Question: My image is low contrast and the tissue and fiducial markers are hard to detect. Can I mark the spots covered by tissue manually?
Answer: Here is an example of a low contrast image that may not work with automatic alignment in Space Ranger because the fiducial markings are barely visible:
You can use the manual alignment using the instructions provided on the 10x Support Website.
First, align the image by identifying the fiducial markers:
Second, identify the spots underneath the tissue using the lasso or brush tools: This will allow you to manually mark the spots covered by the tissue. In this particular image, there are 2657 spots underneath the tissue.
The next step will allow you to export the alignment to a .json file, which you will need to provide as input to the Space Ranger software. Save this file to a location where you can access it later:
The instructions on this 10x Support page show you how to run Space Ranger.
You will upload this .json file to the directory on the server where your FASTQ files are located using a file transfer program or scp:
scp /path/to/manual_alignment.json [email protected]:/path/to/destination/spaceranger-folder/
Then, proceed with running Space Ranger, including this file called out in bold below:
spaceranger count --id=sample345 \ --transcriptome=/opt/refdata/GRCh38-3.0.0 \ --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ --sample=mysample \ --image=/home/jdoe/runs/images/sample345.tif \ --slide=V19J01-123 \ --area=A1 \ --loupe-alignment=manual_alignment.json \ --localcores=8 \ --localmem=64