{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T08:10:43Z","timestamp":1779351043794,"version":"3.51.4"},"reference-count":18,"publisher":"Oxford University Press (OUP)","issue":"10","license":[{"start":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T00:00:00Z","timestamp":1779321600000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["Ku563\/17-1"],"award-info":[{"award-number":["Ku563\/17-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"name":"European Molecular Biology Laboratory Heidelberg"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,5,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Custom oligonucleotides (oligos) play a crucial role in molecular biology applications, including sequencing, polymerase chain reaction (PCR), and library construction. For highly sensitive techniques such as next-generation sequencing (NGS) library preparation, oligos must exhibit exceptional purity and accuracy. Here, we provide a perspective on the presence of unrelated, contaminating sequences in custom-made oligos using a case study of an RNA\u2013protein crosslinked immunoprecipitation (CLIP-seq) experiment. These contaminants led to artefactual results in NGS data, rendering their results uninterpretable. Systematic investigation identified a contaminating human immunodeficiency virus (HIV) gag sequence embedded between Illumina-compatible adapter sequences, forming a functional \u2018library-structured\u2019 molecule capable of amplification and sequencing. Additionally, other nonspecific sequences, including fragments from unrelated oligos, were detected. By sharing our analysis strategy and outcomes, we aim to raise awareness within the research community and emphasize the need for more stringent quality controls in oligo synthesis and validation by commercial suppliers, safeguarding academic experimenters\u2019 time and resources. Furthermore, this perspective provides a systematic approach for the identification of the source of contaminating sequences in custom-made oligos designed for NGS library preparations.<\/jats:p>","DOI":"10.1093\/nar\/gkag433","type":"journal-article","created":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T11:37:46Z","timestamp":1776944266000},"source":"Crossref","is-referenced-by-count":0,"title":["Cross-contamination of commercial oligonucleotides with library-structured sequences"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1100-4790","authenticated-orcid":false,"given":"Manouk","family":"Gerritsen","sequence":"first","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]},{"name":"Molecular Medicine Partnership Unit (MMPU) , Im Neuenheimer Feld 350, 69120 Heidelberg ,","place":["Germany"]},{"name":"Collaboration for joint PhD degree between EMBL and Faculty of Biosciences, Heidelberg University , 69120 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8795-3060","authenticated-orcid":false,"given":"Partho Sarothi","family":"Ray","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]},{"name":"Molecular Medicine Partnership Unit (MMPU) , Im Neuenheimer Feld 350, 69120 Heidelberg ,","place":["Germany"]},{"name":"Department of Paediatric Oncology, Haematology and Immunology, Hopp Children\u2019s Cancer Center (KiTZ), Heidelberg University & Clinical Cooperatiuon Unit \"Pediatric Leukemia, German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 430, 69120 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8227-3691","authenticated-orcid":false,"given":"Thileepan","family":"Sekaran","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7697-7000","authenticated-orcid":false,"given":"Thomas","family":"Schwarzl","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4023-7876","authenticated-orcid":false,"given":"Matthias W","family":"Hentze","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]},{"name":"Molecular Medicine Partnership Unit (MMPU) , Im Neuenheimer Feld 350, 69120 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1953-0848","authenticated-orcid":false,"given":"Andreas E","family":"Kulozik","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory (EMBL) , Meyerhofstrasse 1, 69117 Heidelberg ,","place":["Germany"]},{"name":"Molecular Medicine Partnership Unit (MMPU) , Im Neuenheimer Feld 350, 69120 Heidelberg ,","place":["Germany"]},{"name":"Department of Paediatric Oncology, Haematology and Immunology, Hopp Children\u2019s Cancer Center (KiTZ), Heidelberg University & Clinical Cooperatiuon Unit \"Pediatric Leukemia, German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 430, 69120 Heidelberg ,","place":["Germany"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2026,5,21]]},"reference":[{"key":"2026052103575287800_B1","doi-asserted-by":"publisher","first-page":"2204","DOI":"10.1007\/s00044-024-03352-7","article-title":"Oligonucleotides: evolution and innovation","volume":"33","author":"Mohammed","year":"2024","journal-title":"Med Chem Res"},{"key":"2026052103575287800_B2","doi-asserted-by":"publisher","first-page":"146","DOI":"10.3390\/ijms25010146","article-title":"Advances in nucleic acid research: exploring the potential of oligonucleotides for therapeutic applications and biological studies","volume":"25","author":"Moccia","year":"2023","journal-title":"Int J Mol Sci"},{"key":"2026052103575287800_B3","doi-asserted-by":"publisher","first-page":"415","DOI":"10.1007\/s40005-024-00669-8","article-title":"Oligonucleotide therapeutics and their chemical modification strategies for clinical applications","volume":"54","author":"Kim","year":"2024","journal-title":"J. Pharm. Investig."