HIV-PULSE : a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research..
A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11 vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:51 |
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| Enthalten in: |
Nucleic acids research - 51(2023), 20 vom: 10. Nov., Seite e102 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lambrechts, Laurens [VerfasserIn] |
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| Links: |
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Journal Article |
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| Anmerkungen: |
Date Completed 15.11.2023 Date Revised 28.11.2023 published: Print UpdateOf: bioRxiv. 2023 Jan 19;:. - PMID 36711686 Citation Status MEDLINE |
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| doi: |
10.1093/nar/gkad790 |
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| funding: |
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| 520 | |a A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11 vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics | ||
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| 700 | 1 | |a Verstraeten, Rita |e verfasserin |4 aut | |
| 700 | 1 | |a Pardons, Marion |e verfasserin |4 aut | |
| 700 | 1 | |a Noppe, Ytse |e verfasserin |4 aut | |
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| 700 | 1 | |a Cole, Basiel |e verfasserin |4 aut | |
| 700 | 1 | |a Vandekerckhove, Linos |e verfasserin |4 aut | |
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