Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’

Jones, Mathew; Williams, James P.; Gärtner, Kathleen; Phillips, Rodney E.; Hurst, Jacob; Frater, John

doi:10.1016/j.jviromet.2014.02.020

Cited by 142 publications

(129 citation statements)

References 12 publications

Supporting

Mentioning

121

Contrasting

Order By: Relevance

“…More recently, an assay that dispenses with the extraction step has been proposed (70). Recent technical progress has permitted the development of HIV DNA quantification by digital droplet PCR (ddPCR), which does not require an external quantification standard (71)(72)(73)(74)(75)(76)(77). However, some false-positive signals have been observed with ddPCR, which can be a problem when this method is used for diagnosis (76).…”

Section: Dna Load In Resting Cd4mentioning

confidence: 99%

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

et al. 2016

View full text Add to dashboard Cite

SUMMARYHIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis.

show abstract

Section: Dna Load In Resting Cd4mentioning

confidence: 99%

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Currently, only a few methods have been described for threshold setting in ddPCR reactions [7,9]. According to the instructions provided by the manufacturer of the ddPCR QX100 platform (Bio-Rad), it is possible to set a manual threshold, allowing the investigator to make a visual interpretation of the background, rain, and false-positive droplets, but this may also introduce investigator specific bias.…”

Section: Ddpcr Data Analysis Toolsmentioning

confidence: 99%

“…Apart from the QuantaSoft software, first attempts to enable a more robust automated threshold setting were made by Strain et al and more recently by Jones et al [7,9]. These methods use clustering algorithms to identify the positive and negative droplet populations and do not use one threshold but define the 95/99 % confidence intervals for the negative and positive population.…”

Section: Ddpcr Data Analysis Toolsmentioning

confidence: 99%

“…A final consideration concerning available data analysis methods is that both clustering and MTg methods assume a normal distribution of the fluorescent amplitude of the droplet fluorescence and do not correct for possible shifts in baseline fluorescence between NTCs [7,9]. This may have a major influence on the correct allocation of droplets in case the distribution of fluorescent amplitudes does not follow a normal distribution.…”

Section: Ddpcr Data Analysis Toolsmentioning

confidence: 99%

See 1 more Smart Citation

ddpcRquant: threshold determination for single channel droplet digital PCR experiments

et al. 2015

View full text Add to dashboard Cite

Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

show abstract

“…ddPCR is an PCR-based method for absolute quantification offering higher accuracy compared to conventional quantitative real-time PCR (qPCR) 7,8 . ddPCR also provides higher resolving power and accuracy for the detection of rare mutations in DNA templates, enabling more informative cancer research and diagnosis 9 . Additional advantages of ddPCR over conventional qPCR include its enhanced sensitivity and accuracy when studying low template copy numbers [10][11][12] .…”

Section: Introductionmentioning

confidence: 99%

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Rajasekaran

Oh²,

Kim

et al. 2015

JoVE

View full text Add to dashboard Cite

AbstractddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples. Video LinkThe video component of this article can be found at

show abstract

Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’

Cited by 142 publications

References 12 publications

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications

ddpcRquant: threshold determination for single channel droplet digital PCR experiments

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Contact Info

Product

Resources

About