{"doi":"10.1093/nar/gkaf558","title":"Novel enzyme-based reduced representation method for DNA methylation profiling with low inputs","abstract":"Commonly used bisulfite-based procedures for DNA methylation sequencing can degrade DNA, worsening signal-to-noise ratios in samples with low DNA input. Enzymatic methylation sequencing (EM-seq) has been proposed as a less biased alternative for methylation profiling with greater genome coverage. Reduced representation approaches enrich samples for CpG-rich genomic regions, thereby enhancing throughput and cost effectiveness. We hypothesized that enzyme-based technology could be adapted for reduced representation methylation sequencing to enable DNA methylation profiling of low-input samples. We leveraged the well-established differences in methylation profile between mouse CD4+ T cell populations to compare the performance of our reduced representation EM-seq (RREM-seq) procedure against an established reduced representation bisulfite sequencing (RRBS) protocol. While the RRBS method failed to generate reliable DNA libraries when using <2 ng of DNA, the RREM-seq method successfully generated reliable DNA libraries from 1-25 ng of mouse and human DNA. Low-input (≤2-ng) RREM-seq libraries demonstrated superior regulatory genomic element coverage compared with RRBS libraries with >10-fold higher DNA input. RREM-seq also successfully detected lineage-defining methylation differences between alveolar conventional T and regulatory T cells obtained from patients with severe SARS-CoV-2 pneumonia. Our RREM-seq method enables single-nucleotide resolution methylation profiling using low-input samples, including from clinical sources.","journal":"Nucleic Acids Research","year":2025,"id":545026,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9539,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":267590,"name":"Kathryn A. Helmin","orcid":"0009-0006-8857-9943","position":1,"is_corresponding":false},{"id":1435467,"name":"Zachary D. Dortzbach","orcid":"0009-0006-7974-4871","position":2,"is_corresponding":false},{"id":864205,"name":"Carla P. Reyes Flores","orcid":"0000-0001-9016-3257","position":3,"is_corresponding":false},{"id":289275,"name":"Manuel A. Torres Acosta","orcid":"0000-0002-9939-7401","position":4,"is_corresponding":false},{"id":1173998,"name":"Jonathan Gurkan","orcid":"0000-0002-0149-3463","position":5,"is_corresponding":false},{"id":614993,"name":"Anthony M. Joudi","orcid":"0000-0003-1232-3256","position":6,"is_corresponding":false},{"id":1028170,"name":"Nurbek Mambetsariev","orcid":"0000-0002-7332-6947","position":7,"is_corresponding":false},{"id":289274,"name":"Luisa Morales‐Nebreda","orcid":"0000-0002-7126-0518","position":8,"is_corresponding":false},{"id":553695,"name":"Mengjia Kang","orcid":"0000-0002-1679-9473","position":9,"is_corresponding":false},{"id":394676,"name":"Luke V. Rasmussen","orcid":"0000-0002-4497-8049","position":10,"is_corresponding":false},{"id":1225958,"name":"Xóchitl G. Pérez-Leonor","orcid":"0009-0006-6617-7927","position":11,"is_corresponding":false},{"id":232815,"name":"Hiam Abdala‐Valencia","orcid":"0000-0003-3384-5299","position":12,"is_corresponding":false},{"id":267595,"name":"Benjamin D. Singer","orcid":"0000-0001-5775-8427","position":13,"is_corresponding":false},{"id":1103310,"name":"Qianli Liu","orcid":"0000-0002-7629-5648","position":0,"is_corresponding":true}],"reference_count":21,"raw_metadata":null,"created_at":"2026-07-19T02:53:17.418915Z","pmid":"40586301","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}