{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:04:03Z","timestamp":1788433443166,"version":"build-2803163510"},"reference-count":22,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2023,1,13]],"date-time":"2023-01-13T00:00:00Z","timestamp":1673568000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Tropical Med Int Health"],"published-print":{"date-parts":[[2023,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Objectives<\/jats:title><jats:p>Low\u2010capital\u2010layout sequencing options from Oxford Nanopore Technologies (<jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content>) could assist in expanding <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content> drug resistance testing to resource\u2010limited settings. <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content> drug resistance mutations often occur as mixtures, but current <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> sequence file without intervention from skilled bioinformaticists. We therefore investigated <jats:styled-content style=\"fixed-case\">Nano\u2010RECall<\/jats:styled-content>, which provides seamless drug resistance interpretation while requiring low\u2010read coverage <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> sequence data from affordable Flongle or <jats:styled-content style=\"fixed-case\">MinION<\/jats:styled-content> flow cells and which provides mutation mixtures similar to Sanger Sequencing.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We compared Sanger sequencing to <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> sequencing of the same <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content>\u20101 subtype C polymerase chain reaction (<jats:styled-content style=\"fixed-case\">PCR<\/jats:styled-content>) amplicons, respectively using <jats:styled-content style=\"fixed-case\">RECall<\/jats:styled-content> and the novel <jats:styled-content style=\"fixed-case\">Nano\u2010RECall<\/jats:styled-content> bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content>\u20101 Genotyping Kit (<jats:styled-content style=\"fixed-case\">ThermoFisher<\/jats:styled-content>) spanning <jats:italic>protease (<jats:styled-content style=\"fixed-case\">PR<\/jats:styled-content>)<\/jats:italic> to <jats:italic>reverse transcriptase (<jats:styled-content style=\"fixed-case\">RT<\/jats:styled-content>) (<jats:styled-content style=\"fixed-case\">PR\u2010RT<\/jats:styled-content>)<\/jats:italic> (<jats:italic>n<\/jats:italic>\u00a0=\u200946) and (b) homebrew <jats:italic>integrase (<jats:styled-content style=\"fixed-case\">IN<\/jats:styled-content>)<\/jats:italic> (<jats:italic>n<\/jats:italic>\u00a0=\u200921). The agreement between Sanger sequences and <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> sequences was assessed at nucleotide level, and at codon level for Stanford <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content> drug resistance database mutations at an optimal <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> read depth of 400 reads only.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The average sequence similarity between <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> and Sanger sequences was 99.3% (95% <jats:styled-content style=\"fixed-case\">CI<\/jats:styled-content>: 99.1%\u201399.4%) for <jats:italic>PR\u2010RT<\/jats:italic> and 99.6% (95% <jats:styled-content style=\"fixed-case\">CI<\/jats:styled-content>: 99.4%\u201399.7%) for <jats:italic>INT.<\/jats:italic> Drug resistance mutations did not differ for 21 <jats:styled-content style=\"fixed-case\"><jats:italic>IN<\/jats:italic><\/jats:styled-content> specimens; 8 mutations were detected by both <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content>\u2010 and Sanger sequencing. For the 46 <jats:styled-content style=\"fixed-case\"><jats:italic>PR<\/jats:italic><\/jats:styled-content> and <jats:styled-content style=\"fixed-case\"><jats:italic>RT<\/jats:italic><\/jats:styled-content> specimens, 245 mutations were detected by either <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> or Sanger, of these 238 (97.1%) were detected by both.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>The <jats:styled-content style=\"fixed-case\">Nano\u2010RECall<\/jats:styled-content> pipeline, freely available as a downloadable application on a Windows computer, provides Sanger\u2010equivalent <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content> drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on <jats:styled-content style=\"fixed-case\">ONT<\/jats:styled-content> flow\u2010cells would contribute to making <jats:styled-content style=\"fixed-case\">HIV<\/jats:styled-content> drug resistance sequencing feasible for resource\u2010limited settings.<\/jats:p><\/jats:sec>","DOI":"10.1111\/tmi.13851","type":"journal-article","created":{"date-parts":[[2023,1,5]],"date-time":"2023-01-05T04:00:00Z","timestamp":1672891200000},"page":"186-193","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["<scp>Nano\u2010RECall<\/scp> provides an integrated pipeline for <scp>HIV<\/scp>\u20101 drug resistance testing from Oxford Nanopore sequence data"],"prefix":"10.1111","volume":"28","author":[{"given":"Kayla Eileen","family":"Delaney","sequence":"first","affiliation":[{"name":"Division of Medical Virology Stellenbosch University  Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Trevor","family":"Ngobeni","sequence":"additional","affiliation":[{"name":"Division of Medical Virology Stellenbosch University  Cape Town South Africa"},{"name":"Tygerberg Business Unit National Health Laboratory Service  Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Conan K.","family":"Woods","sequence":"additional","affiliation":[{"name":"University of British Columbia  Vancouver British Columbia Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carli","family":"Gordijn","sequence":"additional","affiliation":[{"name":"Division of Medical Virology Stellenbosch University  Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mathilda","family":"Claassen","sequence":"additional","affiliation":[{"name":"Division of Medical Virology Stellenbosch University  Cape Town South Africa"},{"name":"Tygerberg Business Unit National Health Laboratory Service  Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Urvi","family":"Parikh","sequence":"additional","affiliation":[{"name":"University of Pittsburgh  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P. Richard","family":"Harrigan","sequence":"additional","affiliation":[{"name":"University of British Columbia  Vancouver British Columbia Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gert Uves","family":"van Zyl","sequence":"additional","affiliation":[{"name":"Division of Medical Virology Stellenbosch University  Cape Town South Africa"},{"name":"Tygerberg Business Unit National Health Laboratory Service  Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,1,13]]},"reference":[{"key":"e_1_2_8_2_1","unstructured":"UNAIDS.Fast\u2010track: ending the AIDS epidemic by 2030. 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