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Abstract Background Spatial transcriptomics (ST) is advancing our understanding of complex tissues and organisms. However, building a robust clustering algorithm to define spatially coherent regions in a single tissue slice and aligning or integrating multiple tissue slices originating from diverse sources for essential downstream analyses remains challenging. Numerous clustering, alignment, and integration methods have been specifically designed for ST data by leveraging its spatial information. The absence of comprehensive benchmark studies complicates the selection of methods and future method development. Results In this study, we systematically benchmark a variety of state-of-the-art algorithms with a wide range of real and simulated datasets of varying sizes, technologies, species, and complexity. We analyze the strengths and weaknesses of each method using diverse quantitative and qualitative metrics and analyses, including eight metrics for spatial clustering accuracy and contiguity, uniform manifold approximation and projection visualization, layer-wise and spot-to-spot alignment accuracy, and 3D reconstruction, which are designed to assess method performance as well as data quality. The code used for evaluation is available on our GitHub. Additionally, we provide online notebook tutorials and documentation to facilitate the reproduction of all benchmarking results and to support the study of new methods and new datasets. Conclusions Our analyses lead to comprehensive recommendations that cover multiple aspects, helping users to select optimal tools for their specific needs and guide future method development.

Abstract Background Large-scale single-cell transcriptomic datasets generated using different technologies contain batch-specific systematic variations that present a challenge to batch-effect removal and data integration. With continued growth expected in scRNA-seq data, achieving effective batch integration with available computational resources is crucial. Here, we perform an in-depth benchmark study on available batch correction methods to determine the most suitable method for batch-effect removal. Results We compare 14 methods in terms of computational runtime, the ability to handle large datasets, and batch-effect correction efficacy while preserving cell type purity. Five scenarios are designed for the study: identical cell types with different technologies, non-identical cell types, multiple batches, big data, and simulated data. Performance is evaluated using four benchmarking metrics including kBET, LISI, ASW, and ARI. We also investigate the use of batch-corrected data to study differential gene expression. Conclusion Based on our results, Harmony, LIGER, and Seurat 3 are the recommended methods for batch integration. Due to its significantly shorter runtime, Harmony is recommended as the first method to try, with the other methods as viable alternatives.