Question: Why were the Sample Index PCR conditions updated for Visium Spatial Gene Expression library preparation?
Answer: In Revision D of the Visium Spatial Gene Expression Reagent Kits User Guide (CG000239), the annealing temperature of the Sample Index PCR was increased from 54 °C to 67 °C and the number of PCR cycles was increased by 3.
This optimized Sample Index PCR protocol enhances the quality of Visium Spatial Gene Expression libraries by reducing background noise. This improves the signal-to-noise ratio for spatially-enriched genes.
As shown in the figure below, background UMI counts for three genes with spatially-enriched expression patterns in the mouse brain decrease in Visium Spatial Gene Expression libraries prepared using the optimized Sample Index PCR protocol (Rev D).
This optimized Sample Index PCR protocol was adopted to ensure compatibility of Visium Spatial Gene Expression libraries with the Targeted Gene Expression assay.
In the Targeted Gene Expression assay, if background fragments are present in the parent whole transcriptome library, they can become enriched during the hybridization and capture workflow. This can lead to increased noise in the Targeted Gene Expression library.
To maximize the signal-to-noise ratios in Targeted Gene Expression libraries, it is very important to minimize noise in the parent libraries. Therefore, it is essential to follow the optimized Sample Index PCR protocol when preparing Visium Spatial Gene Expression libraries that will be used as input into the Targeted Gene Expression assay.
Product: Visium for fresh-frozen, Targeted Gene Expression