Question: Why are the "Reads Mapped Confidently to the Probe Set" or "Reads Mapped Confidently to the Filtered Probe Set" metrics in my Fixed RNA Profiling web summary file low?
Answer: In a typical Fixed RNA Profiling web summary file, we expect to see the “Reads Mapped Confidently to the Probe Set” and "Reads Mapped Confidently to the Filtered Probe Set" metrics to be >50%. Lower values of these metrics are unexpected and are typically associated with the presence of single unligated probes, mismatched probe pairs, or non-specific probes in the final library. Outlined below are the common root causes of these metrics being low and mitigation strategies to maximize the likelihood of success with the Fixed RNA Profiling workflow.
Low Fraction Reads Mapped Confidently to the Probe Set:
Observing a “Low Fraction Reads Mapped Confidently to the Probe Set” flag in the web summary file generally suggests that there was a workflow error; such as suboptimal Post-Hybridization/Ligation Washes (e.g. deviation in the temperature, timing, the number of washes, or excess supernatant carryover). Lower mapping rates can be further compounded by lower RNA quality. Depending on the fraction of usable reads and complexity of the sample of interest, viable data may still be generated with lower “Fraction Reads Mapped Confidently to the Probe Set” values.
Low Fraction Reads Mapped Confidently to the Filtered Probe Set:
The Probe Set CSV reference consists of probes for genes that are either included in the analysis (designated as “TRUE”) or excluded (“FALSE”) by default. Genes with the "FALSE" label have one or more probes that have predicted off-target activity.. Probes with the “DEPRECATED” prefix are always excluded from the analysis. Genes with off-target probes can be included in analysis with a flag in the config csv file for Cell Ranger Multi. Observing a “Low Fraction Reads Mapped Confidently to the Filtered Probe Set” flag in the web summary file suggests that the sample(s) expresses transcripts that off-target probes (labeled as “FALSE”) detect. Depending on the fraction of usable reads and complexity of the sample of interest, viable data may still be generated with lower “Fraction Reads Mapped Confidently to the Filtered Probe Set” values. The difference between “Fraction Reads Mapped Confidently to the Probe Set” and “Fraction Reads Mapped Confidently to the Filtered Probe Set” comes from the reads mapped to deprecated probes and probes with predicted off-target activity.
Products: Fixed RNA Profiling