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Single Genome Sequencing for HIV Cure Research
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HIV-1 Reservoir Characterization

Single Genome Sequencing for
HIV Cure Research

Three validated sequencing approaches for proviral reservoir characterization

If your cure research program requires sequence-level data on the HIV-1 proviral reservoir – distinguishing intact from defective proviruses, mapping clonal expansions, or identifying drug-resistance mutations – Accelevir’s Baltimore-based, CAP/CLIA-certified laboratory offers three validated sequencing approaches (NFL-SGS, bulk consensus sequencing, and EnvLRS) for proviral reservoir characterization, so your study generates the precise, regulatory-ready data needed to advance HIV cure strategies.
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Overview

HIV cure researchers need different levels of resolution depending on the scientific question. Accelevir provides three distinct sequencing services, each addressing specific aspects of proviral reservoir biology - from complete genome intactness assessment to focused envelope characterization.

Request a Technical Consultation to discuss which sequencing approach fits your study design.
Sequencing Portfolio
NFL-SGS · bulk consensus sequencing · EnvLRS · HIV-focused bioinformatics

Three validated approaches under one roof

Why Accelevir

Why HIV Cure
Researchers Trust
Accelevir

Regulated Baltimore Facility

CAP/CLIA-certified, BSL-2+ bioanalytical laboratory in Baltimore, MD capable of handling infectious disease specimens.

Ultra-Sensitive Proviral Expertise

Deep specialization in ultra-sensitive HIV proviral analysis since founding, with established workflows supporting translational and clinical cure research programs.

Submission-Ready Data Packages

Validated processes designed to generate data packages suitable for IND/BLA regulatory submissions.

Cross-Sector Track Record

Proven track record serving academic investigators, biotech sponsors, and pharma partners conducting HIV-1 persistence studies across multiple clinical phases.
Why Choose Accelevir

Why Choose Accelevir for Single Genome Sequencing

Accelevir’s differentiation stems from workflows optimized for the rarity and diversity of the HIV proviral reservoir.

Ultra-rare target recovery protects low-abundance intact proviruses from PCR dropout and amplification bias.

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Ultra-Rare Target Detection Expertise

The proviral reservoir harbors intact sequences at extremely low frequencies – often less than 10% of total proviral DNA. Accelevir’s workflows are optimized for recovering and characterizing these rare targets at single genome resolution, ensuring that low-abundance intact proviruses are not lost to PCR dropout or amplification bias.

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Three Specialized Approaches Under One Roof

Accelevir offers a comprehensive panel of HIV sequencing services to support your unique study question, each validated and optimized for its specific analytical purpose: NFL-SGS for intactness and clonality, bulk consensus sequencing for population-level variant calling, and targeted EnvLRS for envelope characterization.

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Integration with Quantitative Proviral Assays

Single genome sequencing pairs directly with the IPDA® to combine quantitative reservoir sizing with sequence-level characterization.

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Plan Your Sequencing Study with a Scientist

Tell us about your cohort, sample types, and study question – we will recommend the right sequencing approach and a validated workflow for your program.
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Our Sequencing Services

Near-Full-Length Single Genome Sequencing (NFL-SGS)

NFL-SGS amplifies and sequences individual proviral templates at limiting dilution, producing near-full-length sequences (9kb for intact HIV-1) from individual provirus genomes.
This single genome sequencing approach ensures each amplicon derives from an individual proviral molecule, eliminating the recombination artifacts and resampling errors inherent in bulk methods. The primary difference between single genome sequencing and traditional methods is resolution: NFL-SGS determines whether a specific provirus is intact or defective, identifies the exact nature and number of defects (large deletions, stop codons, APOBEC hypermutation, psi packaging signal disruptions), and reveals clonally expanded genetically identical cells carrying identical proviral sequences.

Published data illustrate the power of this approach. In a study of 9 ART-suppressed individuals, 195 proviral sequences were recovered and differentiated as intact or harboring major defects including deletions in critical ORFs and/or regulatory elements, and hypermutation-mediated introduction of stop codons in critical genome regions. Multiple populations of expanded proviral clones were identified, supporting the hypothesis that proliferation of infected cells contributes to reservoir persistence in individuals on long-term ART. Critically, the choice of bioinformatics pipeline for defect classification significantly affected how many sequences were deemed intact versus defective – underscoring the importance of standardized, validated data analysis workflows like those Accelevir employs.

NFL-SGS is essential for cure intervention studies that need to measure whether a therapy impacts the composition of the HIV proviral landscape, alters clonal composition of the proviral reservoir, or selects for drug-resistance mutations on specific provirus populations in suppressed individuals.

