Question: Is the Targeted Gene Expression assay compatible with nuclei?
Answer: The Targeted Gene Expression assay is compatible with Whole Transcriptome Analysis (WTA) libraries prepared from single cells or from single nuclei.
When comparing targeted libraries prepared from single-cell and single nuclei WTA libraries, both perform well with respect to the recovered complexity of the targeted libraries, and with respect to the correlation of UMI counts between targeted libraries and their parent WTA libraries.
However, the fraction of reads mapped to the targeted transcriptome is typically lower for targeted libraries prepared from single nuclei WTA libraries as compared to their whole-cell counterparts. This is mainly because single nuclei WTA libraries typically have higher levels of reads mapping antisense to genes than single-cell WTA libraries.
Due to the double-stranded nature of libraries, antisense reads are enriched as efficiently as sense reads in the targeted assay. Therefore, both sense and antisense reads corresponding to targeted genes can be observed in the targeted library. The vast majority of antisense reads in targeted libraries map to targeted genes.
During cellranger count, antisense reads are filtered out and do not contribute to the final UMI count for reads mapping to the targeted transcriptome. Therefore, a higher level of antisense reads leads to a lower fraction of reads mapping to the targeted transcriptome.
Note regarding intronic reads and the use of a pre-mRNA reference:
- WTA libraries prepared from nuclei typically have higher levels of intronic reads as compared to their whole-cell counterparts.
- Intronic reads are not counted as mapping to the transcriptome and therefore do not contribute to final UMI counts. If you would like to include intronic reads in the UMI counts, a “pre-mRNA” transcriptome reference can be used for cellranger count. Please see: Generating a Cell Ranger compatible "pre-mRNA" Reference Package.
- In the Targeted Gene Expression assay, baits are designed to capture exons, not introns. Therefore, the fraction of intronic reads tend to be lower in targeted libraries, even if high levels of intronic reads were present in the parent WTA library.
- When analyzing targeted libraries prepared from single nuclei WTA libraries, a pre-mRNA reference package can be used. However, given that intronic reads are not enriched in targeted libraries, using a pre-mRNA reference instead of the standard reference will typically not dramatically change the final UMI counts.
- However, we do not recommend the use of a pre-mRNA reference when using cellranger targeted-compare to compare a targeted and its parent WTA library.
- If a pre-mRNA reference is used, intronic reads will be included in UMI counts in the WTA library. Given that baits are designed to capture exons, most of these intronic reads will not be recovered in the targeted library. Therefore, metrics related to UMI recovery will be apparently lower when using a pre-mRNA reference as compared to using the standard reference.
Products: Targeted Gene Expression, Single Cell Gene Expression, Single Cell Immune Profiling