{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,26]],"date-time":"2026-02-26T20:34:33Z","timestamp":1772138073858,"version":"3.50.1"},"reference-count":38,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2025,4,23]],"date-time":"2025-04-23T00:00:00Z","timestamp":1745366400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004359","name":"Swedish Research Council","doi-asserted-by":"publisher","award":["2021\u201304000"],"award-info":[{"award-number":["2021\u201304000"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,5,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Motivation<\/jats:title>\n                    <jats:p>Long-read sequencing techniques can sequence transcripts from end to end, greatly improving our ability to study the transcription process. Although there are several well-established tools for long-read transcriptome analysis, most are reference-based. This limits the analysis of organisms without high-quality reference genomes and samples or genes with high variability (e.g. cancer samples or some gene families). In such settings, analysis using a reference-free method is favorable. The computational problem of clustering long reads by region of common origin is well-established for reference-free transcriptome analysis pipelines. Such clustering enables large datasets to be split roughly by gene family and, therefore, an independent analysis of each cluster. There exist tools for this. However, none of those tools can efficiently process the large amount of reads that are now generated by long-read sequencing technologies.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>We present isONclust3, an improved algorithm over isONclust and isONclust2, to cluster massive long-read transcriptome datasets into gene families. Like isONclust, isONclust3 represents each cluster with a set of minimizers. However, unlike other approaches, isONclust3 dynamically updates the cluster representation during clustering by adding high-confidence minimizers from new reads assigned to the cluster and employs an iterative cluster-merging step. We show that isONclust3 yields results with higher or comparable quality to state-of-the-art algorithms but is 10\u2013100 times faster on large datasets. Also, using a 256\u2009Gb computing node, isONclust3 was the only tool that could cluster 37 million PacBio reads, which is a typical throughput of the recent PacBio Revio sequencing machine.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Availability and implementation<\/jats:title>\n                    <jats:p>https:\/\/github.com\/aljpetri\/isONclust3.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btaf207","type":"journal-article","created":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T07:40:57Z","timestamp":1745221257000},"source":"Crossref","is-referenced-by-count":1,"title":["De novo clustering of large long-read transcriptome datasets with isONclust3"],"prefix":"10.1093","volume":"41","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-9397-0341","authenticated-orcid":false,"given":"Alexander J","family":"Petri","sequence":"first","affiliation":[{"name":"Department of Mathematics, Science for Life Laboratory, Stockholm University , Stockholm 106 91,","place":["Sweden"]},{"name":"Department of Computer Science, University of Helsinki , Helsinki 00014,","place":["Finland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7378-2320","authenticated-orcid":false,"given":"Kristoffer","family":"Sahlin","sequence":"additional","affiliation":[{"name":"Department of Mathematics, Science for Life Laboratory, Stockholm University , Stockholm 106 91,","place":["Sweden"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2025,4,23]]},"reference":[{"key":"2025051307512211200_btaf207-B2","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1186\/s13059-022-02715-w","article-title":"Rattle: reference-free reconstruction and quantification of transcriptomes from nanopore sequencing","volume":"23","author":"de la Rubia","year":"2022","journal-title":"Genome Biol"},{"key":"2025051307512211200_btaf207-B3","doi-asserted-by":"crossref","first-page":"e10805","DOI":"10.7717\/peerj.10805","article-title":"Syncmers are more sensitive than minimizers for selecting conserved k-mers in biological sequences","volume":"9","author":"Edgar","year":"2021","journal-title":"PeerJ"},{"key":"2025051307512211200_btaf207-B4","doi-asserted-by":"crossref","first-page":"2460","DOI":"10.1093\/bioinformatics\/btq461","article-title":"Search and clustering orders of magnitude faster than blast","volume":"26","author":"Edgar","year":"2010","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B5","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1101\/gr.8.3.175","article-title":"Base-calling of automated sequencer traces usingphred. I. Accuracy assessment","volume":"8","author":"Ewing","year":"1998","journal-title":"Genome Res"},{"key":"2025051307512211200_btaf207-B6","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1186\/1471-2105-12-271","article-title":"Dnaclust: accurate and efficient clustering of phylogenetic marker