Question: Can I use the Targeted Gene Expression assay to detect fusion transcripts?
Answer: We have not performed any in-house tests to determine whether the Targeted Gene Expression assay can be used to detect fusion transcripts. In addition, identification of fusion transcripts in Targeted Gene Expression data will require additional bioinformatic expertise and is not part of the Cell Ranger pipeline.
In theory, baits targeting the fusion event could be designed by entering a FASTA sequence that spans the fusion event as an “exogenous sequence” in the 10x Genomics Custom Panel Designer. However, our bait design algorithm was not optimized for designing baits for fusion genes. The algorithm tiles baits in 120-nt multiples. Therefore, by chance, the actual fusion site may not be covered in the bait design.
From the assay perspective, it may be difficult to specifically enrich for fusions over the two independent transcripts. As suggested in a report from Twist Biosciences, hybridization-based target enrichment assays have some tolerance for stretches of unbound bait. Therefore, fusion-specific baits may be somewhat non-discriminatory. Even if enrichment is not as specific, as long as fusion and non-fusion transcripts are both enriched to some level, this would allow recovery of the fusion transcript in the final Targeted Gene Expression library.
Another point to consider is that the 3' or 5' Single Cell Gene Expression libraries that are used as input into the Targeted Gene Expression assay are generated by capturing the 3’ and 5' ends of transcripts, respectively. Therefore, if a fusion event lies far from the 3' or 5' end of the transcript, it may not be represented in the Single Cell Gene Expression library, and therefore would also not be represented in the Targeted Library.
Products: Targeted Gene Expression