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

Publisher: MDPI Country of Publication: Switzerland NLM ID: 101509722 Publication Model: Electronic Cited Medium: Internet ISSN: 1999-4915 (Electronic) Linking ISSN: 19994915 NLM ISO Abbreviation: Viruses Subsets: MEDLINE

Original Publication: Basel, Switzerland : MDPI

High-Throughput Nucleotide Sequencing*/methods ; RNA-Directed DNA Polymerase*/genetics ; RNA-Directed DNA Polymerase*/metabolism ; Retroviridae*/genetics ; Retroviridae*/enzymology; RNA, Viral/genetics ; Humans ; Reverse Transcription ; Mutation

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.

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PID2022-136725OB-I00/AEI/10.13039/501100011033 Ministerio de Ciencia, Innovación y Universidades

Keywords: cDNA synthesis; fidelity of DNA synthesis; next-generation sequencing; retrovirus; reverse transcriptase

EC 2.7.7.49 (RNA-Directed DNA Polymerase)
0 (RNA, Viral)

Date Created: 20250226 Date Completed: 20250226 Latest Revision: 20250228

20250228

PMC11861041

10.3390/v17020173

40006928