{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T18:19:45Z","timestamp":1788459585109,"version":"build-2803163510"},"reference-count":17,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100019186","name":"HORIZON EUROPE Innovative Europe","doi-asserted-by":"publisher","award":["Horizon Europe grants DNAMIC 101115389"],"award-info":[{"award-number":["Horizon Europe grants DNAMIC 101115389"]}],"id":[{"id":"10.13039\/100019186","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100019186","name":"HORIZON EUROPE Innovative Europe","doi-asserted-by":"publisher","award":["NEO 101115317"],"award-info":[{"award-number":["NEO 101115317"]}],"id":[{"id":"10.13039\/100019186","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"\n Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually\n \n \n \n \n \n \u2264<\/mml:mo>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides \u2014 particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis \u2014 remain inadequate. To address this gap,\n OligoSeq is presented<\/jats:italic>\n as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than\n \n \n \n \n \n \u2265<\/mml:mo>\n <\/mml:mrow>\n <\/mml:math>\n <\/jats:alternatives>\n <\/jats:inline-formula>\n 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method.\n OligoSeq<\/jats:italic>\n can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC\/MS.\n <\/jats:p>","DOI":"10.1371\/journal.pone.0355056","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:47:06Z","timestamp":1788457626000},"page":"e0355056","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides"],"prefix":"10.1371","volume":"21","author":[{"given":"Robert","family":"Ramirez-Garcia","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9170-7733","authenticated-orcid":true,"given":"Akashaditya","family":"Das","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrycja","family":"Sokolowska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7470-2123","authenticated-orcid":true,"given":"Thomas","family":"Heinis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"issue":"22","key":"pone.0355056.ref001","doi-asserted-by":"crossref","first-page":"10700","DOI":"10.1073\/pnas.90.22.10700","article-title":"Generation and screening of an oligonucleotide-encoded synthetic peptide library","volume":"90","author":"MC Needels","year":"1993","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0355056.ref002","article-title":"Direct mutagenesis of thousands of genomic targets using microarray-derived oligonucleotides","author":"MT Bonde","year":"2014","journal-title":"Proceedings of the National Academy of Sciences of the United States of America"},{"key":"pone.0355056.ref003","article-title":"Adaptive sampling in nanopore sequencing for PCR-free random access in DNA data storage","author":"R Sokolovskii","year":"2024","journal-title":"bioRxiv"},{"issue":"5","key":"pone.0355056.ref004","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1038\/88107","article-title":"Creation of genome-wide protein expression libraries using random activation of gene expression","volume":"19","author":"JJ Harrington","year":"2001","journal-title":"Nat Biotechnol"},{"issue":"8","key":"pone.0355056.ref005","doi-asserted-by":"crossref","first-page":"3173","DOI":"10.1021\/ac900100x","article-title":"Sequencing of single and double stranded RNA oligonucleotides by acid hydrolysis and MALDI mass spectrometry","volume":"81","author":"U Bahr","year":"2009","journal-title":"Anal Chem"},{"issue":"17","key":"pone.0355056.ref006","doi-asserted-by":"crossref","first-page":"11154","DOI":"10.1021\/acs.analchem.9b01858","article-title":"Sequencing of Phosphoramidate Oligonucleotides by Acid Hydrolysis and Mass Spectrometry","volume":"91","author":"S Pourshahian","year":"2019","journal-title":"Anal Chem"},{"issue":"10","key":"pone.0355056.ref007","doi-asserted-by":"crossref","first-page":"2490","DOI":"10.1021\/jasms.4c00281","article-title":"Sequencing of Phosphorodiamidate Morpholino Oligomers by Hydrophilic Interaction Chromatography Coupled to Tandem Mass Spectrometry","volume":"35","author":"M Wang","year":"2024","journal-title":"J Am Soc Mass Spectrom"},{"key":"pone.0355056.ref008","unstructured":"Pacific Biosciences. Pacific Biosciences Template Preparation and Sequencing. 2014."},{"key":"pone.0355056.ref009","author":"Oxford Nanopore Technologies","year":"2022"},{"issue":"1","key":"pone.0355056.ref010","article-title":"Best Practices for Illumina Library Preparation","volume":"102","author":"IF Bronner","year":"2019","journal-title":"Curr Protoc Hum Genet"},{"issue":"1","key":"pone.0355056.ref011","doi-asserted-by":"crossref","first-page":"421","DOI":"10.2144\/btn-2019-0161","article-title":"Emulsion PCR made easy","volume":"69","author":"V Verma","year":"2020","journal-title":"Biotechniques"},{"key":"pone.0355056.ref012","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/978-1-4939-6343-0_5","article-title":"SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution","volume":"1472","author":"A Currin","year":"2017","journal-title":"Methods Mol Biol"},{"issue":"7082","key":"pone.0355056.ref013","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1038\/nature04586","article-title":"Folding DNA to create nanoscale shapes and patterns","volume":"440","author":"Rothemund PWK","year":"2006","journal-title":"Nature"},{"issue":"8","key":"pone.0355056.ref014","first-page":"2567","article-title":"Target amplification by the polymerase chain reaction","volume":"83","author":"KB Mullis","year":"1986","journal-title":"Proceedings of the National Academy of Sciences"},{"key":"pone.0355056.ref015","first-page":"263","article-title":"Specific enzymatic amplification of DNA in vitro","volume-title":"Cold Spring Harbor Symposia on Quantitative Biology","author":"KB Mullis","year":"1986"},{"issue":"1","key":"pone.0355056.ref016","doi-asserted-by":"crossref","first-page":"9663","DOI":"10.1038\/s41598-019-45832-6","article-title":"A Characterization of the DNA Data Storage Channel","volume":"9","author":"R Heckel","year":"2019","journal-title":"Sci Rep"},{"issue":"7","key":"pone.0355056.ref017","doi-asserted-by":"crossref","first-page":"2272","DOI":"10.1093\/bioinformatics\/btz921","article-title":"Logomaker: beautiful sequence logos in Python","volume":"36","author":"A Tareen","year":"2020","journal-title":"Bioinformatics"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0355056","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T17:47:10Z","timestamp":1788457630000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0355056"}},"subtitle":[],"editor":[{"given":"Zheng","family":"Yuan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":17,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9,3]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0355056","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}