Question: Can I perform shallow sequencing to assess the quality of Visium Spatial Gene Expression for Fresh Frozen libraries?
Answer: Shallow sequencing can be performed to obtain an initial assessment of library quality before performing deeper sequencing.
Metrics including “Valid Barcodes”, “Valid UMIs”, “Fraction Reads in Spots Under Tissue”, and “Reads Mapped Confidently to Transcriptome” are accurate even at low sequencing depths (Figure 1).
Metrics that may not be accurate at low sequencing depths include ‘Median UMIs per Spot”, “Median Genes per Spot”, and “Total Genes Detected”. The number of clusters present can also change depending on the sequencing depth. Therefore, it is important to review all of the metrics that are considered accurate at low depths when deciding whether to proceed with deeper sequencing.
Tissue plots colored by UMI count or clustering can be used to gauge whether a sample has been compromised. High quality samples reveal high UMI counts in regions of high cell density or highly expressing cells, whereas in compromised samples, UMIs across the tissue do not mirror the overall tissue morphology. Similarly, tissue plots colored by clustering in which clusters mirror the tissue morphology indicate results expected from a typical sample. Clustering across the tissue may not agree with the tissue morphology in a compromised sample. Using these outputs to ascertain overall sample quality can be performed even at read depths as low as 1k mean reads per spot under tissue. An example of a typical sample is shown in Figure 2.
Figure 1. Downsampled metrics from mouse olfactory bulb of typical quality across sequencing depths.
Figure 2. Tissue plots colored by UMI count or clustering at standard and low read depth in a sample of typical quality. UMI count and clustering mirrors the morphology of the tissue irrespective of read depth.
Product: Visium for Fresh-Frozen