I have recently followed adopted the Harvard Chan Bioinformatics Core guidelines for SC QC/Normalization/Clustering (https://hbctraining.github.io/scRNA-seq_online/schedule/links-to-lessons.html). I have integrated CD4+/CD8+ T cells from two time points.
I recently received feedback that my integrated dimension reduction plot clustering looked problematic. Specifically, the small clusters peripheral (splash/star?) and the number of distinct clusters.
Data was normalized using SCTransform, variables regressed were mitochondrial ratio and G2M-S phase score difference, as suggested for differentiating cell types. Alternative Workflow: https://satijalab.org/seurat/articles/cell_cycle_vignette.html
My clusters were called at 40 PC's w/ 0.6 resolution.
As for the number of clusters, TCR B VDJ subgenes were identified as strong conserved markers in several clusters. I wonder if it is worth excluding VDJ markers from analysis?
Any comment on the appearance of the dim plot and implications would be appreciated. Thank you!