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: The single cell 3' RNA-seq assay and Cell Ranger software are not designed to distinguish between different isoforms of a gene. Cell Ranger counts UMIs per gene (as defined by the exons in the reference) and not per CDS (coding sequence) or transcript.
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, each isoform must be annotated as a unique gene in the reference. This can be done by generating a custom reference, instructions for which are here.
Note that Cell Ranger only uses confidently mapped reads that align to a single gene for UMI counting. See here for more details. Therefore, reads aligned to the common regions of different isoforms will be discarded for UMI counting, which may lead to suppression of expression level (UMI counts) for both isoforms.
Please note that detecting isoforms from 3' Single Cell product is currently not a supported application and any ideas/code provided are for instructional purposes only.
Products: Single Cell Gene Expression