Question: Using the 10x Genomics Chromium Single Cell 3' Gene Expression Solution, we have identified gene X enriched in a cell type. However, this gene has multiple transcript isoforms. Can we use the single cell 3' RNA-seq data to determine the expression level of different isoforms for this gene X?
Answer: Combining long-read sequencing with single cell assays can enable the unambiguous identification of alternative splicing at single cell resolution. More detail can be found here: Can I detect alternative transcript isoforms using 10x assays?
The single cell 3' Gene Expression assay (with short-read sequencing) and Cell Ranger software are not designed to distinguish between different isoforms of a gene. Cell Ranger counts UMIs at gene-level, not for each transcripts. However, if two isoforms are sufficiently different, reads that align to the distinct parts of the isoforms may help distinguish between them. To achieve this, isoforms of interests must be annotated as a unique genes in the reference. This will require editing specific gene/transcripts in GTF, and then generating a custom reference. Note that Cell Ranger only uses confidently mapped reads that align to a single gene for UMI counting. Therefore, in this case, reads aligned to the common regions of different isoforms could be discarded for UMI counting, which may lead to undercounting of UMIs for both isoforms.
Please note that detecting isoforms from 3' Single Cell product with short-read sequencing is not a supported application and any ideas/code provided are for instructional purposes only.
Products: Single Cell Gene Expression