We recently reported the distribution of N4-acetylcytidine (ac4C) in HeLa mRNA at base resolution through chemical reduction and the induction of C:T mismatches in sequencing (RedaC:T-seq). Our results contradicted an earlier report from Schwartz and colleagues utilizing a similar method termed ac4C-seq. Here, we revisit both datasets and reaffirm our findings. Through RedaC:T-seq reanalysis, we establish a low basal error rate at unmodified nucleotides that is not skewed to any specific mismatch type and a prominent increase in C:T substitutions as the dominant mismatch type in both treated wild-type replicates, with a high degree of reproducibility across replicates. In contrast, through ac4C-seq reanalysis, we uncover significant data quality issues including insufficient depth, with one wild-type replicate yielding 2.7 million reads, inconsistencies in reduction efficiencies between replicates, and an overall increase in mismatches involving thymine that could obscure ac4C detection. These analyses bolster the detection of ac4C in HeLa mRNA through RedaC:T-seq.

我们最近报道了通过化学还原和测序中 C:T 错配的诱导 (RedaC:T-seq)，N4-乙酰胞苷 (ac4C) 在 HeLa mRNA 中碱基分辨率的分布。我们的结果与 Schwartz 及其同事使用类似方法 ac4C-seq 的早期报告相矛盾。在这里，我们重新审视这两个数据集并重申我们的发现。通过 RedaC:T-seq 重新分析，我们在未修饰的核苷酸上建立了较低的基础错误率，不偏向于任何特定的错配类型，并且 C:T 取代显着增加，作为两个处理的野生型重复中的主要错配类型，重复的高度再现性。相比之下，通过 ac4C-seq 重新分析，我们发现了重大的数据质量问题，包括深度不足（一次野生型重复产生 270 万个读数）、重复之间还原效率不一致，以及涉及胸腺嘧啶的错配总体增加，这可能会掩盖 ac4C 检测。这些分析支持通过 RedaC:T-seq 检测 HeLa mRNA 中的 ac4C。