A Pan-HIV Strategy for Complete Genome Sequencing

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e.,switchingmechanismat 5′ end ofRNAtranscript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

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Application of next generation sequencing in dual HIV infection studies

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-153 ◽

2022 ◽

Vol 98 (6) ◽

pp. 627-638

Author(s):

I. A. Lapovok ◽

P. B. Baryshev ◽

D. V. Saleeva ◽

A. A. Kirichenko ◽

A. V. Shlykova (Murzakova) ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hiv Infection ◽

Routine Practice ◽

Next Generation ◽

Genetic Characteristics ◽

Synonymous Mutations ◽

Operational Taxonomic Units ◽

Subtype B ◽

Generation Sequencing ◽

Hiv 1

Introduction. The aim of the study was to use comparative analysis for assessing efficiency of detection and confirmation of dual HIV infection, using conventional population sequencing (PS) and next generation sequencing (NGS) for an HIV-1 pol gene fragment, which encompasses protease and partially reverse transcriptase (positions 2253–3368).Materials and methods. The study was performed on intersubtype dual HIV infection model samples containing viruses of HIV-1 subtype B, sub-subtype A6 and recombinant form CRF63_02A1. Viruses were mixed pairwise in proportions from 10 to 90% to obtain 3 groups of model samples: CRF63vsB, CRF63vsA6, and A6vsB. The nucleotide sequences obtained by using PS and NGS technologies having 5, 10, 15, and 20% sensitivity thresholds for minor virus variants (NGS5–NGS20, respectively) were used to estimate the number of degenerate nucleotides or the degenerate base (DB) count and the number of synonymous mutations (SM) or the SM count. The fragment of the studied region (positions 2725–2981) was used for the analysis of operational taxonomic units.Results. The application of NGS5 proved highly efficient for detection of dual HIV infection in the model samples. The statistically significant (p < 0.01) increase in DB and SM counts was demonstrated by NGS5 compared to PS. As a result, NGS5 helped detect dual HIV infection in 25 out of 27 model samples, while with PS it was detected only in 15 samples. The analysis of operational taxonomic units confirmed dual HIV infection in all the groups of model samples.Discussion. The efficiency of detection and confirmation of dual HIV infection depends both on the content of each virus in the sample and on genetic characteristics of these viruses. Conclusion. Using NGS genetic testing in routine practice will be instrumental for efficient identification of genetic characteristics of infectious agents and for thorough analysis of the epidemiological situation.

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Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Journal of Virology ◽

10.1128/jvi.00522-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8540-8555 ◽

Cited By ~ 77

Author(s):

Shuntai Zhou ◽

Corbin Jones ◽

Piotr Mieczkowski ◽

Ronald Swanstrom

Keyword(s):

Next Generation Sequencing ◽

Error Rate ◽

Consensus Sequence ◽

Next Generation ◽

Sampling Depth ◽

Data Set ◽

Sequencing Errors ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Hiv 1

ABSTRACTValidating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS.IMPORTANCEAlthough next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.

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Fact and Fiction about 1%: Next Generation Sequencing and the Detection of Minor Drug Resistant Variants in HIV-1 Populations with and without Unique Molecular Identifiers

Viruses ◽

10.3390/v12080850 ◽

2020 ◽

Vol 12 (8) ◽

pp. 850 ◽

Cited By ~ 1

Author(s):

Shuntai Zhou ◽

Ronald Swanstrom

Keyword(s):

Next Generation Sequencing ◽

High Throughput Sequencing ◽

Consensus Sequence ◽

Viral Population ◽

Drug Resistant ◽

Next Generation ◽

Data Set ◽

Method Error ◽

Generation Sequencing ◽

Hiv 1

Next generation sequencing (NGS) platforms have the ability to generate almost limitless numbers of sequence reads starting with a PCR product. This gives the illusion that it is possible to analyze minor variants in a viral population. However, including a PCR step obscures the sampling depth of the viral population, the key parameter needed to understand the utility of the data set for finding minor variants. Also, these high throughput sequencing platforms are error prone at the level where minor variants are of interest, confounding the interpretation of detected minor variants. A simple strategy has been applied in multiple applications of NGS to solve these problems. Prior to PCR, individual molecules are “tagged” with a unique molecular identifier (UMI) that can be used to establish the actual sample size of viral genomes sequenced after PCR and sequencing. In addition, since PCR generates many copies of each sequence tagged to a specific UMI, a template consensus sequence (TCS) can be created from the many reads of each template, removing virtually all of the method error. From this perspective we examine our own use of a UMI, called Primer ID, in the detection of minor drug resistant variants in HIV-1 populations.

