A comparative study of ddPCR and sanger sequencing for quantitative detection of low-frequency mutation rate

TLR7 and 8 regulate B cell immunity, but the precise details of the mechanism are still unclear. Here, we studied the kinetics of both heavy and light chains (IgKL) of B-cell receptor (BCR) repertoire responding to the TLR7/8 stimulation in two geniuses of non-human primates (NHPs), African green monkeys (AGMs) and rhesus macaques (RMs). We evaluated the activation of lymphocytes by flow cytometry and studied characteristics of BCR repertoire in terms of gene usage, repertoire diversity, and the number of lineages. Although AGMs had a weaker activation than RMs, and a different responding kinetic, both AGMs and RMs presented an increased IgKL repertoire diversity and lineages expansion. It suggested that the responding time rather than initiation of TLR7/8-induced IgKL repertoire response related to B cell activation. Expanded IgKL lineages with frequency from 0.001% to 1% had an elevated mutation rate and expanded IgH lineages used more IgA/G/E, suggesting that the TLR7/8 stimulation expanded low-frequent but high-mutated lineages. Besides, most of expanded IgKL lineages were lambda isotype. In conclusion, TLR7/8 selectively expands IgKL lineages with a high mutation rate, low frequency, and lambda isotype. The selective effect of TLR7/8 on BCR repertoire allows TLR7/8 agonists to be adjuvant for selectively accelerating antibody maturation.

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Comparative study of 0.1Hz very-low frequency withstand voltage test method and traditional method

Journal of Physics Conference Series ◽

10.1088/1742-6596/2005/1/012175 ◽

2021 ◽

Vol 2005 (1) ◽

pp. 012175

Author(s):

Dong Sun ◽

Jinyu Gao ◽

Yongxin Piao ◽

Bo Guo ◽

Chengjia Qu ◽

...

Keyword(s):

Comparative Study ◽

Traditional Method ◽

Low Frequency ◽

Test Method ◽

Very Low Frequency

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Comparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00785-13 ◽

2013 ◽

Vol 51 (8) ◽

pp. 2592-2598 ◽

Cited By ~ 12

Author(s):

K.-H. Chan ◽

K. K. W. To ◽

B. W. K. Chan ◽

C. P. Y. Li ◽

S. S. Chiu ◽

...

Keyword(s):

Mycoplasma Pneumoniae ◽

Sanger Sequencing ◽

Melting Curve ◽

Low Frequency ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Respiratory Specimens

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Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00038-16 ◽

2016 ◽

Vol 60 (6) ◽

pp. 3380-3397 ◽

Cited By ~ 26

Author(s):

Fred Kyeyune ◽

Richard M. Gibson ◽

Immaculate Nankya ◽

Colin Venner ◽

Samar Metha ◽

...

Keyword(s):

Drug Resistance ◽

Treatment Failure ◽

Sanger Sequencing ◽

Antiretroviral Treatment ◽

Low Frequency ◽

Drug Resistant ◽

Viral Quasispecies ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Hiv 1

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.

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