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

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.

CENPL, ISG20L2, LSM4, MRPL3 are four novel hub genes and may serve as diagnostic and prognostic markers in breast cancer.

As a highly prevalent disease among women worldwide, breast cancer remains in urgent need of further elucidation its molecular mechanisms to improve the patient outcomes. Identifying hub genes involved in the pathogenesis and progression of breast cancer can potentially help to unveil mechanism and also provide novel diagnostic and prognostic markers. In this study, we integrated multiple bioinformatic methods and RNA in situ detection technology to identify and validate hub genes. EZH2 was recognized as a key gene by PPI network analysis. CENPL, ISG20L2, LSM4, MRPL3 were identified as four novel hub genes through the WGCNA analysis and literate search. Among these, many studies on EZH2 in breast cancer have been reported, but no studies are related to the roles of CENPL, ISG20L2, MRPL3 and LSM4 in breast cancer. These four novel hub genes were up-regulated in tumor tissues and associated with cancer progression. The receiver operating characteristic analysis and Kaplan-Meier survival analysis indicated that these four hub genes are promising candidate genes that can serve as diagnostic and prognostic biomarkers for breast cancer. Moreover, these four newly identified hub genes as aberrant molecules in the maintenance of breast cancer development, their exact functional mechanisms deserve further in-depth study.