Luca Tosti, Calum Mould, Imogen Gatehouse, Anthony C Smith, Krystian Ubych, Valentina Miano, Peter W Laird, Jack Kennefick, Robert K Neely
{"title":"使用甲基转移酶定向标记和富集对未修饰DNA进行全基因组分析。","authors":"Luca Tosti, Calum Mould, Imogen Gatehouse, Anthony C Smith, Krystian Ubych, Valentina Miano, Peter W Laird, Jack Kennefick, Robert K Neely","doi":"10.1016/j.crmeth.2025.101187","DOIUrl":null,"url":null,"abstract":"<p><p>We present \"Active-Seq\" (azide click tagging for in vitro epigenomic sequencing), a base-conversion-free technology that enables the isolation of DNA containing unmodified CpG sites using a mutated bacterial methyltransferase enzyme and a synthetically prepared cofactor analog. Active-Seq is a robust epigenomic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation and compatible with DNA input quantities as low as 1 ng. We establish a baseline for the performance of Active-Seq using model DNA oligos and further validate it against gold-standard whole-genome bisulfite sequencing data. We show robust performance of the platform across tissue-derived DNA and demonstrate enrichment of DNA at unmethylated, cell-type-specific marker regions of the epigenome, laying the foundation for the future application of this technology in tissue deconvolution applications. Finally, we apply the technology to cell-free DNA samples, outlining an approach for tumor-informed disease profiling in patients with colorectal cancer.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":" ","pages":"101187"},"PeriodicalIF":4.5000,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment.\",\"authors\":\"Luca Tosti, Calum Mould, Imogen Gatehouse, Anthony C Smith, Krystian Ubych, Valentina Miano, Peter W Laird, Jack Kennefick, Robert K Neely\",\"doi\":\"10.1016/j.crmeth.2025.101187\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>We present \\\"Active-Seq\\\" (azide click tagging for in vitro epigenomic sequencing), a base-conversion-free technology that enables the isolation of DNA containing unmodified CpG sites using a mutated bacterial methyltransferase enzyme and a synthetically prepared cofactor analog. Active-Seq is a robust epigenomic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation and compatible with DNA input quantities as low as 1 ng. We establish a baseline for the performance of Active-Seq using model DNA oligos and further validate it against gold-standard whole-genome bisulfite sequencing data. We show robust performance of the platform across tissue-derived DNA and demonstrate enrichment of DNA at unmethylated, cell-type-specific marker regions of the epigenome, laying the foundation for the future application of this technology in tissue deconvolution applications. Finally, we apply the technology to cell-free DNA samples, outlining an approach for tumor-informed disease profiling in patients with colorectal cancer.</p>\",\"PeriodicalId\":29773,\"journal\":{\"name\":\"Cell Reports Methods\",\"volume\":\" \",\"pages\":\"101187\"},\"PeriodicalIF\":4.5000,\"publicationDate\":\"2025-09-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cell Reports Methods\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1016/j.crmeth.2025.101187\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Reports Methods","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.crmeth.2025.101187","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Genome-wide profiling of unmodified DNA using methyltransferase-directed tagging and enrichment.
We present "Active-Seq" (azide click tagging for in vitro epigenomic sequencing), a base-conversion-free technology that enables the isolation of DNA containing unmodified CpG sites using a mutated bacterial methyltransferase enzyme and a synthetically prepared cofactor analog. Active-Seq is a robust epigenomic profiling platform with a simple and streamlined workflow, performed in tandem with sequencing library preparation and compatible with DNA input quantities as low as 1 ng. We establish a baseline for the performance of Active-Seq using model DNA oligos and further validate it against gold-standard whole-genome bisulfite sequencing data. We show robust performance of the platform across tissue-derived DNA and demonstrate enrichment of DNA at unmethylated, cell-type-specific marker regions of the epigenome, laying the foundation for the future application of this technology in tissue deconvolution applications. Finally, we apply the technology to cell-free DNA samples, outlining an approach for tumor-informed disease profiling in patients with colorectal cancer.