Abstract
Single-cell RNA sequencing (scRNA-seq) has emerged as a transformative technology for elucidating cellular heterogeneity at unprecedented resolution. However, technical limitations such as limited sequencing depth and mRNA capture efficiency often result in zero counts, commonly referred to as "dropout zeros" in scRNA-seq data. These zeros pose significant challenges to downstream analysis, as they can distort the interpretation of cellular transcriptomes. While numerous computational methods have been developed to address this challenge, existing approaches frequently suffer from either insufficient imputation of zeros (under-imputation) or excessive modification of zeros (over-imputation). Here, we propose a precisely balanced imputation (PbImpute) method designed to achieve optimal equilibrium between dropout recovery and biological zero preservation in scRNA-seq data. PbImpute employs a multistage approach: (1) Initial discrimination between technical dropouts and biological zeros through parameter optimization of a new zero-inflated negative binomial (ZINB) distribution model, followed by initial imputation; (2) Application of a uniquely designed static repair algorithm to enhance data fidelity; (3) Secondary dropout identification based on gene expression frequency and partition-specific coefficient of variation; (4) Graph-embedding neural network-based imputation; and (5) Implementation of a uniquely designed dynamic repair mechanism to mitigate over-imputation effects. PbImpute distinguishes itself by uniquely integrating ZINB modeling with static and dynamic repair. This advantageous combined approach achieves a balance between over- and under-imputation, while simultaneously preserving true biological zeros and reducing signal distortion. Comprehensive evaluation using both simulated and real scRNA-seq data sets demonstrated that PbImpute achieves superior performance (F1 Score = 0.88 at 83% dropout rate, ARI = 0.78 on PBMC) in discriminating between technical dropouts and biological zeros compared to state-of-the-art methods. The method significantly improves gene-gene and cell-cell correlation structures, enhances differential expression analysis sensitivity, optimizes clustering resolution and dimensional reduction visualization, and facilitates more accurate trajectory inference. Ablation studies confirmed the essential contribution of both the imputation and repair modules to the method's performance. The code is available at https://github.com/WyBioTeam/PbImpute. By enhancing the accuracy of scRNA-seq data imputation, PbImpute can improve the identification of cell subpopulations and the detection of differentially expressed genes, thereby facilitating more precise analyses of cellular heterogeneity and advancing disease research.