Question: Can I perform shallow sequencing to assess the quality of Visium Spatial Gene Expression for FFPE libraries?
Answer: Shallow sequencing can be performed to obtain an initial assessment of library quality prior to performing deeper sequencing.
Metrics including “Valid Barcodes”, “Valid UMIs”, “Fraction Reads in Spots Under Tissue”, and “Reads Mapped Confidently to the Filtered Probe Set” are accurate even at low sequencing depths for both samples of typical quality and compromised samples (Fig. 1).
Metrics that may not be accurate at low sequencing depths include ‘Median UMIs per Spot,” “Median Genes per Spot,” and “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. Examples of a typical sample (Fig. 2) and a compromised sample with tissue detachment (Fig. 3) are shown below.
Figure 1: Downsamples metrics from human breast ductal carcinoma samples of typical quality (left) or compromised/detached (right) samples across sequencing depths.
Figure 2. Tissue plots showing H&E staining (left), UMI count (middle), or clustering (right) at standard (top) or low (bottom) read depth in a typical sample. UMI count and clustering mirrors the morphology of the tissue irrespective of read depth.
Figure 3: Tissue plots showing H&E staining (left), UMI count (middle), or clustering (right) at standard (top) or low (bottom) read depth in a compromised/detached sample. UMI count and clustering across the tissue does not agree with tissue morphology irrespective of read depth.
Products: Visium for FFPE