A Label-Free Multi-Metric Pipeline for Benchmarking Single-Cell RNA-Sequencing Clustering and Testing the Reproducibility of Cell-Type Heterogeneity

A discovered sub-population from single-cell transcriptomic data is only meaningful if it is reproducible, yet clustering is usually done with one method on one embedding and rarely tested. We present a label- free, multi-metric pipeline that reframes clustering as an auditable, methods-blind decision and separates two notions of stability that are commonly conflated: reproducibility under cell resampling (bootstrap) and reproducibility under re-embedding (retraining the representation). The pipeline evaluates seven clustering configurations across cluster counts using five non-redundant quality metrics. As a whole- dataset control on a mouse retinal atlas, it recovers an eight-cell-type annotation at 96.3% accuracy (adjusted Rand index, ARI = 0.91) without labels. We then validate the discovery mode on two cell types with opposite ground truth. On bipolar cells, which have well-established subtypes, the pipeline accepts the sub-structure: across-embedding reproducibility rises with cluster number to a high plateau (mean pairwise ARI ∼0.93 near the ∼15 known bipolar subtypes), with quality metrics improving in parallel. On rod photoreceptors, treated as homogeneous, it rejects over-clustering: the metric-selected partition passes a bootstrap-stability check but is not reproducible when the embedding is retrained (mean pairwise ARI = 0.69), and the metrics do not improve with cluster number. On synthetic data, the test recovers real structure down to a 5% subpopulation while rejecting null data (high sensitivity and specificity).

Bootstrap stability alone is therefore insufficient evidence for sub-population; the across-embedding test discriminates real sub-structure from over-clustering and applies to any cell type as a reproducible alternative to single-method, single-embedding clustering.