Years of experiments and databases filled with RNA-seq results belie the simple reality that, until fairly recently, it was impossible to analyze RNA directly. The RNA-seq studies performed with conventional sequencing platforms are based entirely on cDNA, an imperfect proxy for RNA that requires scientists to infer knowledge about the original RNA molecule rather than observe it directly.
It is understandable that the scientific community accepted this compromise. Single-stranded RNA is dynamic and highly structured, folding back on itself in ways that make it impossible for the polymerases used in most sequencing technologies to sequence an entire transcript. Repetitive sequences, hairpin structures, modifications, and vulnerability to degradation—RNA harbors a number of challenges that collectively prevent direct sequencing.
While cDNA-seq has been a necessary and important approximation for learning more about RNA, it is past…
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