Evaluation of GS Junior and MiSeq next-generation sequencing technologies as an alternative to Trugene population sequencing in the clinical HIV laboratory

Ram, Daniela; Leshkowitz, Dena; Gonzalez, Dimitri; Forer, Relly; Levy, Itzchak; Chowers, Michal; Lorber, Margalit; Hindiyeh, Musa; Mendelson, Ella

doi:10.1016/j.jviromet.2014.11.003

Cited by 22 publications

(20 citation statements)

References 33 publications

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“…More importantly, unique barcoding of samples allows sequences from individual samples to be recovered, which allows individual-level clinical decisions to be made from pooled sequencing data. The relatively large depth of coverage (Ͼ9,900-fold) obtained for the majority of samples tested suggests that minority variant detection using the wide sequencing approach is possible, as the level of coverage achieved is comparable to that presented in earlier studies of HIV resistance testing by deep sequencing (20,21). However, the clinical relevance of any minority variants identified by this method requires further investigation.…”

Section: Discussionmentioning

confidence: 74%

“…In this deep sequencing approach, HIV RNA is PCR amplified and thousands of templates per sample are clonally sequenced. The depth of coverage obtained (typically several thousands to tens of thousands of reads per sample [20,21]) can be used to detect lowfrequency variants and to study within-host HIV evolution (22). Several studies have clearly demonstrated that the presence of low-frequency drug-resistant variants, particularly those with NNRTI resistance, can negatively affect treatment outcomes (20,(23)(24)(25)(26)(27).…”

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confidence: 99%

“…Furthermore, the greatly increased sequencing capacity of the newest NGS instruments offers additional benefits. Samples could be sequenced to greater depth, allowing more sensitive detection of rare variants (21). Alternatively, a correspondingly greater number of samples could be sequenced in a single run, a strategy that we refer to as "wide" sequencing.…”

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confidence: 99%

“…Alternatively, a correspondingly greater number of samples could be sequenced in a single run, a strategy that we refer to as "wide" sequencing. To our knowledge, however, NGS resistance testing methods have primarily been demonstrated using low sample numbers per run (21,(28)(29)(30)(31).…”

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confidence: 99%

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HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Lapointe

Dong

Lee

et al. 2015

Antimicrob Agents Chemother

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f Limited access to HIV drug resistance testing in low-and middle-income countries impedes clinical decision-making at the individual patient level. An efficient protocol to address this issue must be established to minimize negative therapeutic outcomes for HIV-1-infected individuals in such settings. This is an observational study to ascertain the potential of newer genomic sequencing platforms, such as the Illumina MiSeq instrument, to provide accurate HIV drug resistance genotypes for hundreds of samples simultaneously. Plasma samples were collected from Canadian patients during routine drug resistance testing (n ‫؍‬ 759) and from a Ugandan study cohort (n ‫؍‬ 349). Amplicons spanning HIV reverse transcriptase codons 90 to 234 were sequenced with both MiSeq sequencing and conventional Sanger sequencing methods. Sequences were evaluated for nucleotide concordance between methods, using coverage and mixture parameters for quality control. Consensus sequences were also analyzed for disparities in the identification of drug resistance mutations. Sanger and MiSeq sequencing was successful for 881 samples (80%) and 892 samples (81%), respectively, with 832 samples having results from both methods. Most failures were for samples with viral loads of <3.0 log 10 HIV RNA copies/ml. Overall, 99.3% nucleotide concordance between methods was observed. MiSeq sequencing achieved 97.4% sensitivity and 99.3% specificity in detecting resistance mutations identified by Sanger sequencing. Findings suggest that the Illumina MiSeq platform can yield high-quality data with a high-multiplex "wide" sequencing approach. This strategy can be used for multiple HIV subtypes, demonstrating the potential for widespread individual testing and annual population surveillance in resource-limited settings.A dvances in highly active antiretroviral therapy (HAART) in recent decades have resulted in sustained decreases in HIVrelated morbidity and mortality rates. HIV-infected individuals who receive treatment now have nearly normal life expectancies, such that HIV is now considered a manageable chronic disease (1, 2); antiretroviral therapy (ART) not only provides benefits at the individual patient level but also results in a population-level advantage through HAART-induced suppression of HIV replication and the inherent prevention of onward transmission of the virus, termed "treatment as prevention" (3-6).Drug resistance testing is an essential complement to HAART, enabling clinicians to identify patients infected with drug-resistant HIV and to prescribe the appropriate antiretroviral regimens (7). Lack of access to HIV drug resistance testing acts as a major barrier to long-term treatment success, either through prescription of ineffective regimens in the case of transmitted resistance or through decreased ability of physicians to identify causes of treatment failure (7-9). Cases of unsuppressed viremia allow continued transmission and can compromise the management of HIV on both the patient and population levels. These challenges are particula...

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Section: Discussionmentioning

confidence: 74%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Lapointe

Dong

Lee

et al. 2015

Antimicrob Agents Chemother

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“…However, these limitations are inherent in STRPs studies due to highly variable and non-unique nature of the STRs (Brahmachary et al 2014). In future research, this obstacle may be overcome with the development of long-read next-generation sequencing technologies (Li et al 2015; Ram et al 2015). …”

Section: Discussionmentioning

confidence: 99%

Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines

et al. 2015

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Inter-individual variation in cytosine modifications has been linked to complex traits in humans. Cytosine modification variation is partially controlled by single nucleotide polymorphisms (SNPs), known as modified cytosine quantitative trait loci (mQTL). However, little is known about the role of short tandem repeat polymorphisms (STRPs), a class of structural genetic variants, in regulating cytosine modifications. Utilizing the published data on the International HapMap Project lymphoblastoid cell lines (LCLs), we assessed the relationships between 721 STRPs and the modification levels of 283540 autosomal CpG sites. Our findings suggest that, in contrast to the predominant cis-acting mode for SNP-based mQTL, STRPs are associated with cytosine modification levels in both cis-acting (local) and trans-acting (distant) modes. In local scans within the +/− 1Mb windows of target CpGs, 21, 9, and 21 cis-acting STRP-based mQTL were detected in CEU (Caucasian residents from Utah, USA), YRI (Yoruba people from Ibadan, Nigeria), and combined samples, respectively. In contrast, 139420, 76817, and 121866 trans-acting STRP-based mQTL were identified in CEU, YRI, and combined samples, respectively. A substantial proportion of CpG sites detected with local STRP-based mQTL were not associated with SNP-based mQTL, suggesting that STRPs represent an independent class of mQTL. Functionally, genetic variants neighboring CpG-associated STRPs are enriched with genome-wide association study (GWAS) loci for a variety of complex traits and diseases, including cancers, based on the National Human Genome Research Institute (NHGRI) GWAS Catalog. Therefore, elucidating these STRP-based mQTL in addition to SNP-based mQTL can provide novel insights into the genetic architectures of complex traits.

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Performance comparison of deep sequencing platforms for detecting HIV‐1 variants in the pol gene

Nicot

Jeanne

Raymond

et al. 2018

Journal of Medical Virology

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The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.

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Evaluation of GS Junior and MiSeq next-generation sequencing technologies as an alternative to Trugene population sequencing in the clinical HIV laboratory

Cited by 22 publications

References 33 publications

HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

HIV Drug Resistance Testing by High-Multiplex “Wide” Sequencing on the MiSeq Instrument

Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines

Performance comparison of deep sequencing platforms for detecting HIV‐1 variants in the pol gene

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