Poly(A) selection limits detection of long and alternatively spliced transcripts compared with rRNA depletion in RNA-Sequencing

The eukaryotic transcriptome diversity arises largely from alternative splicing. One of the widely used high-throughput methods to study this diversity is RNA sequencing. RNA sequencing has become a cornerstone of both basic biology and precision medicine, facilitating the quantification of gene and transcript expression, as well as the characterization of alternative splicing events and regulatory biological pathways in these studies. As there is a wide interest in studying non-ribosomal RNAs, which constitute about 20% of cellular RNAs, it is common to either select for poly(A) + RNAs or to deplete ribosomal RNAs during the library preparation stage of RNA sequencing. Using blood and skeletal muscle transcriptomics data, we show that poly(A) + enriched RNA library data inefficiently detects long transcripts, with lengths larger than 5kb constituting to ~ 16.5% of isoforms in Gencode v39, and predominantly detects the 3′ end compared to the 5′ end of these transcripts. In contrast, rRNA depletion provides a more uniform 5′-3′ coverage, an improved detection of splicing events, and a robust detection of long disease-relevant transcripts. Furthermore, we show that the improved performance of rRNA depleted RNA sequencing, compared to poly(A)+, is particularly evident in the detection of extremely large transcripts, such as the sarcomeric genes OBSCN (~ 39kb) and TTN (> 100 kb). Our findings reveal the advantages of using rRNA depletion over the more commonly used poly(A) + selection for both research and diagnostic applications, especially where RNA-Seq is employed to analyse long muscle transcripts and detect pathogenic splicing defects and refine variant interpretation.