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Limiting-dilution amplification ensures each sequence derives from a single proviral molecule.

Our Sequencing Services

Bulk Consensus Sequencing

When your study requires population-level variant frequencies, longitudinal diversity tracking, or broad mutation surveillance across large cohorts and multiple time points, bulk consensus sequencing provides a scalable, cost-effective solution.

This approach amplifies proviral DNA (or plasma RNA) from cell populations using sensitive, high-throughput PCR and next generation sequencing platforms, generating consensus sequences that capture the dominant variants and detect minority species above defined frequency thresholds.

Bulk sequencing reveals how HIV reservoir sequence diversity contributes to sustained control of the virus in some individuals, even in the absence of ART. In an analysis of 18 post-treatment controllers and non-controllers, bulk proviral sequencing analysis revealed lower sequence diversity in key CD8+ T cell epitopes in post-treatment controllers, demonstrating that viral reservoir characteristics such as epitope conservation contribute to virologic control in the absence of ART.

Bulk consensus sequencing integrates naturally with other molecular assays in Accelevir’s portfolio, including dPCR-based quantification and viral load measurements, enabling comprehensive reservoir profiling within a single bioanalytical services program.

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Scalable, high-throughput sequencing captures dominant and minority variants across cohorts.

Our Sequencing Services

Targeted Sequencing (EnvLRS)

For programs focused on envelope biology – tropism determination, broadly neutralizing antibody (bNAb) epitope mapping, glycosylation pattern analysis, or escape mutation tracking – Env long-read sequencing (EnvLRS) provides full-length env gene characterization using long-read platforms.

This targeted approach offers high-fidelity, single-molecule level sequence resolution without the cost and throughput challenges of limit dilution PCR.

Recent advances in long-read accuracy have made EnvLRS increasingly practical. Oxford Nanopore sequencing duplex reads with SUP basecalling can now achieve >99.9% basecall accuracy (>Q30) from approximately 500,000–750,000 raw reads per dataset. Genotypic tropism concordance between Nanopore and Sanger sequencing reaches 98.1%, validating the platform for clinical-grade tropism assessment. PacBio SMRT circular consensus sequencing offers a complementary approach, with studies reporting a median of 6,799 high-quality full-length env CCS reads per time point at >99.7% accuracy.

 

EnvLRS is particularly valuable for therapeutic programs developing bNAbs or envelope-based immunogens, where understanding the diversity and evolution of the target antigen directly informs candidate selection and resistance monitoring.

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Long-read platforms deliver full-length env resolution for tropism and epitope analysis.

Workflow

How Our HIV Proviral Sequencing Assays Work

From sample receipt to final report, Accelevir's workflow is designed to maximize recovery of proviral genomes, ensuring the maximum depth of reservoir coverage, key for detection of rare sequence variants and unbiased representation of intact HIV provirus in resulting datasets.

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High-integrity DNA extraction protects rare proviral targets.

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Step 1

Step 1

Sample Processing and DNA Extraction

High-quality input material is foundational. Accelevir’s PBMC processing services isolate CD4+ T cells from whole blood or leukapheresis products, preserving the high cell viability critical to the quality of downstream molecular applications. DNA isolation protocols are optimized to recover pure, unfragmented DNA molecules, essential to unbiased representation of intact proviral populations and rare sequence variants in resulting NGS datasets.
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Limiting-dilution amplification ensures single-template resolution.

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Step 2

Step 2

Amplification and Library Preparation

For NFL-SGS, HIV provirus genomes are amplified at limit dilution concentrations – calibrated so that approximately 30% of polymerase chain reaction wells yield positive amplification. This ensures by Poisson statistics that most positives derive from a single proviral template. Nested PCR with high-fidelity polymerase then amplifies near-full-length proviral sequences spanning the 5′ psi element and gag, pol, and env to the 3′ LTR. Primer design accounts for known sequence variation across global HIV-1 subtypes to minimize assay drop-out on samples from diverse geographic origins. Highly multiplexed NGS library preparation workflows are employed, allowing for a scalable workflow even on large numbers of samples, reducing sequencing costs, and increasing assay throughput so high quality results are delivered faster.
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HIV-optimized bioinformatics is critical – sub-optimal pipelines can mis-call intact sequences and rare variants.
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Step 3

