Question: Why do my Visium CytAssist samples have poor mapping metrics? Is there a problem with my data?
Answer: All probe based 10x assays must be run with a transcriptome reference as well as the corresponding probeset. It is important to ensure that the correct probeset is chosen according to version and species. Failure to select the correct probeset may lead to low/poor mapping metrics.
Using a probeset with a poly-A based assay will erroneously invoke and display metrics for probe-based mapping.
After ensuring that analysis was run correctly, you can also examine the BAM file to get further information on the mapping metrics and their breakdown using BAM tags (BAM tags in spaceranger)
The MAPQ score is a measure of the mapping quality and there are several tags and values which indicate what has gone on in the mapping. For probe based assays we have the following
||Both read halves map to the same probe.|
||Each read half maps to a different probe.|
||One read half maps to a probe and the other half does not.|
||Neither read half maps to a probe.|
Looking into the distribution of reads with the MAPQ score can give additional context for further troubleshooting. A high percentage of reads with score of 3 indicates a high number of chimeric reads.
Note: If you have designed custom probes, you will need to ensure that the probeset and reference have been updated as necessary.
Products: Visium Spatial Gene Expression for FFPE, Visium CytAssist Spatial Gene and Protein Expression, Visium CytAssist Gene Expression