Question: Is performance of the Targeted Gene Expression assay comparable for 3’ and 5’ Gene Expression libraries?
Answer: Both 3’ and 5’ Single Cell Gene Expression libraries can be used as Whole Transcriptome Analysis (WTA) input libraries for the Targeted Gene Expression assay. As outlined in our Technical Note on Targeted Gene Expression Pre-designed Panel Performance Metrics, targeted libraries prepared from 3’ or 5’ WTA libraries display similar metrics with respect to the recovered complexity of the targeted libraries and with respect to the correlation of UMI counts between targeted libraries and parent WTA libraries.
However, the fraction of reads mapped to the targeted transcriptome is typically lower for targeted libraries prepared from 5’ WTA libraries as compared to 3’ WTA libraries. This is because 5’ WTA libraries typically have higher levels of reads mapping antisense to genes than 3’ 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 are not counted as mapping to the targeted transcriptome. Therefore, libraries with higher levels of antisense reads typically have a lower fraction of reads mapping to the targeted transcriptome.
Products: Targeted Gene Expression