Does spatial barcode diffusion affect localization accuracy?

Answer:
Limited local diffusion of spatial barcodes may occur within tissue, but overall diffusion is controlled by the experimental system.

SeekSpace spatial reconstruction does not rely on a single barcode. Instead, localization is determined through integrated analysis of:

• Multi-barcode enrichment patterns
• Neighborhood spatial relationships
• Single-nucleus transcriptomic clustering
• Spatial consistency constraints

As a result, limited local diffusion typically does not disrupt overall spatial architecture.

Additional analytical approaches further reduce diffusion-related effects, including:

• Dominant barcode enrichment analysis
• Neighborhood consistency analysis
• Spatial clustering constraints

Cell center determination

Cell center assignment is based on the distribution of barcode-associated UMIs within each cell barcode.

During analysis:

• Bin100 is used as the basic spatial analysis unit.
• The total UMI count from the top 25 bins with the highest UMI abundance is compared with the total UMI count from the next highest 25 bins.
• If: ＞2, then the cell is considered to have a unique spatial center, indicating confident spatial localization.

In practical terms, the location with the strongest spatial barcode signal is considered the most likely position of that cell.