
Accepted Date: 2023-02-02
Rev Recd Date: 2023-01-30
Publish Date: 2023-02-15

Abstract

Abstract

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling. We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed.
- Spatial transcriptomics,
- In situ sequencing,
- Single cell,
- Gene expression profiling,
- Imaging

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References(33)

References

[1]	Chen, K.H., Boettiger, A.N., Moffitt, J.R., Wang, S.,Zhuang, X., 2015. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science, 348, aaa6090.
[2]	Chen, Y., McAndrews, K.M.,Kalluri, R., 2021. Clinical and therapeutic relevance of cancer-associated fibroblasts. Nat. Rev. Clin. Oncol., 18, 792-804.
[3]	Codeluppi, S., Borm, L.E., Zeisel, A., La Manno, G., van Lunteren, J.A., Svensson, C.I.,Linnarsson, S., 2018. Spatial organization of the somatosensory cortex revealed by osmFISH. Nat. Methods, 15, 932-935.
[4]	Drmanac, R., Sparks, A.B., Callow, M.J., Halpern, A.L., Burns, N.L., Kermani, B.G., Carnevali, P., Nazarenko, I., Nilsen, G.B., Yeung, G., et al., 2010. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science, 327, 78-81.
[5]	Efremova, M., Vento-Tormo, M., Teichmann, S.A.,Vento-Tormo, R., 2020. CellPhoneDB: inferring cell-cell communication from combined expression of multi-subunit ligand-receptor complexes. Nat. Protoc., 15, 1484-1506.
[6]	Emmert-Buck, M.R., Bonner, R.F., Smith, P.D., Chuaqui, R.F., Zhuang, Z., Goldstein, S.R., Weiss, R.A.,Liotta, L.A., 1996. Laser capture microdissection. Science, 274, 998-1001.
[7]	Eng, C.L., Lawson, M., Zhu, Q., Dries, R., Koulena, N., Takei, Y., Yun, J., Cronin, C., Karp, C., Yuan, G.C., et al., 2019. Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH. Nature, 568, 235-239.
[8]	Jian, F., Yanhong, J., Limeng, W., Guoping, N., Yiqing, T., Hao, L.,Zhaoji, P., 2022. TIMP2 is associated with prognosis and immune infiltrates of gastric and colon cancer. Int. Immunopharm., 110, 109008.
[9]	Ghislat, G., Cheema, A.S., Baudoin, E., Verthuy, C., Ballester, P.J., Crozat, K., Attaf, N., Dong, C., Milpied, P., Malissen, B., et al., 2021. NF-κB-dependent IRF1 activation programs cDC1 dendritic cells to drive antitumor immunity. Sci. Immunol., 6, 3570.
[10]	Giorello, M.B., Borzone, F.R., Labovsky, V., Piccioni, F.V.,Chasseing, N.A., 2021. Cancer-associated fibroblasts in the breast tumor microenvironment. J. Mammary Gland Biol. Neoplasia, 26, 135-155.
[11]	Gisina, A., Novikova, S., Kim, Y., Sidorov, D., Bykasov, S., Volchenko, N., Kaprin, A., Zgoda, V., Yarygin, K.,Lupatov, A., 2021. CEACAM5 overexpression is a reliable characteristic of CD133-positive colorectal cancer stem cells. Cancer Biomarkers., 32, 85-98.
[12]	Gyllborg, D., Langseth, C.M., Qian, X., Choi, E., Salas, S.M., Hilscher, M.M., Lein, E.S.,Nilsson, M., 2020. Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue. Nucleic Acids Res., 48, e112.
[13]	Kanzaki, R.,Pietras, K., 2020. Heterogeneity of cancer-associated fibroblasts: opportunities for precision medicine. Cancer Sci., 111, 2708-2717.
[14]	Ke, R., Mignardi, M., Hauling, T.,Nilsson, M., 2016. Fourth generation of next-generation sequencing technologies: promise and consequences. Hum. Mutat., 37, 1363-1367.
[15]	Ke, R., Mignardi, M., Pacureanu, A., Svedlund, J., Botling, J., Wahlby, C.,Nilsson, M., 2013. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857-860.
[16]	Koboldt, D.C., Steinberg, K.M., Larson, D.E., Wilson, R.K.,Mardis, E.R., 2013. The next-generation sequencing revolution and its impact on genomics. Cell, 155, 27-38.
