Robust integration of multiple single-cell RNA sequencing datasets using a single reference space

In many biological applications of single-cell RNA sequencing (scRNA-seq), an integrated analysis of data from multiple batches or studies is necessary. Current methods typically achieve integration using shared cell types or covariance correlation between datasets, which can distort biological signals. Here we introduce an algorithm that uses the gene eigenvectors from a reference dataset to establish a global frame for integration. Using simulated and real datasets, we demonstrate that this approach, called Reference Principal Component Integration (RPCI), consistently outperforms other methods by multiple metrics, with clear advantages in preserving genuine cross-sample gene expression differences in matching cell types, such as those present in cells at distinct developmental stages or in perturbated versus control studies. Moreover, RPCI maintains this robust performance when multiple datasets are integrated. Finally, we applied RPCI to scRNA-seq data for mouse gut endoderm development and revealed temporal emergence of genetic programs helping establish the anterior–posterior axis in visceral endoderm. Integration of multiple single-cell RNA sequencing datasets is improved by creating a common reference space using a new algorithm.