RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant – and sometimes non-informative – RNA species.
Researchers at Ghent University have developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of this method, they applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. The researchers demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.
Overview of targeted transcripts and investigated library preparation methods
A Visual representation of the evaluated RNA sequencing library preparation methods with the associated blocked transcripts. B–F Coverage plots of standard library prep RNA-sequencing data for the targeted region. The height of the bars represents the relative number of reads mapping to that position in the transcript. The LNA oligonucleotide and its binding location are shown in red/green under the coverage plot. The targeted transcripts and their chromosomal location are indicated at the bottom of each plot. G Schematic overview of blocking during RT and PCR amplification
This method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
Everaert C, Verwilt J, Verniers K, Vandamme N, Marcos Rubio A, Vandesompele J, Mestdagh P. (2023) Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation. Biol Proced Online 25(1):7. [article].