What is the recommended number reads when your sample tissue does not cover the entire Tile area?

Recommended Reads and Saturation Metrics for Partial Tile Coverage

For a 3x3 tile that is fully covered by the tissue section, we suggest 30 million reads for shallow sequencing or 200 million reads for deep sequencing based on data generated with mouse hippocampus. If your tissue section does not cover the entire tile area, you can lower the number of reads based on estimated % of coverage. For example, if your tissue only covers 50% of the tile you can start with 15 million reads for shallow sequencing. 

However, the optimal number of reads is also dependent on the transcriptionally activity of your tissue. For example, if your tissue is tumorigenic, it’s highly transcriptionally active and may require 2-3x more read-depth to reach saturation if the section is fully covering the tile.

The HTML sample report generated from our primary analysis pipeline provides valuable metrics to assess sequencing saturation. A few of these metrics include the total number of raw fastq readpairs and the median reads per UMI. By analyzing internal benchmarking experiments, we have determined that a median reads per UMI value between 8 and 10 indicates sequencing saturation. This means that you have sequenced deeply enough to fully comprehend the complexity of your library. If the median reads per UMI value is less than 8, we suggest sequencing at a deeper level before proceeding with a secondary analysis.