How are spatial barcodes integrated with single-cell RNA data for reconstruction, and how is reconstruction accuracy maintained?

Answer:
Spatial barcodes and single-nucleus RNA data are jointly analyzed in SeekSpace Tools pipeline. The analysis workflow integrates:

• Spatial barcode enrichment patterns
• Single-nucleus RNA clustering results
• Histological image information

This enables probabilistic assignment of each nucleus to a reconstructed spatial location, resulting in cell-centered spatial reconstruction.

Cell center determination

Cell center assignment is based on barcode-associated UMI distribution within the same cell barcode.

During analysis:

• Bin100 is used as the basic analysis unit.
• The total UMI count from the top 25 bins is compared with the total UMI count from the next highest 25 bins.
• If the ratio exceeds 2, the cell is considered to possess a unique spatial center and is regarded as confidently localized.

In simple terms, the location containing the strongest barcode enrichment is interpreted as the cell’s spatial position.

Mechanisms supporting reconstruction accuracy

SeekSpace improves reconstruction robustness through:

• Multi-barcode integration rather than reliance on single barcodes
• Spatial clustering constraints that reduce the influence of local barcode diffusion
• Histology-guided removal of abnormal barcode distributions
• High RNA quality and sufficient nuclei counts, both of which substantially improve spatial localization stability