Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations

Retroviruses, like other RNA viruses, mutate at very high rates and exist as genetically heterogeneous populations. The error-prone activity of viral reverse transcriptase (RT) is largely responsible for the observed variability, most notably in HIV-1. In addition, RTs are widely used in biotechnolo...

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Autores: Martínez del Río, Javier, Menéndez-Arias, Luis
Tipo de recurso: artículo
Fecha de publicación:2025
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/379511
Acceso en línea:http://hdl.handle.net/10261/379511
Access Level:acceso abierto
Palabra clave:Reverse transcriptase
Next-generation sequencing
Fidelity of DNA synthesis
Retrovirus
cDNA synthesis
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spelling Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and LimitationsMartínez del Río, JavierMenéndez-Arias, LuisReverse transcriptaseNext-generation sequencingFidelity of DNA synthesisRetroviruscDNA synthesisRetroviruses, like other RNA viruses, mutate at very high rates and exist as genetically heterogeneous populations. The error-prone activity of viral reverse transcriptase (RT) is largely responsible for the observed variability, most notably in HIV-1. In addition, RTs are widely used in biotechnology to detect RNAs and to clone expressed genes, among many other applications. The fidelity of retroviral RTs has been traditionally analyzed using enzymatic (gel-based) or reporter-based assays. However, these methods are laborious and have important limitations. The development of next-generation sequencing (NGS) technologies opened the possibility of obtaining reverse transcription error rates from a large number of sequences, although appropriate protocols had to be developed. In this review, we summarize the developments in this field that allowed the determination of RNA-dependent DNA synthesis error rates for different RTs (viral and non-viral), including methods such as PRIMER IDs, REP-SEQ, ARC-SEQ, CIR-SEQ, SMRT-SEQ and ROLL-SEQ. Their advantages and limitations are discussed. Complementary DNA (cDNA) synthesis error rates obtained in different studies, using RTs and RNAs of diverse origins, are presented and compared. Future improvements in methodological pipelines will be needed for the precise identification of mutations in the RNA template, including modified bases.This research was funded by the Spanish Ministry of Science, Innovation and Universities (grant PID2022-136725OB-I00/AEI/10.13039/501100011033) and FEDER “A way to make Europe”, and an institutional grant of Fundación Ramón Areces (Madrid, Spain) to the CBM. The CBM has been certified as Severo Ochoa Center of Excellence by AEI/MCI/10.13039/501100011033 since 2023. L.M.-A. is a member of the Global Virus Network.Peer reviewedMultidisciplinary Digital Publishing InstituteMinisterio de Ciencia, Innovación y Universidades (España)Agencia Estatal de Investigación (España)Fundación Ramón ArecesEuropean CommissionMartínez del Río, Javier [0000-0002-9970-7596]Menéndez-Arias, Luis [0000-0002-1251-6640]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2025202520252025info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501application/pdfhttp://hdl.handle.net/10261/379511reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2022-136725OB-I00https://doi.org/10.3390/v17020173Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3795112026-05-22T06:33:51Z
dc.title.none.fl_str_mv Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
title Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
spellingShingle Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
Martínez del Río, Javier
Reverse transcriptase
Next-generation sequencing
Fidelity of DNA synthesis
Retrovirus
cDNA synthesis
title_short Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
title_full Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
title_fullStr Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
title_full_unstemmed Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
title_sort Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations
dc.creator.none.fl_str_mv Martínez del Río, Javier
Menéndez-Arias, Luis
author Martínez del Río, Javier
author_facet Martínez del Río, Javier
Menéndez-Arias, Luis
author_role author
author2 Menéndez-Arias, Luis
author2_role author
dc.contributor.none.fl_str_mv Ministerio de Ciencia, Innovación y Universidades (España)
Agencia Estatal de Investigación (España)
Fundación Ramón Areces
European Commission
Martínez del Río, Javier [0000-0002-9970-7596]
Menéndez-Arias, Luis [0000-0002-1251-6640]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Reverse transcriptase
Next-generation sequencing
Fidelity of DNA synthesis
Retrovirus
cDNA synthesis
topic Reverse transcriptase
Next-generation sequencing
Fidelity of DNA synthesis
Retrovirus
cDNA synthesis
description Retroviruses, like other RNA viruses, mutate at very high rates and exist as genetically heterogeneous populations. The error-prone activity of viral reverse transcriptase (RT) is largely responsible for the observed variability, most notably in HIV-1. In addition, RTs are widely used in biotechnology to detect RNAs and to clone expressed genes, among many other applications. The fidelity of retroviral RTs has been traditionally analyzed using enzymatic (gel-based) or reporter-based assays. However, these methods are laborious and have important limitations. The development of next-generation sequencing (NGS) technologies opened the possibility of obtaining reverse transcription error rates from a large number of sequences, although appropriate protocols had to be developed. In this review, we summarize the developments in this field that allowed the determination of RNA-dependent DNA synthesis error rates for different RTs (viral and non-viral), including methods such as PRIMER IDs, REP-SEQ, ARC-SEQ, CIR-SEQ, SMRT-SEQ and ROLL-SEQ. Their advantages and limitations are discussed. Complementary DNA (cDNA) synthesis error rates obtained in different studies, using RTs and RNAs of diverse origins, are presented and compared. Future improvements in methodological pipelines will be needed for the precise identification of mutations in the RNA template, including modified bases.
publishDate 2025
dc.date.none.fl_str_mv 2025
2025
2025
2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/379511
url http://hdl.handle.net/10261/379511
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2021-2023/PID2022-136725OB-I00
https://doi.org/10.3390/v17020173
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
publisher.none.fl_str_mv Multidisciplinary Digital Publishing Institute
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
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repository.mail.fl_str_mv
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