[17]	Le, P., Ahmed, N.,Yeo, G.W., 2022. Illuminating RNA biology through imaging. Nat. Cell Biol., 24, 815-824.
[18]	Lee, H., Marco Salas, S., Gyllborg, D.,Nilsson, M., 2022. Direct RNA targeted in situ sequencing for transcriptomic profiling in tissue. Sci. Rep., 12, 7976.
[19]	Lee, H.-O., Hong, Y., Etlioglu, H.E., Cho, Y.B., Pomella, V., Van den Bosch, B., Vanhecke, J., Verbandt, S., Hong, H., Min, J.-W., et al., 2020. Lineage-dependent gene expression programs influence the immune landscape of colorectal cancer. Nat. Genet., 52, 594-603.
[20]	Lee, J.H., Daugharthy, E.R., Scheiman, J., Kalhor, R., Yang, J.L., Ferrante, T.C., Terry, R., Jeanty, S.S., Li, C., Amamoto, R., et al., 2014a. Highly multiplexed subcellular RNA sequencing in situ. Science, 343, 1360-1363.
[21]	Lee, S.Y., Kim, J.M., Cho, S.Y., Kim, H.S., Shin, H.S., Jeon, J.Y., Kausar, R., Jeong, S.Y., Lee, Y.S.,Lee, M.A., 2014b. TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signalling. Biochem. J., 459, 565-576.
[22]	Lin, C., Jiang, M., Liu, L., Chen, X., Zhao, Y., Chen, L., Hong, Y., Wang, X., Hong, C., Yao, X., et al., 2021. Imaging of individual transcripts by amplification-based single-molecule fluorescence in situ hybridization. Nat. Biotechnol., 61, 116-123.
[23]	Liu, S., Punthambaker, S., Iyer, E.P.R., Ferrante, T., Goodwin, D., Furth, D., Pawlowski, A.C., Jindal, K., Tam, J.M., Mifflin, L., et al., 2021. Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses. Nucleic Acids Res., 49, e58.
[24]	Moses, L.,Pachter, L., 2022. Museum of spatial transcriptomics. Nat. Methods, 19, 534-546.
[25]	Pham, D., Tan, X., Xu, J., Grice, L.F., Lam, P.Y., Raghubar, A., Vukovic, J., Ruitenberg, M.J.,Nguyen, Q., 2020. stLearn: integrating spatial location, tissue morphology and gene expression to find cell types, cell-cell interactions and spatial trajectories within undissociated tissues. bioRxiv, 2020.2005.2031.125658.
[26]	Qian, X., Harris, K.D., Hauling, T., Nicoloutsopoulos, D., Munoz-Manchado, A.B., Skene, N., Hjerling-Leffler, J.,Nilsson, M., 2020. Probabilistic cell typing enables fine mapping of closely related cell types in situ. Nat. Methods, 17, 101-106.
[27]	Song, G., Xu, S., Zhang, H., Wang, Y., Xiao, C., Jiang, T., Wu, L., Zhang, T., Sun, X., Zhong, L., et al., 2016. TIMP1 is a prognostic marker for the progression and metastasis of colon cancer through FAK-PI3K/AKT and MAPK pathway. J. Exp. Clin. Cancer Res., 35, 148.
[28]	Stahl, P.L., Salmen, F., Vickovic, S., Lundmark, A., Navarro, J.F., Magnusson, J., Giacomello, S., Asp, M., Westholm, J.O., Huss, M., et al., 2016. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science, 353, 78-82.
[29]	Stringer, C., Wang, T., Michaelos, M.,Pachitariu, M., 2021. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods, 18, 100-106.
[30]	Svedlund, J., Strell, C., Qian, X., Zilkens, K.J.C., Tobin, N.P., Bergh, J., Sieuwerts, A.M.,Nilsson, M., 2019. Generation of in situ sequencing based OncoMaps to spatially resolve gene expression profiles of diagnostic and prognostic markers in breast cancer. EBioMedicine, 48, 212-223.
[31]	Wang, F., Flanagan, J., Su, N., Wang, L.C., Bui, S., Nielson, A., Wu, X., Vo, H.T., Ma, X.J.,Luo, Y., 2012. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn., 14, 22-29.
[32]	Wang, Y.S.,Guo, J., 2021. Multiplexed single-cell in situ RNA profiling. Front. Mol. Biosci., 8, 775410.
[33]	Yu, Y., Zeng, Z., Xie, D., Chen, R., Sha, Y., Huang, S., Cai, W., Chen, W., Li, W., Ke, R., et al., 2021. Interneuron origin and molecular diversity in the human fetal brain. Nat. Neurosci., 24, 1745-1756.