{"doi":"10.1101/2024.06.03.597111","title":"PT-seq: A method for metagenomic analysis of phosphorothioate epigenetics in complex microbial communities","abstract":"ABSTRACT Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the dnd, ssp , and brx genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3’-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":498208,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9597,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":495906,"name":"Michael S. DeMott","orcid":"0000-0002-8575-7277","position":1,"is_corresponding":false},{"id":437634,"name":"Shane R. Byrne","orcid":"0000-0002-2136-305X","position":2,"is_corresponding":false},{"id":1345038,"name":"the Global Microbiome Conservancy","orcid":null,"position":3,"is_corresponding":false},{"id":289582,"name":"Peter C. Dedon","orcid":"0000-0003-0011-3067","position":4,"is_corresponding":false},{"id":388659,"name":"Yifeng Yuan","orcid":"0000-0002-9549-2698","position":0,"is_corresponding":true}],"reference_count":42,"raw_metadata":null,"created_at":"2026-07-19T02:09:38.543544Z","pmid":"38895297","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":[]}