Molecular phenotyping through shallow 3’-end RNA-sequencing workflows is increasingly applied in the context of large-scale chemical or genetic perturbation screens to study disease biology or support drug discovery. While these workflows enable accurate quantification of the most abundant genes, they are less effective for applications that require expression profiling of low abundant transcripts, like long non-coding RNAs (lncRNAs), or selected gene panels. To tackle these issues, researchers from the Cancer Research Institute Ghent describe a workflow combining 3’-end library preparation with 3’-end hybrid capture probes and shallow RNA-sequencing for cost-effective, targeted quantification of subsets of (low abundant) genes across hundreds to thousands of samples. To assess the performance of the method, the researchers designed a capture probe set for more than 100 mRNA and lncRNA target genes and applied the workflow to a cohort of 360 samples. When compared to standard 3’-end RNA-sequencing, 3’-end capture sequencing resulted in a more than 100-fold enrichment of target gene abundance while conserving relative inter-gene and inter-sample abundances. 3’-end RNA capture sequencing enables accurate targeted gene expression profiling at extremely shallow sequencing depth.
Comparison of the classic 3’-end sequencing workflow and combination
with targeted capture sequencing
A. Distribution of mean counts for lncRNAs (red) and mRNAs (blue) by shallow 3’end RNA sequencing across a cohort of 360 samples. B. Number of mRNA (blue) and lncRNA (red) genes with a mean count of at least 10 in the same cohort. C. In the standard 3’end sequencing approach, after introducing a sample barcode at the cDNA synthesis step, libraries are sequenced at low depth resulting in sufficient coverage for the most abundant genes. A subset of (low abundant) genes can be enriched from these pools to avoid reads being consumed by the most abundant genes or simply being allocated to irrelevant genes.