Liu Ruolin, Darbeheshti Farzaneh, Walsh Laurel, Li Rachel, Bae Jin H, Zeggar Hayet Radia, Narayan Azeet, Xiong Kan, Makrigiorgos G Mike, Adalsteinsson Viktor A
Broad Institute of MIT and Harvard, Cambridge, MA 02215, United States.
Dana-Farber Cancer Institute, Boston, MA 02142, United States.
Nucleic Acids Res. 2025 May 22;53(10). doi: 10.1093/nar/gkaf482.
DNA mutations and methylation often contribute to disease development in a synergistic manner. While duplex sequencing is the most accurate method for detecting DNA mutations, it typically lacks the ability to simultaneously assess methylation or requires many reads. Here, we developed Methyl-CODEC to enable simultaneous methylation sequencing and duplex sequencing using single read pairs. To achieve this, Methyl-CODEC links an enzymatically deaminated sense strand to the reverse complement of the antisense strand, which is protected from conversion by using conversion-resistant dCTPs in the strand linking step. Methyl-CODEC shows high concordance with standard enzymatic or bisulfite-based whole genome methylation sequencing, while also uniquely preserving the original DNA sequence. We show that hydroxy-methyl-dCTP is superior in this regard relative to other conversion-resistant dCTPs. Methyl-CODEC improves genetic sequencing accuracy, enables better read alignment for next-generation sequencing, and distinguishes C > T mutations from unmethylated Cs. It also identifies rare mutations including those producing methylated Cs, which are enriched in CpG contexts. Methyl-CODEC opens new horizons for enhanced detection of biomarkers in cancer and molecular medicine.
DNA突变和甲基化通常以协同方式促进疾病发展。虽然双链测序是检测DNA突变最准确的方法，但它通常缺乏同时评估甲基化的能力，或者需要大量读数。在这里，我们开发了Methyl-CODEC，以实现使用单读对同时进行甲基化测序和双链测序。为了实现这一点，Methyl-CODEC将酶促脱氨的有义链与反义链的反向互补序列连接起来，在链连接步骤中使用抗转化的dCTP来保护反义链不被转化。Methyl-CODEC与基于标准酶促或亚硫酸氢盐的全基因组甲基化测序具有高度一致性，同时还独特地保留了原始DNA序列。我们表明，在这方面，羟甲基-dCTP相对于其他抗转化dCTP具有优势。Methyl-CODEC提高了基因测序的准确性，使下一代测序的读段比对更好，并区分了C>T突变与未甲基化的C。它还能识别罕见突变，包括那些产生甲基化C的突变，这些突变在CpG环境中富集。Methyl-CODEC为增强癌症和分子医学中生物标志物的检测开辟了新的视野。
