Ellis Thomas James, Nizhynska Viktoria, Pisupati Rahul, Mollá-Morales Almudena, Nordborg Magnus
Gregor Mendel Institute, Austrian Academy of Sciences, Vienna BioCenter, Vienna, Austria.
PLoS One. 2025 May 21;20(5):e0322539. doi: 10.1371/journal.pone.0322539. eCollection 2025.
'Tagmentation' approaches to bisulphite sequencing use a transposase to simultaneously make double-stranded breaks and ligate adaptors to the resulting fragments, allowing for higher throughput with less starting material. However, it has also been noted that certain tagmentation protocols have an unusually high number unmethylated cytosines that are not converted to thymine. Here we describe this phenomenon in detail, and find that results are consistent with single strand nicks by the transposase, followed by strand displacement of part or all of the DNA fragment, leading to erroneous incorporation of methylated cytosines. Nevertheless we show that these errors can be accounted for in downstream analysis and need not impede biological conclusions. We provide a Python package to allow users to implement this framework. Ultimately the additional effort of accounting for errors must be traded off against the scalability of the protocol in planning experiments.
亚硫酸氢盐测序的“转座酶标签法”利用转座酶同时产生双链断裂并将接头连接到所得片段上，从而在起始材料较少的情况下实现更高的通量。然而，也有人指出，某些转座酶标签法方案存在异常高数量未甲基化的胞嘧啶，这些胞嘧啶未转化为胸腺嘧啶。在这里，我们详细描述了这种现象，并发现结果与转座酶造成的单链切口一致，随后部分或全部DNA片段发生链置换，导致甲基化胞嘧啶的错误掺入。尽管如此，我们表明这些错误可以在下游分析中得到解释，并不一定会妨碍生物学结论的得出。我们提供了一个Python软件包，供用户实施此框架。最终，在规划实验时，必须在考虑错误的额外工作与该方案的可扩展性之间进行权衡。
