In vitro transcribed (IVT) mRNA therapeutics offer a promising strategy for preventing and treating various diseases, including infectious diseases and cancer, by delivering specific nucleic acid sequences. Assessing the stability and equivalence of mRNA sequences and poly(A) tail lengths is crucial for minimizing adverse effects and ensuring the efficacy of the drugs. However, precise measurement of homopolymer stretches, such as poly(A) tails, is technically challenging, and a specialized method for IVT mRNA is lacking. Here, we introduce 3AIM-seq, a high-accuracy sequencing technique optimized both experimentally and analytically for measuring poly(A) tail lengths in IVT mRNA. We used a ligation-free adapter followed by 3-prime end amplification to prepare high-throughput sequencing libraries to generate high-quality data. The 3AIM-seq algorithm then employed a sliding window approach to base quality scores for precise poly(A) length measurement. Using calibration curves that correlate mean absolute error with poly(A) lengths from in silico synthetic standard spike-ins experiment, the method estimated the homogeneity of poly(A) lengths at the individual DNA molecule levels to evaluate fidelity. Notably, the method provided significantly more accurate length measurements than the base-calling method, which showed considerable inaccuracies. While poly(A) stretches of up to 70 bases were accurately estimated as IVT synthesized, stretches exceeding 100 bases exhibited a high proportion of non-designed lengths, highlighting the importance of careful and warranted assessment. Therefore, 3AIM-seq offers a reliable method for evaluating the structural integrity of IVT mRNA products, ensuring quantitative equivalence before clinical use.