},{"key":"2026052103575287800_B4","doi-asserted-by":"publisher","first-page":"354","DOI":"10.1016\/j.molcel.2018.01.005","article-title":"Advances in CLIP technologies for studies of protein\u2013RNA interactions","volume":"69","author":"Lee","year":"2018","journal-title":"Mol Cell"},{"key":"2026052103575287800_B5","doi-asserted-by":"publisher","first-page":"1212","DOI":"10.1126\/science.1090095","article-title":"CLIP identifies Nova-regulated RNA networks in the brain","volume":"302","author":"Ule","year":"2003","journal-title":"Science"},{"key":"2026052103575287800_B6","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1038\/s43586-021-00018-1","article-title":"CLIP and complementary methods","volume":"1","author":"Hafner","year":"2021","journal-title":"Nat Rev Methods Primers"},{"key":"2026052103575287800_B7","doi-asserted-by":"publisher","first-page":"7921","DOI":"10.1038\/ncomms8921","article-title":"Improved binding site assignment by high-resolution mapping of RNA\u2013protein interactions using iCLIP","volume":"6","author":"Hauer","year":"2015","journal-title":"Nat Commun"},{"key":"2026052103575287800_B8","doi-asserted-by":"publisher","DOI":"10.1101\/125724v1","article-title":"Index switching causes \u201cspreading-of-signal\u201d among multiplexed samples in Illumina HiSeq 4000 DNA sequencing","author":"Sinha","year":"2017"},{"key":"2026052103575287800_B9","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1186\/s12864-017-4428-5","article-title":"Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing","volume":"19","author":"MacConaill","year":"2018","journal-title":"BMC Genomics [Electronic Resource]"},{"key":"2026052103575287800_B10","doi-asserted-by":"publisher","first-page":"3137","DOI":"10.1093\/nar\/gkae068","article-title":"Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides","volume":"52","author":"Arakawa","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"2026052103575287800_B11","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1038\/s41596-022-00680-z","article-title":"Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq","volume":"17","author":"Blue","year":"2022","journal-title":"Nat Protoc"},{"key":"2026052103575287800_B12","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1186\/s13100-015-0041-9","article-title":"Repbase Update, a database of repetitive elements in eukaryotic genomes","volume":"6","author":"Bao","year":"2015","journal-title":"Mobile DNA"},{"key":"2026052103575287800_B13","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1016\/j.tim.2012.11.006","article-title":"HIV Gag polyprotein: processing and early viral particle assembly","volume":"21","author":"Bell","year":"2013","journal-title":"Trends Microbiol"},{"key":"2026052103575287800_B14","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/978-1-4939-7204-3_14","article-title":"Robust, cost-effective profiling of RNA binding protein targets with single-end enhanced crosslinking and immunoprecipitation (seCLIP)","volume":"1648","author":"Van\u00a0Nostrand","year":"2017","journal-title":"Methods Mol Biol"},{"key":"2026052103575287800_B15","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb0211s42","article-title":"Synthesis and purification of oligonucleotides","author":"Ellington","year":"2001","journal-title":"Curr Protoc Mol Biol"},{"key":"2026052103575287800_B16","article-title":"A new oligonucleotide synthesis process for high quality oligonucleotides with superior performance in NGS applications","author":"Max\u00a0Leopold"},{"key":"2026052103575287800_B17","doi-asserted-by":"publisher","first-page":"662","DOI":"10.1128\/MCB.00970-14","article-title":"The overlooked fact: fundamental need for spike-in control for virtually all genome-wide analyses","volume":"36","author":"Chen","year":"2016","journal-title":"Mol Cell Biol"},{"key":"2026052103575287800_B18","doi-asserted-by":"publisher","first-page":"814","DOI":"10.1016\/j.pathol.2020.08.002","article-title":"Contamination of SARS-CoV-2 RT-PCR probes at the oligonucleotide manufacturer","volume":"52","author":"Wang","year":"2020","journal-title":"Pathology (Phila)"}],"container-title":["Nucleic Acids Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/10\/gkag433\/68350711\/gkag433.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/nar\/article-pdf\/54\/10\/gkag433\/68350711\/gkag433.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T07:58:00Z","timestamp":1779350280000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/nar\/article\/doi\/10.1093\/nar\/gkag433\/8688744"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,20]]},"references-count":18,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2026,5,20]]}},"URL":"https:\/\/doi.org\/10.1093\/nar\/gkag433","relation":{},"ISSN":["0305-1048","1362-4962"],"issn-type":[{"value":"0305-1048","type":"print"},{"value":"1362-4962","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,6,10]]},"published":{"date-parts":[[2026,5,20]]},"article-number":"gkag433"}}