Question: How can I determine the percentage of the capture area covered, so that I can calculate the recommended sequencing depth for Visium HD Spatial Gene Expression libraries?
Answer: You can estimate the percentage of capture area covered using the manual workflow in Loupe Browser (v8.0 and later), which you can download from the support site at this link.
The following directions uses Loupe Browser v8.0.
To calculate the percentage of the capture area covered for a 6.5 mm HD Capture Area:
1. Open Loupe Browser and select “Launch Visium Image Alignment” in the lower left corner (highlighted by a red box in the image below).
2. Choose “Visium HD Manual Alignment” (highlighted by a red box in the image below).
3. Complete the first four steps in the Manual Alignment workflow:
a. Upload the 1st image (CytAssist image) then select "Next" (bottom right).
b. Fill in the slide information (Slide Serial Number & Capture Area),then select "Next" (bottom right). The fields you need to fill in are highlighted in the image below by red boxes. It is also acceptable to provide the slide layout file, or select that you do not have the Visium slide serial number or slide layout file in this step.
c. Identify the fiducial corner markers and then select "Next" (bottom right).
d. Adjust the fiducials, select "Auto-refine" ( highlighted in the image below by a red box) to refine fiducial alignment and apply the perspective correction, and then select "Next" (bottom right).
4. In the “Identify tissue” step, select “Select all bins” to determine the total number of 8 µm x 8 µm bins. There are 702,244 bins. The field that contains the number of bins is highlighted by a red box in the image below.
5. Select “Deselect all bins” to clear the capture area of selected bins and then use the lasso, paint brush and eraser to select tissue-associated bins. This can be a highly accurate measurement or a rough estimate. In this example, 609,037 tissue-associated bins were identified.
6. Calculate the percentage of the capture area covered using the total bins from Step 4 and the tissue-associated bins in Step 5 as follows:
Percentage of the capture area covered = (Number of selected tissue-associated bins) / (Total number of bins in the capture area)*100
Using the information from above this calculation would be:
Percentage of the capture area covered = (609,037 selected tissue-associated bins)/(702,244 bins in the capture area)*100=86.7%
7. To determine the minimum recommended sequencing depth, multiply the minimum sequencing depth recommendation for 100% capture area coverage by the percentage of the capture area covered as determined above.
Using the previous result for the percentage of the capture ares covered the recommended sequencing depth would be:
Recommended minimum number of read pairs = (Number of reads for 100% of the capture area coverage)*(Percentage of the capture area covered)
Recommended minimum number of read pairs = (275 million read pairs * .867) = 238 million read pairs
Note: In Step 5, it's preferable to overestimate rather than underestimate the number of tissue-associated bins.
Related Links: What are the recommended sequencing specifications for Visium HD libraries?
Product: Visium HD Spatial Gene Expression
Updated May 01, 2024