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The hypothesis guiding this study is that the 3D proximity of genes correlates with their functional interactions and relevance in prokaryotes. We introduced 3D-GeneNet, an innovative software tool that utilizes high-throughput sequencing data from chromosome conformation capture techniques and integrates topological metrics to construct gene association networks. Through a series of comparative analyses focused on spatial versus linear distances, we explored various dimensions such as topological structure, functional enrichment levels, distribution patterns of linear distances among gene pairs, and the area under the receiver operating characteristic curve by utilizing model organism Escherichia coli K-12. Furthermore, 3D-GeneNet was shown to maintain good accuracy compared to multiple algorithms (neighbourhood, co-occurrence, coexpression, and fusion) across multiple bacteria, including E. coli, Brucella abortus, and Vibrio cholerae. In addition, the accuracy of 3D-GeneNet\u2019s prediction of long-distance gene interactions was identified by bacterial two-hybrid assays on E. coli K-12 MG1655, where 3D-GeneNet not only increased the accuracy of linear genomic distance tripled but also achieved 60% accuracy by running alone. Finally, it can be concluded that the applicability of 3D-GeneNet will extend to various bacterial forms, including Gram-negative, Gram-positive, single-, and multi-chromosomal bacteria through Hi-C sequencing and analysis. Such findings highlight the broad applicability and significant promise of this method in the realm of gene association network. 3D-GeneNet is freely accessible at https:\/\/github.com\/gaoyuanccc\/3D-GeneNet.<\/jats:p>","DOI":"10.1093\/bib\/bbae320","type":"journal-article","created":{"date-parts":[[2024,7,8]],"date-time":"2024-07-08T12:31:53Z","timestamp":1720441913000},"source":"Crossref","is-referenced-by-count":1,"title":["Spatial proximity and gene function: a new dimension in prokaryotic gene association network analysis with 3D-GeneNet"],"prefix":"10.1093","volume":"25","author":[{"given":"Yuan","family":"Gao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"Hubei 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Hongshan District , Wuhan 430070, Hubei , China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huimin","family":"Gong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"Hubei Provincial Key Laboratory of Preventive Veterinary Medicine, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District , Wuhan 430070, Hubei , China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aohan","family":"Guan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"College of Veterinary Medicine, Huazhong Agricultural University , No. 1 Shizishan Street, Hongshan District, Wuhan 430070, Hubei , China"},{"name":"Hubei Provincial Key Laboratory of Preventive Veterinary Medicine, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District , Wuhan 430070, Hubei , China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kexin","family":"Hua","sequence":"additional","affiliation":[{"name":"Swine Genome and Breeding Team, Yazhouwan National Laboratory , No. 8 Huanjin Road, Yazhou District, Sanya City, Hainan Province 572024 , China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul R","family":"Langford","sequence":"additional","affiliation":[{"name":"Section of Paediatric Infectious Disease, Imperial College London, St Mary\u2019s Campus, Norfolk Place , London W2 1PG , United 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