{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T19:31:41Z","timestamp":1780515101441,"version":"3.54.1"},"reference-count":18,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2024,9,14]],"date-time":"2024-09-14T00:00:00Z","timestamp":1726272000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100021856","name":"Ministero dell\u2019Universit\u00e0 e della Ricerca","doi-asserted-by":"crossref","award":["202074AA2B"],"award-info":[{"award-number":["202074AA2B"]}],"id":[{"id":"10.13039\/501100021856","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. Materials and Methods: We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. Results: The samples had a median 4.8 log [IQR 4.4\u20135.2] HIV-1 RNA copies\/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2\/28) compared with AD4SEQ (13\/28) (p &lt; 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13\/28 and 9\/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. Conclusions: Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage.<\/jats:p>","DOI":"10.3390\/v16091465","type":"journal-article","created":{"date-parts":[[2024,9,16]],"date-time":"2024-09-16T04:14:07Z","timestamp":1726460047000},"page":"1465","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A Comparison of Sanger Sequencing and Amplicon-Based Next Generation Sequencing Approaches for the Detection of HIV-1 Drug Resistance Mutations"],"prefix":"10.3390","volume":"16","author":[{"given":"Camilla","family":"Biba","sequence":"first","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lia","family":"Fiaschi","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilenia","family":"Varasi","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1018-9545","authenticated-orcid":false,"given":"Chiara","family":"Paletti","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2314-1434","authenticated-orcid":false,"given":"Niccol\u00f2","family":"Bartolini","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0344-6281","authenticated-orcid":false,"given":"Maurizio","family":"Zazzi","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4306-2960","authenticated-orcid":false,"given":"Ilaria","family":"Vicenti","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9934-377X","authenticated-orcid":false,"given":"Francesco","family":"Saladini","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e202","DOI":"10.1016\/S2352-3018(22)00327-7","article-title":"We Should Not Stop Considering HIV Drug Resistance Testing at Failure of First-Line Antiretroviral Therapy","volume":"10","author":"Kantor","year":"2022","journal-title":"Lancet HIV"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"13596535231201162","DOI":"10.1177\/13596535231201162","article-title":"HIV Drug Resistance in the Era of Contemporary Antiretroviral Therapy: A Clinical Perspective","volume":"28","author":"Carr","year":"2023","journal-title":"Antivir. Ther."},{"key":"ref_3","first-page":"56","article-title":"Genetic Barrier to Resistance for Dolutegravir","volume":"17","author":"Llibre","year":"2015","journal-title":"AIDS Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e733","DOI":"10.1016\/S2352-3018(23)00228-X","article-title":"HIV-1 Drug Resistance in People on Dolutegravir-Based Antiretroviral Therapy: A Collaborative Cohort Analysis","volume":"10","author":"Loosli","year":"2023","journal-title":"Lancet HIV"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6824","DOI":"10.1128\/AAC.01490-15","article-title":"HIV Drug Resistance Testing by High-Multiplex \u201cWide\u201d Sequencing on the MiSeq Instrument","volume":"59","author":"Lapointe","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6711","DOI":"10.2147\/IDR.S387215","article-title":"HIV-1 Genotypic Resistance Testing Using Sanger and Next-Generation Sequencing in Adults with Low-Level Viremia in China","volume":"15","author":"Li","year":"2022","journal-title":"Infect. Drug Resist."},{"key":"ref_7","first-page":"1000106","article-title":"Accuracy of Next Generation Sequencing Platforms","volume":"1","author":"Fox","year":"2014","journal-title":"Next Gener Seq. Appl."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4","DOI":"10.20892\/j.issn.2095-3941.2018.0055","article-title":"Next-Generation Sequencing and Its Clinical Application","volume":"16","author":"Qin","year":"2019","journal-title":"Cancer Biol. Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Ode, H., Matsuda, M., Matsuoka, K., Hachiya, A., Hattori, J., Kito, Y., Yokomaku, Y., Iwatani, Y., and Sugiura, W. (2015). Quasispecies Analyses of the HIV-1 Near-Full-Length Genome with Illumina MiSeq. Front. Microbiol., 6.","DOI":"10.3389\/fmicb.2015.01258"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.virusres.2016.12.008","article-title":"Promises and Pitfalls of Illumina Sequencing for HIV Resistance Genotyping","volume":"239","author":"Brumme","year":"2017","journal-title":"Virus Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e44","DOI":"10.1093\/nar\/gku739","article-title":"COMET: Adaptive Context-Based Modeling for Ultrafast HIV-1 Subtype Identification","volume":"42","author":"Struck","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"\u00c1vila-R\u00edos, S., Parkin, N., Swanstrom, R., Paredes, R., Shafer, R., Ji, H., and Kantor, R. (2020). Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations. Viruses, 12.","DOI":"10.3390\/v12060617"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Bonifacio, M.A., Genchi, C., Lagioia, A., Talamo, V., Volpe, A., and Mariggi\u00f2, M.A. (2022). Analytical Assessment of the Vela Diagnostics NGS Assay for HIV Genotyping and Resistance Testing: The Apulian Experience. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052727"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Armenia, D., Carioti, L., Micheli, V., Bon, I., Allice, T., Bonura, C., Bruzzone, B., Bracchitta, F., Cerutti, F., and Giammanco, G.M. (2024). Comparison of Different HIV-1 Resistance Interpretation Tools for Next-Generation Sequencing in Italy. Viruses, 16.","DOI":"10.3390\/v16091422"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3510","DOI":"10.1093\/jac\/dkaa352","article-title":"Performance of a High-Throughput next-Generation Sequencing Method for Analysis of HIV Drug Resistance and Viral Load","volume":"75","author":"Fogel","year":"2020","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3627","DOI":"10.1002\/jmv.26582","article-title":"Investigation of Drug Resistance against Protease, Reverse Transcriptase, and Integrase Inhibitors by next-Generation Sequencing in HIV-Positive Patients","volume":"93","author":"Tekin","year":"2021","journal-title":"J. Med. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e00382-20","DOI":"10.1128\/JCM.00382-20","article-title":"A Comprehensive Genomics Solution for HIV Surveillance and Clinical Monitoring in Low-Income Settings","volume":"58","author":"Bonsall","year":"2020","journal-title":"J. Clin. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1152","DOI":"10.1093\/jac\/dky003","article-title":"HIV-1 Genotypic Resistance Testing Using the Vela Automated next-Generation Sequencing Platform","volume":"73","author":"Raymond","year":"2018","journal-title":"J. Antimicrob. Chemother."}],"updated-by":[{"DOI":"10.3390\/v17081059","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2024,9,14]],"date-time":"2024-09-14T00:00:00Z","timestamp":1726272000000}}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/9\/1465\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,8]],"date-time":"2025-08-08T08:13:56Z","timestamp":1754640836000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/16\/9\/1465"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,14]]},"references-count":18,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["v16091465"],"URL":"https:\/\/doi.org\/10.3390\/v16091465","relation":{"correction":[{"id-type":"doi","id":"10.3390\/v17081059","asserted-by":"object"}]},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,9,14]]}}}