Despite significant advances in parallel single-cell RNA sequencing revealing astonishing cellular heterogeneity in many tissue types, the spatial information in the tissue context remains missing. Spatial transcriptome sequencing technology is designed to distinguish the gene expression of individual cells in their original location. The technology is important for the identification of tissue function, tracking developmental processes, and pathological and molecular detection. Encoding the position information is the key to spatial transcriptomics because different methods have different encoding efficiencies and application scenarios. In this review, we focus on the latest technologies of single-cell spatial transcriptomics, including technologies based on microwell plates, barcoded bead arrays, microdissection, in situ hybridization, and barcode in situ targeting, as well as mixed separation-based technologies. Moreover, we compare these encoding methods for use as a reference when choosing the appropriate technology.

Gene set analysis using signaling pathway has become a popular downstream analysis following differential expression analysis. From a biological point of view, only some portions of a pathway are expected to be altered; however, a few approaches using the different importance of genes in signaling pathways, which encompass the constitutive functional nonequivalent roles of genes in real pathways, have been proposed and none of them tries to associate the importance of genes with the related disease. In this paper, we developed an extended method of signaling pathway impact analysis (SPIA), called gwSPIA, by incorporating three signaling pathway-based gene weight merits that reflect the importance of genes from different aspects and attempt to associate the importance of genes with the related diseases. By applying the gwSPIA to the gene expression data sets in comparison with other seven methods in three measures, sensitivity, prioritization, and specificity, we show that the gwSPIA ranks in the second place in both sensitivity and prioritization. Furthermore, the specificity of the gwSPIA is better than SPIA, which is lower than 25%. The results also suggest that the gene weight used in the gwSPIA can reflect the association between the genes and the related diseases. The R package of the gwSPIA can be accessed from https://github.com/sterding/gwSPIA. INDEX TERMS Differentially expressed genes, gene weights, gwSPIA, signaling pathways analysis.

Signaling pathway analysis has become a routine task after differentially expressed gene (DEG) studies across disease conditions, drug treatments, or developmental stages. A signaling pathway can be represented by a graph that consists of Genes and interactions (the genetic regulation) between them. However, existing signaling pathway analysis methods ignore the strength variations of interactions in signaling pathways under different conditions. Here, we developed a novel method named SPACI (Signaling Pathway Analysis Combined with the strength variations of Interactions between genes in signaling pathways under different conditions) to improve signaling pathway analysis after DEG studies. To further evaluate the performance of SPACI, we compared SPACI with nine other methods by using a benchmark of 28 gene expression datasets in two standard measures: sensitivity and prioritization. The False positive rate (FPR) of SPACI was also compared with five methods. The results show that SPACI is the second-ranked method in terms of prioritization and the third-ranked method in terms of sensitivity. SPACI is the top method when compared in terms of the sum value of the two ranks. Also, the FPR of SPACI is modest compared with the classic methods. Furthermore, the strength variation of the interaction is demonstrated as coherent with the biological problem. The interactions with high strength variations under different conditions can help improve the discovery of the underlying biological information. The R package of SPACI can be accessed at https://github.com/ZhenshenBao/SPACI.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

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