Blocking abundant RNA transcripts by high-affinity oligonucleotides during transcriptome library preparation

Abstract

RNA sequencing has become the gold standard for transcriptome analysis but comes with an inherent limitation with respect to quantification of low abundant transcripts. In contrast to microarray technology, RNA-sequencing reads are proportionally divided across transcripts. Therefore, low abundant RNAs can be out-competed by highly abundant--and sometimes non-informative--RNA species. We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR-amplification of specific RNA transcripts, hereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different RNA molecules and library preparation strategies, including YRNAs in small RNA-sequencing of human plasma samples, mitochondrial rRNAs in both 3-prime end sequencing and long-read sequencing, and MALAT1 in single-cell 3-prime end sequencing. We demonstrate that the blocking strategy is extremely efficient, reproducible and specific, and generally results in better transcriptome coverage and complexity. Our method does not require modifications of the library preparation procedure apart from adding blocking oligonucleotides to the RT reaction and can thus be easily integrated in virtually any RNA sequencing library preparation protocol.

LNA blockers for improved amplification selectivity

“…The higher melting temperatures (T m s) of oligonucleotides that include LNA bases provide greater specificity and new functions. They have been used successfully as PCR primers/probes 4-6 , as antisense reagents 7-9 , as selective binders for distinguishing single-nucleotide variants [10][11][12][13][14][15][16][17][18] , as agents for selective capture/ degradation 19,20 , and as polymerization/splicing blockers [21][22][23][24][25][26][27][28] . Each of these roles requires that the LNA bind tighter than the corresponding pure DNA, but some functions may also require additional attributes that could be affected by the number and location of LNA bases within the oligonucleotide.…”

“…Furthermore, proteins may interact with LNAs differently than with standard DNAs, dictating whether LNA or DNA should occupy a particular position.While LNA blockers have been used in multiple situations, those studies have little commonality to provide insight into preferred designs. Some use chimeric [11][12][13]15,21,23,26 or pure 10,17,27 LNAs of 16 nt or shorter, while others use chimeric LNAs of 20 nt or longer 16,22,24 For 20-mers, there are more than one million possible LNA-DNA configurations for each of the more than 1 trillion possible sequences. In addition, functional predictions of how different LNAs will perform as primers, blockers, or in other roles are even less well characterized because additional factors beyond the well-studied T m 29-33 may play a critical role in how different LNAs perform [34][35][36] .…”