{"doi":"10.1101/2021.11.03.467190","title":"Semi-quantitative detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct and long-read sequencing","abstract":"Abstract We developed and applied a semi-quantitative method for high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identified many known sites of pseudouridylation and uncovered new uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites were validated using 1,000-mer synthetic RNA controls bearing a single pseudouridine in the center position which demonstrate systematical under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our pipeline allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data as well as multiple modifications on the same strand.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":214441,"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":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9531,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":808489,"name":"Mohammad Nabizadehmashhadtoroghi","orcid":null,"position":1,"is_corresponding":false},{"id":662079,"name":"Amr Makhamreh","orcid":"0000-0002-4615-5820","position":2,"is_corresponding":false},{"id":488621,"name":"Howard Gamper","orcid":"0000-0001-9007-8546","position":3,"is_corresponding":false},{"id":807911,"name":"Caroline A. McCormick","orcid":"0009-0000-0670-6707","position":4,"is_corresponding":false},{"id":808490,"name":"Neda K. Rezapour","orcid":null,"position":5,"is_corresponding":false},{"id":17829,"name":"Ya-Ming Hou","orcid":"0000-0001-6546-2597","position":6,"is_corresponding":false},{"id":572586,"name":"Meni Wanunu","orcid":"0000-0002-9837-0004","position":7,"is_corresponding":false},{"id":807912,"name":"Sara H. Rouhanifard","orcid":"0000-0002-6991-4877","position":8,"is_corresponding":false},{"id":807910,"name":"Sepideh Tavakoli","orcid":"0000-0002-0802-7051","position":0,"is_corresponding":true}],"reference_count":49,"raw_metadata":null,"created_at":"2026-07-18T23:52:46.604690Z","pmid":null,"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":[]}