Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Mol Cell. 2024 Apr 18;84(8):1611-1625.e3. doi: 10.1016/j.molcel.2024.03.018.
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.
我们最近通过化学还原和测序中 C:T 错配的诱导（RedaC:T-seq），以碱基分辨率报告了 N4-乙酰胞苷（ac4C）在 HeLa mRNA 中的分布。我们的结果与 Schwartz 及其同事使用类似方法（称为 ac4C-seq）的早期报告相矛盾。在这里，我们重新审视了这两个数据集，并再次确认了我们的发现。通过 RedaC:T-seq 重新分析，我们确定了未修饰核苷酸的低基础错误率，并且没有偏向任何特定的错配类型，并且在两种处理的野生型重复中，C:T 取代作为主要错配类型显著增加，在重复之间具有高度的可重复性。相比之下，通过 ac4C-seq 重新分析，我们发现了一些严重的数据质量问题，包括深度不足，一个野生型重复仅产生 270 万个读数，重复之间的还原效率不一致，以及整体涉及胸腺嘧啶的错配增加，这可能会掩盖 ac4C 的检测。这些分析支持通过 RedaC:T-seq 检测 HeLa mRNA 中的 ac4C。