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FDA authorizes marketing of first next-generation sequencing test for detecting HIV-1 drug resistance mutations

Case Medical Research ◽

10.31525/cmr-2106c4c ◽

2019 ◽

Author(s):

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Next Generation ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Generation Sequencing ◽

Hiv 1

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Universal Amplification, Next-Generation Sequencing, and Assembly of HIV-1 Genomes

Journal of Clinical Microbiology ◽

10.1128/jcm.01516-12 ◽

2012 ◽

Vol 50 (12) ◽

pp. 3838-3844 ◽

Cited By ~ 85

Author(s):

A. Gall ◽

B. Ferns ◽

C. Morris ◽

S. Watson ◽

M. Cotten ◽

...

Keyword(s):

Next Generation Sequencing ◽

Next Generation ◽

Generation Sequencing ◽

Hiv 1

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Clinical Application of Metagenomic Next-Generation Sequencing in Patients with Hematologic Malignancies Suffering from Sepsis

Microorganisms ◽

10.3390/microorganisms9112309 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2309

Author(s):

Wang-Da Liu ◽

Ting-Yu Yen ◽

Po-Yo Liu ◽

Un-In Wu ◽

Prerana Bhan ◽

...

Keyword(s):

Next Generation Sequencing ◽

Hematologic Malignancies ◽

Blood Cultures ◽

Diagnostic Methods ◽

Next Generation ◽

Viral Loads ◽

A Prospective Study ◽

Positive Results ◽

Serology Testing ◽

Generation Sequencing

Background: Sepsis remains a common but fatal complication among patients with immune suppression. We aimed to investigate the performance of metagenomic next-generation sequencing (mNGS) compared with standard microbiological diagnostics in patients with hematologic malignancies. Methods: We performed a prospective study from June 2019 to December 2019. Adult patients with hematologic malignancies and a clinical diagnosis of sepsis were enrolled. Conventional diagnostic methods included blood cultures, serum galactomannan for Aspergillus, cryptococcal antigen and cytomegalovirus (CMV) viral loads. Blood samples for mNGS were collected within 24 h after hypotension developed. Results: Of 24 patients enrolled, mNGS and conventional diagnostic methods (blood cultures, serology testing and virus RT-PCR) reached comparable positive results in 9 cases. Of ten patients, mNGS was able to identify additional pathogens compared with conventional methods; most of the pathogens were virus. Conclusion: Our results show that mNGS may serve as adjunctive diagnostic tool for the identification of pathogens of hematologic patients with clinically sepsis.

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Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay

Journal of Clinical Virology ◽

10.1016/j.jcv.2019.104207 ◽

2019 ◽

Vol 121 ◽

pp. 104207 ◽

Cited By ~ 2

Author(s):

Enagnon Kazali Alidjinou ◽

Pauline Coulon ◽

Christophe Hallaert ◽

Olivier Robineau ◽

Agnès Meybeck ◽

...

Keyword(s):

Drug Resistance ◽

Next Generation Sequencing ◽

Resistance Testing ◽

Next Generation ◽

Proviral Dna ◽

Drug Resistance Testing ◽

Generation Sequencing ◽

Hiv 1

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Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa352 ◽

2020 ◽

Vol 75 (12) ◽

pp. 3510-3516 ◽

Cited By ~ 1

Author(s):

Jessica M Fogel ◽

David Bonsall ◽

Vanessa Cummings ◽

Rory Bowden ◽

Tanya Golubchik ◽

...

Keyword(s):

Drug Resistance ◽

Viral Load ◽

Next Generation Sequencing ◽

High Throughput ◽

Whole Genome ◽

Hiv Viral Load ◽

Viral Loads ◽

Viral Load Assay ◽

Generation Sequencing ◽

Hiv 1

Abstract Objectives To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load. Methods Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV). Results HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000–5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%–30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142). Conclusions The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.

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