Step 3

Sequencing and Analysis

Sequencing data are generated on platforms matched to the assay: Illumina short-read NGS for NFL-SGS and bulk consensus sequencing, and long-read platforms (PacBio SMRT, Oxford Nanopore) for EnvLRS. Accelevir’s bioinformatics pipelines for sequencing data analysis are developed in-house with HIV reservoir analysis applications in mind. NFL-SGS bioinformatics pipelines classify each proviral sequence by multiple defect criteria: large internal deletions, frameshifts or premature stop codons in coding regions, APOBEC-driven hypermutation, disruptions in the psi packaging signal, major splice donor site integrity, and LTR symmetry. HIV-optimized pipelines are critical – sub-optimal pipelines can significantly alter the proportion of sequences classified as intact and mis-call rare sequence variants, making validated, standardized analytical workflows a key differentiator. Final reports include analysis of HIV genome intactness, defect classification, variant frequency, clonality, and can be custom-built to specific data package requirements.
Research Applications

Answers for
HIV Cure Programs

Proviral genome sequencing supports diverse applications in HIV cure research. Accelevir’s sequencing services address four principal application areas:

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HIV Cure and Reservoir Studies

NFL-SGS characterizes with single base resolution the intactness of HIV proviruses, identifies expanded proviral clones, and maps the spectrum of genome defects found in the integrated proviral landscape. Combined with IPDA® quantification, this provides both the size and the sequence-level character of the latent reservoir – data critical for evaluating whether a cure intervention meaningfully reduces replication-competent proviruses.

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Clinical Trial Bioanalysis

For sponsors advancing cure candidates, Accelevir’s fit-for-purpose proviral sequencing workflows generate the data packages required for IND/BLA submissions.

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Proviral Diversity and Evolution Studies

Longitudinal bulk consensus sequencing tracks how the reservoir evolves under ART pressure, monitoring shifts in drug resistance mutations, viral fitness landscapes, and immune escape.

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Drug Resistance Monitoring and Therapeutic Development

Distinguishing DRMs present on intact proviruses from those on defective templates is essential for predicting rebound virus characteristics. Proviral sequencing excels at uncovering rare mutations, and single-genome sequencing provides precise information on structural variations and mutations that may influence response to novel HIV therapeutics. EnvLRS specifically supports bNAb and envelope immunogen programs through detailed tropism and epitope evolution data.
FAQ

Frequently Asked Questions

Study-specific planning aligns assay selection, cell input, and reporting before sequencing begins.
What sample types and volumes do you require for proviral genome sequencing?
NFL-SGS and bulk consensus sequencing typically require high-quality PBMCs or isolated CD4+ T cells. Because intact proviruses are rare, large cell inputs (≥5 x 10⁶ CD4+ cells) improve recovery of intact proviral templates. Accelevir’s PBMC processing services support fresh and cryopreserved samples with validated shipping and storage protocols. For EnvLRS, both proviral DNA and plasma RNA inputs are accepted. Technical limitations affect the quality of sequencing data, so input quality specifications are discussed during study planning to optimize yield.
How does single genome sequencing complement other HIV assays like IPDA®?
The IPDA® provides rapid, quantitative measurement of HIV reservoir size by differentiating intact versus defective proviral DNA at two short genomic regions. Single genome sequencing then characterizes the full sequence context of those proviruses: complete defect profiles, clonality, DRM presence on intact templates, and envelope diversity. Together, they answer both “how large is the reservoir” and “what does it contain” at different scales of data resolution.
What turnaround times and study sizes can you accommodate?

Project timelines depend on assay type, sample number, and analytical complexity. NFL-SGS requires longer turnaround than bulk consensus due to limiting-dilution amplification and single-template verification. Accelevir supports studies ranging from small academic cohorts to multi-site clinical trials with hundreds of time points. Capacity and timeline estimates are provided during initial consultation and refined during assay development.

Do you provide regulatory support for clinical trial applications?
Yes. Accelevir’s fit-for-purpose workflows are designed to generate validated data suitable for IND/BLA submissions. This includes documented assay validation, quality-controlled standard operating procedures, and comprehensive reporting formats aligned with regulatory expectations. Our bioanalytical services team works directly with sponsors to ensure sequencing data meets submission requirements.
Start Your Proviral Sequencing Project

Match the Sequencing Strategy to Your Scientific Question

Whether your program needs NFL-SGS to assess reservoir intactness after a cure intervention, bulk consensus sequencing for longitudinal diversity monitoring, or EnvLRS for envelope characterization supporting bNAb development, Accelevir provides the specialized sequencing expertise and regulatory-ready infrastructure to move your HIV cure research forward.
What we'll cover
  • Assay selection
  • Sample and cell-input planning
  • Integrated IPDA® strategy
  • Reporting requirements

Scientific consultation

Baltimore, Maryland · CAP/CLIA-certified laboratory · HIV reservoir specialists
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