Question: Is the Targeted Gene Expression assay more sensitive than a whole transcriptome assay?
Answer: When sequencing a targeted library generated using the Targeted Gene Expression assay, targeted transcripts can typically be detected at a lower sequencing depth than would be required to detect those transcripts in a whole transcriptome analysis (WTA) library.
Therefore, at a given sequencing depth, the targeted assay can be more sensitive than a WTA assay for the genes in the targeted panel. Note that the sensitivity of the targeted assay is largely determined by the sequencing depth used when compared to that of the WTA library. This is demonstrated in the figure below.
However, given that the targeted assay enriches for library fragments already present in the WTA parent library, if a particular transcript is not represented in the parent library, it will also not be represented in the targeted library. Therefore, if a transcript is expressed at very low levels and is not represented in the parent library, it would also not be detected in the targeted library. See also: Can low abundance transcripts be detected?
The sensitivity of the targeted assay can be demonstrated by comparing the per-gene UMI recovery in a targeted library and its parent WTA library:
- In the figure shown below, a Targeted Gene Expression library was generated from a 3’ Single Cell Gene Expression library using the Human Pan-Cancer Panel. The parent library was sequenced to a depth of ~78,000 read pairs per cell, and the targeted library was sequenced to a depth of ~11,000 read pairs per cell.
- As shown in the graph below from the Web Summary File generated using Cell Ranger Targeted Compare, the majority of panel genes had more UMIs in the targeted library as compared to the parent library (median ratio of UMI counts in the targeted library as compared to the parent library was 1.128). Thus, at only a fraction of the sequencing depth, the targeted library showed greater sensitivity for most panel genes as compared to the parent library.
- These results will vary depending on the sequencing depths of the targeted and parent libraries.
Note: The Targeted Gene Expression assay has been discontinued. For alternative options, please see: What options are there for performing a targeted enrichment for my gene expression libraries?
Products: Targeted Gene Expression