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<p>Next-generation RNA sequencing (RNA-seq) is hampered by “primer dimer” (PD) artifacts and its quantitative performance reduced by polymerase fall-off (PF) at RNA modifications and secondary structures. Here we improve RNA-seq efficiency by incorporating (i) a post-reverse-transcription (RT) digestion of excess primers with <i>Escherichia coli</i> exonuclease I for PD mitigation, thus obviating gel purification during RNA-seq library preparation, and (ii) a high-processivity reverse transcriptase to increase full-length reads. A full factorial experimental design is applied to absolute quantification RNA sequencing (AQRNA-seq), the most accurate NGS-based method for quantifying small RNAs, using cDNA libraries constructed from <i>E. coli</i> small RNAs (&gt;85% tRNA) followed by sequencing, data processing, and data analysis. The novel PF and PD mitigation approaches increased AQRNA-seq sensitivity &gt;10-fold by minimizing PF and maximizing target RNA reads. By increasing sensitivity and obviating gel electrophoresis for removing PD, AQRNA-seq and other NGS-based RNA-seq methods can now be automated to increase throughput and reduce RNA sample size.</p>

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Gel-free library preparation for next-generation RNA sequencing and small RNA quantification

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年5月9日

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  • Ruixi Chen,
  • Daniel Yim,
  • Lili Liu,
  • Michael S. DeMott,
  • Bo Cao,
  • Peter C. Dedon

摘要

Next-generation RNA sequencing (RNA-seq) is hampered by “primer dimer” (PD) artifacts and its quantitative performance reduced by polymerase fall-off (PF) at RNA modifications and secondary structures. Here we improve RNA-seq efficiency by incorporating (i) a post-reverse-transcription (RT) digestion of excess primers with Escherichia coli exonuclease I for PD mitigation, thus obviating gel purification during RNA-seq library preparation, and (ii) a high-processivity reverse transcriptase to increase full-length reads. A full factorial experimental design is applied to absolute quantification RNA sequencing (AQRNA-seq), the most accurate NGS-based method for quantifying small RNAs, using cDNA libraries constructed from E. coli small RNAs (>85% tRNA) followed by sequencing, data processing, and data analysis. The novel PF and PD mitigation approaches increased AQRNA-seq sensitivity >10-fold by minimizing PF and maximizing target RNA reads. By increasing sensitivity and obviating gel electrophoresis for removing PD, AQRNA-seq and other NGS-based RNA-seq methods can now be automated to increase throughput and reduce RNA sample size.