genes","volume":"12","author":"Ghodsi","year":"2011","journal-title":"BMC Bioinformatics"},{"key":"2025051307512211200_btaf207-B7","doi-asserted-by":"crossref","first-page":"e0132628","DOI":"10.1371\/journal.pone.0132628","article-title":"Widespread polycistronic transcripts in fungi revealed by single-molecule mrna sequencing","volume":"10","author":"Gordon","year":"2015","journal-title":"PLoS One"},{"key":"2025051307512211200_btaf207-B8","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1007\/BF01908075","article-title":"Comparing partitions","volume":"2","author":"Hubert","year":"1985","journal-title":"J Class"},{"key":"2025051307512211200_btaf207-B10","doi-asserted-by":"crossref","first-page":"e83\u2013e83","DOI":"10.1093\/nar\/gky315","article-title":"Meshclust: an intelligent tool for clustering dna sequences","volume":"46","author":"James","year":"2018","journal-title":"Nucleic Acids Res"},{"key":"2025051307512211200_btaf207-B11","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1186\/s13059-019-1910-1","article-title":"Transcriptome assembly from long-read rna-seq alignments with stringtie2","volume":"20","author":"Kovaka","year":"2019","journal-title":"Genome Biol"},{"key":"2025051307512211200_btaf207-B12","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1093\/bioinformatics\/btv573","article-title":"Rust-Bio: a fast and safe bioinformatics library","volume":"32","author":"K\u00f6ster","year":"2016","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B13","doi-asserted-by":"crossref","first-page":"e13598","DOI":"10.1111\/ppl.13598","article-title":"Molecular phylogeny of fucoxanthin-chlorophyll a\/c proteins from chaetoceros gracilis and lhcq\/lhcf diversity","volume":"174","author":"Kumazawa","year":"2022","journal-title":"Physiol Plant"},{"key":"2025051307512211200_btaf207-B14","doi-asserted-by":"crossref","first-page":"3094","DOI":"10.1093\/bioinformatics\/bty191","article-title":"Minimap2: pairwise alignment for nucleotide sequences","volume":"34","author":"Li","year":"2018","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B15","doi-asserted-by":"crossref","first-page":"1658","DOI":"10.1093\/bioinformatics\/btl158","article-title":"Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences","volume":"22","author":"Li","year":"2006","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B16","doi-asserted-by":"crossref","DOI":"10.1093\/bioinformatics\/btae059","article-title":"Highly efficient clustering of long-read transcriptomic data with geluster","volume":"40","author":"Ma","year":"2024","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B18","author":"Marchet"},{"key":"2025051307512211200_btaf207-B20","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1038\/s41592-022-01730-w","article-title":"Method of the year: long-read sequencing","volume":"20","author":"Marx","year":"2023","journal-title":"Nat Methods"},{"key":"2025051307512211200_btaf207-B21","doi-asserted-by":"crossref","first-page":"33","DOI":"10.12688\/f1000research.29032.2","article-title":"Sustainable data analysis with snakemake","volume":"10","author":"M\u00f6lder","year":"2021","journal-title":"F1000Res"},{"key":"2025051307512211200_btaf207-B22","doi-asserted-by":"crossref","first-page":"2352","DOI":"10.1016\/j.csbj.2023.03.038","article-title":"Benchmarking of nanopore r10. 4 and r9. 4.1 flow cells in single-cell whole-genome amplification and whole-genome shotgun sequencing","volume":"21","author":"Ni","year":"2023","journal-title":"Comput Struct Biotechnol J"},{"key":"2025051307512211200_btaf207-B23","doi-asserted-by":"crossref","first-page":"2940","DOI":"10.1038\/s41467-023-38553-y","article-title":"Reference-free assembly of long-read transcriptome sequencing data with rna-bloom2","volume":"14","author":"Nip","year":"2023","journal-title":"Nat Commun"},{"key":"2025051307512211200_btaf207-B24","doi-asserted-by":"crossref","first-page":"e11","DOI":"10.1093\/nar\/gkac1112","article-title":"Freddie: annotation-independent detection and discovery of transcriptomic alternative splicing isoforms using long-read sequencing","volume":"51","author":"Orabi","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"2025051307512211200_btaf207-B25","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1093\/nar\/gkj515","article-title":"Spectral clustering of protein sequences","volume":"34","author":"Paccanaro","year":"2006","journal-title":"Nucleic Acids Res"},{"key":"2025051307512211200_btaf207-B26","doi-asserted-by":"crossref","first-page":"i222","DOI":"10.1093\/bioinformatics\/btad264","article-title":"Isonform: reference-free transcriptome reconstruction from oxford nanopore data","volume":"39","author":"Petri","year":"2023","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B27","doi-asserted-by":"crossref","first-page":"1415","DOI":"10.1038\/s41596-022-00682-x","article-title":"Rapid in situ identification of biological specimens via dna amplicon sequencing using miniaturized laboratory equipment","volume":"17","author":"Pomerantz","year":"2022","journal-title":"Nat Protoc"},{"key":"2025051307512211200_btaf207-B28","doi-asserted-by":"crossref","first-page":"915","DOI":"10.1038\/s41587-022-01565-y","article-title":"Accurate