A new study published in BMC Genomics by Swethaa Natraj Gayathri et al. evaluates how library preparation methods influence our understanding of the transcriptome. The team systematically compared two widely used RNA-Seq enrichment strategies—poly(A)+ selection and ribosomal RNA (rRNA) depletion—using human blood and skeletal muscle datasets.

The research shows that for transcripts longer than 5 kb, poly(A)+ selection introduces a strong 3′ end coverage bias and reduces splice junction detection. In contrast, rRNA depletion provides highly uniform sequence coverage across the entire transcript body, resulting in more robust detection of complex splice junctions.

These findings are particularly critical for neuromuscular disease research and clinical diagnostics. The authors demonstrated that rRNA depletion significantly improves the detection and statistical confidence of pathogenic, disease-associated splice variants in exceptionally large sarcomeric genes, such as OBSCN (~39 kb) and TTN (>100 kb). This work provides a vital framework for selecting the right sequencing strategy to maximize diagnostic yield in both research and clinical settings.