PT  - JOURNAL ARTICLE
AU  - Laurens Lambrechts
AU  - Noah Bonine
AU  - Rita Verstraeten
AU  - Marion Pardons
AU  - Ytse Noppe
AU  - Sofie Rutsaert
AU  - Filip Van Nieuwerburgh
AU  - Wim Van Criekinge
AU  - Basiel Cole
AU  - Linos Vandekerckhove
TI  - HIV-PULSE: A long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization
AID  - 10.1101/2023.01.18.524396
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.01.18.524396
4099  - http://biorxiv.org/content/early/2023/01/19/2023.01.18.524396.short
4100  - http://biorxiv.org/content/early/2023/01/19/2023.01.18.524396.full
AB  - 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 1,308 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, though 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.Competing Interest StatementL.L. has received a travel grant from Oxford Nanopore Technologies (ONT) to present his findings at a scientific meeting.