Question: What is the maximum supported sequencing depth for Spatial Targeted Gene Expression libraries?
Answer: We recommend sequencing Spatial Targeted Gene Expression libraries to a depth of 5,000 to 10,000 read pairs per tissue-covered spot.
If Spatial Targeted Gene Expression libraries are sequenced beyond a depth of 15,000 “tissue-associated read pairs per tissue-covered spot”, the sequence data will automatically be downsampled in Space Ranger.
The maximum supported read depth occurs when the average number of reads assigned to barcodes that map to tissue is 15,000. This depends on the “fraction of reads in spots under tissue”, which varies based on sample type and sample quality. For example, if a sample has 95% “fraction reads in spots under tissue”, the data would be downsampled in Space Ranger if sequenced beyond 15,789 read pairs per tissue-covered spot.
This read depth upper limit was implemented to ensure optimal signal-to-noise ratios. When Spatial Targeted Gene Expression libraries are analyzed beyond this depth, increased background noise may be observed.
While changing this limit is not recommended, it may be appropriate to increase the limit in order to meet recommendations provided by the targeted-depth tool. In such cases, the maximum depth may be changed using the --rps-limit argument to spaceranger count.
See also: Sequencing Requirements for Spatial Targeted Gene Expression libraries
Product: Targeted Gene Expression, Visium for fresh-frozen