{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T15:59:37Z","timestamp":1791475177653,"version":"4.3.4"},"reference-count":32,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T00:00:00Z","timestamp":1791417600000},"content-version":"vor","delay-in-days":7,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T00:00:00Z","timestamp":1790812800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100000025","name":"National Institute of Mental Health","doi-asserted-by":"publisher","award":["R01 MH121070\u201001A1"],"award-info":[{"award-number":["R01 MH121070\u201001A1"]}],"id":[{"id":"10.13039\/100000025","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    We present a best\u2010practices protocol for targeted DNA methylation sequencing analysis that integrates both bisulfite\u2010 and enzymatic\u2010conversion approaches using Twist Bioscience technology. Building on established tools and vendor\u2010recommended workflows, this protocol provides a standardized, fully reproducible framework that addresses current gaps in the implementation, documentation, and downstream integration of targeted methylation sequencing analyses. The workflow incorporates widely used open\u2010source tools across two main stages: preprocessing (adapter trimming with Trim Galore and quality control via FastQC\/MultiQC) and methylation calling (alignment with BWA\u2010meth, duplicate marking using Picard, and methylation extraction with MethylDackel). Importantly, we extend existing workflows by introducing a structured third stage for downstream statistical analysis in R, including \u03b2\u2010value calculation, differential methylation analysis with methylKit, principal component analysis, and tiling\u2010based regional profiling, all embedded in a unified, reproducible pipeline. A key contribution of this protocol is the integration of multi\u2010stage quality control checkpoints, harmonized parameterization, and explicit reporting of intermediate outputs, enabling transparency and reproducibility across all analytical steps. The modular design allows tool substitution and adaptation to different experimental contexts, including enzyme\u2010based methylation methods, while maintaining consistency in data processing and interpretation. This guide provides step\u2010by\u2010step instructions, troubleshooting strategies, and examples of expected outputs, along with built\u2010in statistical routines that generate comprehensive results, including methylation matrices, correlation analyses, differentially methylated region annotations, and publication\u2010ready visualizations. The complete workflow requires approximately 9\u201327 hr for 10 samples (approximately 134.2 million reads per sample) on Linux systems and assumes moderate proficiency in command\u2010line environments and R. In summary, this protocol offers a practical, transparent, and reproducible framework that standardizes targeted DNA methylation analysis and facilitates its application across diverse biological and clinical research settings. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70464","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T15:33:21Z","timestamp":1791473601000},"source":"Crossref","is-referenced-by-count":0,"title":["A Guide to Enzymatic Targeted Methylation Sequencing Bioinformatics Workflow: Best Practices"],"prefix":"10.1002","volume":"6","author":[{"given":"Vit\u00f3ria Rodrigues Guimar\u00e3es","family":"Alves","sequence":"first","affiliation":[{"name":"Programa de P\u00f3s\u2010Gradua\u00e7\u00e3o em Psiquiatria e Psicologia M\u00e9dica Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Psychiatry Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheila T.","family":"Nagamatsu","sequence":"additional","affiliation":[{"name":"VA Connecticut Healthcare System  West Haven CT USA"},{"name":"Department of Psychiatry Yale University, School of Medicine  New Haven CT USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vanessa Kiyomi","family":"Ota","sequence":"additional","affiliation":[{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Programa de P\u00f3s\u2010gradua\u00e7\u00e3o em Biologia Estrutural e Funcional Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cibele Edom","family":"Bandeira","sequence":"additional","affiliation":[{"name":"Laboratory of Physiological Genomics of Mental Health (PhysioGen Lab) Instituto de Ci\u00eancias Biom\u00e9dicas da Universidade de S\u00e3o Paulo  S\u00e3o Paulo Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janitza L.","family":"Montalvo\u2010Ortiz","sequence":"additional","affiliation":[{"name":"VA Connecticut Healthcare System  West Haven CT USA"},{"name":"Department of Psychiatry Yale University, School of Medicine  New Haven CT USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danilo","family":"Micali","sequence":"additional","affiliation":[{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Carolina Coelho","family":"Milani","sequence":"additional","affiliation":[{"name":"Department of Psychiatry Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Pediatrics Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivaldo","family":"Silva","sequence":"additional","affiliation":[{"name":"Department of Gynaecology Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Applied Health Science, School of Public Health Indiana University Bloomington USA"},{"name":"Stark Neuroscience Research Institute Indiana University School of Medicine  Indianapolis USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristiane Seixas","family":"Duarte","sequence":"additional","affiliation":[{"name":"Department of Psychiatry Columbia University Irving Medical Center and New York State Psychiatric Institute  New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea Parolin","family":"Jackowski","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s\u2010Gradua\u00e7\u00e3o em Psiquiatria e Psicologia M\u00e9dica Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Psychiatry Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Health &amp; Wellness Design, School of Public Health Indiana University  Bloomington USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Posner","sequence":"additional","affiliation":[{"name":"Department of Psychiatry &amp; Behavioral Sciences Duke University  Durham USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina Muniz","family":"Carvalho","sequence":"additional","affiliation":[{"name":"Programa de P\u00f3s\u2010Gradua\u00e7\u00e3o em Psiquiatria e Psicologia M\u00e9dica Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Department of Psychiatry Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sintia Iole","family":"Belangero","sequence":"additional","affiliation":[{"name":"Department of Psychiatry Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"LiNC, Laboratory of Integrative Neuroscience Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"Programa de P\u00f3s\u2010gradua\u00e7\u00e3o em Biologia Estrutural e Funcional Universidade Federal de S\u00e3o Paulo  S\u00e3o Paulo Brasil"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,8]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1186\/gb\u20102012\u201013\u201010\u2010r87"},{"key":"e_1_2_10_3_1","unstructured":"AllPrep DNA\/RNA Mini Handbook \u2010 QIAGEN [WWW Document]. 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