{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T09:10:18Z","timestamp":1783933818427,"version":"3.55.0"},"reference-count":33,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:00Z","timestamp":1783728000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002855","name":"National Key R&D Program of China","doi-asserted-by":"publisher","award":["2022YFF1001400"],"award-info":[{"award-number":["2022YFF1001400"]}],"id":[{"id":"10.13039\/501100002855","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Molecular characterization of the transferred DNA (T-DNA) insertion sites is required for the safety assessment of genetically modified (GM) crops, yet conventional PCR-based methods are labor-intensive and limited in their ability to resolve complex structural variations. We present T-DNA Analyzer, an integrated bioinformatics pipeline that transforms long-read sequencing data (PacBio HiFi or Oxford Nanopore) into a comprehensive insertion site report. The pipeline implements a host-derived read filter that subtracts host-homologous vector regions to eliminate false-positive chimeric read calls; a multi-segment fusion detection algorithm that resolves complex T-DNA integration architectures; and a deletion gap gene impact analysis that identifies genes affected by host genome deletions at the integration site. Validation on maize and cotton datasets demonstrated that the host-derived filter excluded 86.4% of false-positive reads while retaining all true chimeric reads, and the fusion detection algorithm successfully reconstructed a two-copy tandem T-DNA repeat within a single long read. T-DNA Analyzer provides automated, reproducible molecular characterization designed to support regulatory molecular characterization and is freely available as open-source software.<\/jats:p>","DOI":"10.3390\/ijms27146201","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T08:53:58Z","timestamp":1783932838000},"page":"6201","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["T-DNA Analyzer: A Long-Read Sequencing Pipeline for Characterizing T-DNA Insertion Sites in Transgenic Crops"],"prefix":"10.3390","volume":"27","author":[{"given":"Yue","family":"Wan","sequence":"first","affiliation":[{"name":"State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Ya","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-Fan","family":"Yu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhan-Feng","family":"Si","sequence":"additional","affiliation":[{"name":"The Rural Development Academy, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Cheng","family":"Shen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3513-2583","authenticated-orcid":false,"given":"Yu-Xuan","family":"Ye","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Rice Biology and Breeding, Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China"},{"name":"The Rural Development Academy, Zhejiang University, Hangzhou 310058, China"},{"name":"Zhejiang University Zhongyuan Institute, Zhengzhou 450000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"EFSA Panel on Genetically Modified Organisms (GMO) (2010). Guidance on the environmental risk assessment of genetically modified plants. EFSA J., 8, 1879.","DOI":"10.2903\/j.efsa.2010.1879"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kovalic, D., Garnaat, C., Guo, L., Yan, Y., Groat, J., Silvanovich, A., Ralston, L., Huang, M., Tian, Q., and Christian, A. (2012). The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. Plant Genome, 5.","DOI":"10.3835\/plantgenome2012.10.0026"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1080\/21645698.2020.1773198","article-title":"Environmental impacts of genetically modified (GM) crop use 1996\u20132018: Impacts on pesticide use and carbon emissions","volume":"11","author":"Brookes","year":"2020","journal-title":"GM Crops Food"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Gelvin, S.B. (2021). Plant DNA repair and Agrobacterium T\u2212DNA integration. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22168458"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1016\/j.tig.2004.06.004","article-title":"Agrobacterium T-DNA integration: Molecules and models","volume":"20","author":"Tzfira","year":"2004","journal-title":"Trends Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1016\/j.plantsci.2006.03.019","article-title":"Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants","volume":"171","author":"Zhu","year":"2006","journal-title":"Plant Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1046\/j.1365-313X.1999.t01-1-00602.x","article-title":"The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration","volume":"20","author":"Jacobs","year":"1999","journal-title":"Plant J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1023\/A:1023929630687","article-title":"A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome","volume":"52","author":"Forsbach","year":"2003","journal-title":"Plant Mol. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Pucker, B., Kleinb\u00f6lting, N., and Weisshaar, B. (2021). Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genom., 22.","DOI":"10.1186\/s12864-021-07877-8"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"649","DOI":"10.2144\/000112601","article-title":"High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences","volume":"43","author":"Liu","year":"2007","journal-title":"Biotechniques"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1093\/genetics\/120.3.621","article-title":"Genetic applications of an inverse polymerase chain reaction","volume":"120","author":"Ochman","year":"1988","journal-title":"Genetics"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Guo, B., Guo, Y., Hong, H., and Qiu, L.