ABC‐seq Expands Small RNAs Profiling with Successive Nucleic Acid Structure‐Differentiated Enzymatic Recognition

Small RNAs (smRNAs) play crucial roles in gene regulation, but current sequencing methods face a set of tangled challenges, including adapter ligation bias, high adapter dimer byproducts, and amplification variability, which limit their capacity to fully capture smRNA diversity. Here, we develop ABC-seq, a general strategy to enhance smRNA recognition through successive structure-differentiated recognition. We designed two randomized adapters, each containing a restriction enzyme recognition motif or a cleavage site. Then, we operate two product structure-differentiated enzymatic reactions to maximally suppress randomized adapter dimer. Furthermore, the randomized sequence in adapters serves as unique molecular identifiers (UMIs) in smRNA-seq data analysis to correct amplification bias and improves quantification accuracy. Using ABC-seq, we identified a wider range of smRNAs, including miRNAs, scRNAs, and snoRNAs, in hypertrophic cardiomyocytes, detecting 9.4% more miRNAs compared to traditional methods. Additionally, ABC-seq revealed smRNAs associated with cancer drug responses and their roles in immune modulation in non-small cell lung cancer. ABC-seq expands the smRNA landscape and offers new insights into complex gene regulatory networks.