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. 2023 Nov 10;51(20):e102.
doi: 10.1093/nar/gkad790.

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

Laurens Lambrechts  1   2 , Noah Bonine  1   2 , Rita Verstraeten  1   2 , Marion Pardons  1 , Ytse Noppe  1 , Sofie Rutsaert  1 , Filip Van Nieuwerburgh  3 , Wim Van Criekinge  2 , Basiel Cole  1 , Linos Vandekerckhove  1
Affiliations

HIV-PULSE: a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

Laurens Lambrechts et al. Nucleic Acids Res. .

Abstract

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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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
HIV-PULSE methodology overview and performance evaluation. (A) Schematic overview of the HIV-PULSE assay. A PCR reaction with bulk DNA containing multiple HIV-1 templates is pre-amplified using outer HIV-1 primers for a limited number of cycles to improve sensitivity. Next, pre-amplified material is tagged with a dual barcode consisting of a unique molecular identifier (UMI) attached to both ends using an HIV-1 specific inner primer. To generate enough material for long-read library preparation, the tagged material is amplified with synthetic primers in several PCR rounds followed by clean up to prevent length bias. The illustration of the microtube was obtained from SMART (Servier Medical Art; http://smart.servier.com/), licensed under a Creative Common Attribution 3.0 Unported license (https://creativecommons.org/licenses/by/3.0/). (B) Success rate of the HIV-PULSE assay for different input ratios of HIV-1 with varying number of PCR cycles during pre-amplification. Each data point represents the mean of triplicate experiments for each condition. (C) Mean accuracy of HIV-PULSE bin sequences with increasing bin coverage compared to the Illumina reference sequence. The dashed line indicates the Q30 (99.9% accuracy) threshold. (D) Mean number of errors (insertions, deletions and mismatches) found in HIV-PULSE bin sequences of 9.5 kb with increasing bin coverage compared to the Illumina reference sequence.
Figure 2.
Figure 2.
Benchmarking assays: novel HIV-PULSE vs gold standard FLIPS. (A) Donut plots displaying the fraction of unique and presumed clonal proviral sequences detected in each participant for both assays. The number of distinct proviruses generated by each assay is shown in the middle of each donut. The matching colored slices indicate the 6 out of 16 overlapping expansions of identical sequences (EIS) found to be clonal in both assays. A Fisher's exact test was used to compare the proportions of unique proviruses between both assays for each participant, none were significantly different (P = 1.00, P = 0.3716, P = 1.00, P = 1.00 for P03, P12_T1, P12_T2 and P14, respectively). (B) Size distributions of the observed proviral genome lengths for each assay. No significant difference was observed between both assays using a Mann–Whitney U-test (P = 0.08106). Each dot represents a single distinct provirus and is given a color for each participant. (C) For each assay and participant, the percentage of detected proviruses out of the total HIV-1 DNA reservoir size is shown. Assay efficiencies were compared for significance using a Mann–Whitney U-test (P = 0.3429).
Figure 3.
Figure 3.
Proviral reservoir as assayed by the HIV-PULSE assay. (A) The proportions of different proviral classes observed among the distinct proviruses for each participant. On the right the number of total and distinct proviruses is displayed for each participant. (B) A phylogenetic tree including the distinct genome intact sequences. Each participant is shown as different colored dots, empty symbols indicate sequences only found once (unique, white insert) in a PCR replicate. The number of included intact genomes per participant are shown between brackets in the legend.
Figure 4.
Figure 4.
Benchmarking of HIV-PULSE vs STIP-Seq assay. (A) Phylogenetic tree including all distinct proviruses obtained with the HIV-PULSE (excluding sequences with inversions, large deletions and hypermutations) and STIP-Seq assays for eight participants. Symbols reflect the different assays, proviruses only recovered in a single assay are shown in grey while assay overlapping are shown in red (STIP-Seq) or blue (HIV-PULSE). Empty symbols indicate sequences were found once (unique, white insert) in that respective assay. The outer and inner circles indicate for each provirus, respectively, the participant origin and associated HIV-1 genome classification. The number of included genomes per participant are shown between brackets in the legend. (B) UpSet-plot visualizing the number of overlaps between clonal and unique proviruses recovered with each respective assays.
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