-J. (2016). Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method. Front. Plant Sci., 7.","DOI":"10.3389\/fpls.2016.01009"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"100238","DOI":"10.1016\/j.fochms.2024.100238","article-title":"Deciphering the complex molecular architecture of the genetically modified soybean FG72 through paired-end whole genome sequencing","volume":"10","author":"Wang","year":"2025","journal-title":"Food Chem. Mol. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Magembe, E.M., Li, H., Taheri, A., Zhou, S., and Ghislain, M. (2023). Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing. Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1156665"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1038\/s41592-018-0001-7","article-title":"Accurate detection of complex structural variations using single-molecule sequencing","volume":"15","author":"Sedlazeck","year":"2018","journal-title":"Nat. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1038\/s41587-019-0217-9","article-title":"Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome","volume":"37","author":"Wenger","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1186\/s13059-016-1103-0","article-title":"The Oxford Nanopore MinION: Delivery of nanopore sequencing to the genomics community","volume":"17","author":"Jain","year":"2016","journal-title":"Genome Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"31921","DOI":"10.1038\/s41598-024-83403-6","article-title":"Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology","volume":"14","author":"Liu","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"15144","DOI":"10.1038\/s41598-020-71614-6","article-title":"Targeted MinION sequencing of transgenes","volume":"10","author":"Boutigny","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"964","DOI":"10.1111\/pbi.13776","article-title":"LIFE-Seq: A universal L arge I ntegrated DNA F ragment E nrichment Seq uencing strategy for deciphering the transgene integration of genetically modified organisms","volume":"20","author":"Zhang","year":"2022","journal-title":"Plant Biotechnol. J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1186\/s13059-021-02396-x","article-title":"Chromosome-level genome assembly of a regenerable maize inbred line A188","volume":"22","author":"Lin","year":"2021","journal-title":"Genome Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4102","DOI":"10.1111\/pbi.70617","article-title":"Transgenic Lepidopteran-Pests-Resistant and Herbicide-Tolerant Cotton Through Transfer of Cry1Ab-vip3Aa and Cp4-epsps+ bar Genes","volume":"24","author":"Mou","year":"2026","journal-title":"Plant Biotechnol. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1038\/s41588-025-02115-3","article-title":"Post-polyploidization centromere evolution in cotton","volume":"57","author":"Yan","year":"2025","journal-title":"Nat. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"296","DOI":"10.4161\/gmcr.21406","article-title":"Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants","volume":"3","author":"Podevin","year":"2012","journal-title":"GM Crops Food"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2061","DOI":"10.1104\/pp.103.027532","article-title":"T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences","volume":"133","author":"Windels","year":"2003","journal-title":"Plant Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s11248-014-9843-7","article-title":"A comparative analysis of insertional effects in genetically engineered plants: Considerations for pre-market assessments","volume":"24","author":"Schnell","year":"2015","journal-title":"Transgenic Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e20314","DOI":"10.1002\/tpg2.20314","article-title":"Benchmarking Oxford Nanopore read alignment-based insertion and deletion detection in crop plant genomes","volume":"16","author":"Yildiz","year":"2023","journal-title":"Plant Genome"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"722","DOI":"10.1101\/gr.215087.116","article-title":"Canu: Scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation","volume":"27","author":"Koren","year":"2017","journal-title":"Genome Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1038\/s41587-019-0072-8","article-title":"Assembly of long, error-prone reads using repeat graphs","volume":"37","author":"Kolmogorov","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_31","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":"ref_32","doi-asserted-by":"crossref","first-page":"1422","DOI":"10.1093\/bioinformatics\/btp163","article-title":"Biopython: Freely available Python tools for computational molecular biology and bioinformatics","volume":"25","author":"Cock","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1038\/nmeth.1618","article-title":"Points of view: Color blindness","volume":"8","author":"Wong","year":"2011","journal-title":"Nat. Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6201\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T08:56:27Z","timestamp":1783932987000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6201"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,11]]},"references-count":33,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ijms27146201"],"URL":"https:\/\/doi.org\/10.3390\/ijms27146201","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,11]]}}}