Using Primer-ID Deep Sequencing to Detect Recent Human Immunodeficiency Virus Type 1 Infection

Dennis, Ann M.; Zhou, Shuntai; Sellers, Christopher; Learner, Emily R; Potempa, Marc; Cohen, Myron S.; Miller, William C.; Eron, Joseph J.; Swanstrom, Ronald

doi:10.1093/infdis/jiy426

Cited by 16 publications

(15 citation statements)

References 15 publications

(22 reference statements)

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“…A multiplexed Primer ID library that covered partial gag , integrase coding domain, V1/V2 region, partial gp41 and partial nef region was constructed using the previous protocol ( Zhou et al, 2015 ; Dennis et al, 2018 ). In brief, viral RNA was extracted from culture supernatants using QIAmp viral mini kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

The HIV-1 latent reservoir is largely sensitive to circulating T cells

Warren

Zhou²,

et al. 2020

eLife

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HIV-1 specific CD8+ T cells are an important component of HIV-1 curative strategies. Viral variants in the HIV-1 reservoir may limit the capacity of T cells to detect and clear virus-infected cells. We investigated the patterns of T cell escape variants in the replication-competent reservoir of 25 persons living with HIV-1 (PLWH) durably suppressed on ART. We identified all reactive T cell epitopes in the HIV-1 proteome for each participant and sequenced HIV-1 outgrowth viruses from resting CD4+ T cells. All non-synonymous mutations in reactive T cell epitopes were tested for their effect on the size of the T cell response, with a ≥50% loss defined as an escape mutation. The majority (68%) of T cell epitopes harbored no detectable escape mutations. These findings suggest that circulating T cells in PLWH on ART could contribute to control of rebound and could be targeted for boosting in curative strategies.

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Section: Methodsmentioning

confidence: 99%

The HIV-1 latent reservoir is largely sensitive to circulating T cells

Warren

Zhou²,

et al. 2020

eLife

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“…Primer ID MiSeq was used to sequence part of the RT coding region of each sample using primers and methods described previously (41). Input RNA used for each sample was 20 ng.…”

Section: Methodsmentioning

confidence: 99%

Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

Boyer

Melody

Smith

et al. 2019

J Virol

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Two mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation. IMPORTANCE Although antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

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“…The sequence output is first sorted by the Illumina index (i.e., each clinical specimen), then by amplicon region, then by UMI to generate TCS for analysis of drug resistant mutations. In this approach the detected mutations within an amplicon can be interpreted for linkage but those between amplicons cannot [15,16]. However, the multiplexing protocol may lead to a significant loss of sequencing reads of targeted region if the number of multiplexed regions and specimens are not calculated properly.…”

Section: How Does a Unique Molecular Identifier Fix All Of These Probmentioning

confidence: 99%

“…The idea here is that with an input of 1000 RNA copies into the cDNA reaction (and using all of the cDNA product in the PCR), a poor template utilization of between 1.5% (i.e., 15 copies) to 5% (50 copies) will give sampling sensitivity of minor variants in the range of 7-20% abundance within the PCR product (assuming no skewing during PCR). However, with the higher number of templates sequenced (i.e., 50) the confidence interval for detecting something at 20% is 10-34%, significantly better than if something is sampled at 20% with the smaller number of templates (15), which has a wide confidence interval of 4.3-48%.…”

Section: Why 20% Abundance Is the Useful Claim For Ngs Sensitivity (Amentioning

confidence: 99%

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

Zhou

Swanstrom

2020

Viruses

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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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Using Primer-ID Deep Sequencing to Detect Recent Human Immunodeficiency Virus Type 1 Infection

Cited by 16 publications

References 15 publications

The HIV-1 latent reservoir is largely sensitive to circulating T cells

The HIV-1 latent reservoir is largely sensitive to circulating T cells

Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

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

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