isoform discovery with isoquant using long reads","volume":"41","author":"Prjibelski","year":"2023","journal-title":"Nat Biotechnol"},{"key":"2025051307512211200_btaf207-B29","doi-asserted-by":"crossref","first-page":"3363","DOI":"10.1093\/bioinformatics\/bth408","article-title":"Reducing storage requirements for biological sequence comparison","volume":"20","author":"Roberts","year":"2004","journal-title":"Bioinformatics"},{"key":"2025051307512211200_btaf207-B30","first-page":"410","author":"Rosenberg","year":"2007"},{"key":"2025051307512211200_btaf207-B31","author":"Sagniez"},{"key":"2025051307512211200_btaf207-B32","doi-asserted-by":"crossref","first-page":"2080","DOI":"10.1101\/gr.275648.121","article-title":"Effective sequence similarity detection with strobemers","volume":"31","author":"Sahlin","year":"2021","journal-title":"Genome Res"},{"key":"2025051307512211200_btaf207-B33","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1089\/cmb.2019.0299","article-title":"De novo clustering of long-read transcriptome data using a greedy, quality value-based algorithm","volume":"27","author":"Sahlin","year":"2020","journal-title":"J Comput Biol"},{"key":"2025051307512211200_btaf207-B34","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1038\/s41467-020-20340-8","article-title":"Error correction enables use of oxford nanopore technology for reference-free transcriptome analysis","volume":"12","author":"Sahlin","year":"2021","journal-title":"Nat Commun"},{"key":"2025051307512211200_btaf207-B35","doi-asserted-by":"crossref","first-page":"4601","DOI":"10.1038\/s41467-018-06910-x","article-title":"Deciphering highly similar multigene family transcripts from iso-seq data with isocon","volume":"9","author":"Sahlin","year":"2018","journal-title":"Nat Commun"},{"key":"2025051307512211200_btaf207-B36","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.1002\/ece3.7146","article-title":"NGSpeciesID: DNA barcode and amplicon consensus generation from long-read sequencing data","volume":"11","author":"Sahlin","year":"2021","journal-title":"Ecol Evol"},{"key":"2025051307512211200_btaf207-B37","doi-asserted-by":"crossref","first-page":"1026","DOI":"10.1038\/nbt.3988","article-title":"Mmseqs2 enables sensitive protein sequence searching for the analysis of massive data sets","volume":"35","author":"Steinegger","year":"2017","journal-title":"Nat Biotechnol"},{"key":"2025051307512211200_btaf207-B38","doi-asserted-by":"crossref","first-page":"2542","DOI":"10.1038\/s41467-018-04964-5","article-title":"Clustering huge protein sequence sets in linear time","volume":"9","author":"Steinegger","year":"2018","journal-title":"Nat Commun"},{"key":"2025051307512211200_btaf207-B39","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1186\/s13059-019-1883-0","article-title":"Quantifying the benefit offered by transcript assembly with scallop-lr on single-molecule long reads","volume":"20","author":"Tung","year":"2019","journal-title":"Genome Biol"},{"key":"2025051307512211200_btaf207-B40","doi-asserted-by":"crossref","first-page":"6297","DOI":"10.1038\/s41467-024-50239-7","article-title":"Ecological genomics in the Northern krill uncovers loci for local adaptation across ocean basins","volume":"15","author":"Unneberg","year":"2024","journal-title":"Nat Commun"},{"key":"2025051307512211200_btaf207-B41","doi-asserted-by":"crossref","first-page":"1003771","DOI":"10.3389\/fgene.2022.1003771","article-title":"Discovering novel reproductive genes in a non-model fly using de novo gridion transcriptomics","volume":"13","author":"Walter","year":"2022","journal-title":"Front Genet"},{"key":"2025051307512211200_btaf207-B42","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s12859-024-05663-3","article-title":"Clustrast: a short read de novo transcript isoform assembler guided by clustered contigs","volume":"25","author":"Westrin","year":"2024","journal-title":"BMC Bioinformatics"}],"container-title":["Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/bioinformatics\/advance-article-pdf\/doi\/10.1093\/bioinformatics\/btaf207\/62990626\/btaf207.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/41\/5\/btaf207\/62990626\/btaf207.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article-pdf\/41\/5\/btaf207\/62990626\/btaf207.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T07:51:32Z","timestamp":1747122692000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/bioinformatics\/article\/doi\/10.1093\/bioinformatics\/btaf207\/8118158"}},"subtitle":[],"editor":[{"given":"Can","family":"Alkan","sequence":"additional","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,4,23]]},"references-count":38,"journal-issue":{"issue":"5","published-print":{"date-parts":[[2025,5,6]]}},"URL":"https:\/\/doi.org\/10.1093\/bioinformatics\/btaf207","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2024.10.29.620862","asserted-by":"object"}]},"ISSN":["1367-4811"],"issn-type":[{"value":"1367-4811","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,5]]},"published":{"date-parts":[[2025,4,23]]},"article-number":"btaf207"}}