Question: Is trajectory analysis compatible with the Fixed RNA Profiling (Flex) assay, which is probe-based?
Answer:
Trajectory analysis is a general term that may include different types of analysis for cell ordering.
One type of it is RNA velocity analysis. This type of analysis requires the counts from spliced and unspliced RNAs. In our Flex assay, we use probes to detect RNAs. Our probes are designed to target only exons of protein-coding genes. Although some probes overlap with splice junctions, our probes do not cover intronic regions and the majority of the probes do not overlap with a splice junction. Therefore, for many genes it is not possible to tell whether the signal comes from a spliced or unspliced transcript simply by looking at the reads, and it is not recommended to run RNA velocity analysis with the data from our Flex assay, unless custom probes are used.
Another category of trajectory analysis does not require spliced and unspliced counts, so it may be possible to perform trajectory analysis with Flex data. One such tool is monocle, which takes the gene expression data (e.g. sample_filtered_feature_bc_matrix) as input. Theoretically a tool like monocle should work with Flex data, but please keep in mind these are not 10x tools and so are not officially supported. 10x Genomics has not performed tests to confirm they work with Flex data.
Helpful publication to learn more about existing tools for trajectory analysis: Recent advances in trajectory inference from single-cell omics data.
Products: Fixed RNA Profiling (Single Cell Gene Expression Flex)