What is the recommended number of reads when your sample tissue does not cover the entire tile area?
What is the recommended number of 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 2 x 108 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 the estimated percent of tile covered. For example, if your tissue only covers 50% of the tile, you can start with 1.5 x 107 reads for shallow sequencing.
- However, the optimal number of reads is also dependent on the transcriptional activity of your tissue. For example, if your tissue is tumorigenic, it is highly transcriptionally active and may require two- to threefold higher read depth to reach saturation if the section is fully covering the tile.
- The HTML sample report generated from our Seeker Primary Analysis Pipeline provides valuable metrics to assess sequencing saturation. A few of these metrics include the total number of raw FASTQ read pairs 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 secondary analysis.