Elucidation of the mechanism of byproduct DNA synthesis in whole-transcript amplification by poly-A tagging and development of its specific suppression

Poly-A tagging is a whole-transcript amplification method that uses terminal transferase to add poly-A to single-stranded cDNA in a non-template-dependent manner, and is used in highly sensitive single-cell RNA-seq. However, this method has long been reported to amplify byproduct DNA derived from reverse-transcription primers, particularly under conditions of low RNA abundance, leading to experimental complexity and reduced accuracy in quantifying gene expression. In this study, we elucidated the mechanism by which byproduct DNA is amplified at the molecular level and developed a new strategy of suppressing it named Suppression Tagging (SupTag). SupTag makes use of TdT, which has extremely low extension efficiency at the 3' recessive end, to stabilize dA/dT base pairs and inhibit poly-A extension of oligo-dT primers, thereby preventing the formation of precursor DNA for byproduct DNA. We demonstrated that SupTag can specifically suppress byproduct DNA using chemicals such as tetramethylammonium chloride (TMAC), modified nucleotides (e.g., 2F-dATP), and Super T-modified reverse-transcription primers. SupTag improves usability by streamlining purification processes and prevents reductions in sequence read output and quantitative sensitivity caused by byproduct DNA. Additionally, SupTag demonstrated the ability to suppress byproduct DNA amplification without using suppression PCR and is compatible with cDNA amplification introducing different adapter sequences at both ends. This study presents a novel suppression method based on the mechanism of amplification of byproduct DNA in the poly-A tagging method, providing a practical technology enabling high-precision transcriptomic analysis from low-abundance RNA.