ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis

Reja, Valin; Kwok, Avelyn; Stone, Graham N.; Yang, Lin; Missel, Andreas; Menzel, Christoph; Bassam, Brant J.

doi:10.1016/j.ymeth.2010.02.006

Cited by 60 publications

(45 citation statements)

References 8 publications

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“…Bar-HRM is an emerging method that combines DNA barcoding with HRM [81]. The denaturation thermodynamics of individual double-stranded DNA to single strands are based on the binding affinities of individual nucleotide pairs, and the melting pattern will vary due to indels, mutations and methylation.…”

Section: Bar-high Resolution Meltingmentioning

confidence: 99%

DNA Barcoding and Pharmacovigilance of Herbal Medicines

2015

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Pharmacovigilance of herbal medicines relies on the product label information regarding the ingredients and the adherence to good manufacturing practices along the commercialisation chain. Several studies have shown that substitution of plant species occurs in herbal medicines, and this in turn poses a challenge to herbal pharmacovigilance as adverse reactions might be due to adulterated or added ingredients. Authentication of constituents in herbal medicines using analytical chemistry methods can help detect contaminants and toxins, but are often limited or incapable of detecting the source of the contamination. Recent developments in molecular plant identification using DNA sequence data enable accurate identification of plant species from herbal medicines using defined DNA markers. Identification of multiple constituent species from compound herbal medicines using amplicon metabarcoding enables verification of labelled ingredients and detection of substituted, adulterated and added species. DNA barcoding is proving to be a powerful method to assess species composition in herbal medicines and has the potential to be used as a standard method in herbal pharmacovigilance research of adverse reactions to specific products.

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Section: Bar-high Resolution Meltingmentioning

confidence: 99%

DNA Barcoding and Pharmacovigilance of Herbal Medicines

2015

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“…The software also provides the probability that each sample belongs to the assigned (and other) cluster(s) and the typicality of samples or how consistent a sample is within its own group which indicates how well the sample fits within its allocated cluster (for details of the calculation of posterior probabilities and typicalities see Reja et al, 2010). 29 ■ RESULTS AND DISCUSSION Base Modifications and DNA Thermostability. Previous work demonstrated the capacity of HRM to detect and quantify the presence of 5mC directly from DNA samples.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Differential Effect of Three Base Modifications on DNA Thermostability Revealed by High Resolution Melting

et al. 2012

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High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

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“…However, sample and machine variables limit reproducibility and complicate comparison of melt curves [46]. This variability is commonly dealt with in a limited way by temperature shifting, the alignment of similar curves based on input from the investigator [3] or statistical grouping of similar curves [54]. An alternative approach has been the incorporation of internal standards with melting points that flank the curves of interest and against which the test curves can be adjusted [3].…”

Section: Discussionmentioning

confidence: 99%

Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

Mirchevska

Phatak

et al. 2017

PLoS ONE

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Fungal infections are a global problem imposing considerable disease burden. One of the unmet needs in addressing these infections is rapid, sensitive diagnostics. A promising molecular diagnostic approach is high-resolution melt analysis (HRM). However, there has been little effort in leveraging HRM data for automated, objective identification of fungal species. The purpose of these studies was to assess the utility of distance methods developed for comparison of time series data to classify HRM curves as a means of fungal species identification. Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data. Comparison of HRM curves of the rDNA internal transcribed spacer (ITS) region from 51 strains of 18 fungal species using DTW distances allowed accurate classification and clustering of all 51 strains. The utility of DTW distances for species identification was demonstrated by matching HRM curves from 243 previously identified clinical isolates against a database of curves from standard reference strains. The results revealed a number of prior misclassifications, discriminated species that are not resolved by routine phenotypic tests, and accurately identified all 243 test strains. In addition to DTW, several other distance functions, Edit Distance on Real sequence (EDR) and Shape-based Distance (SBD), showed promise. It is concluded that DTW-based distances provide a useful metric for the automated identification of fungi based on HRM curves of the ITS region and that this provides the foundation for a robust and automatable method applicable to the clinical setting.

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ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis

Cited by 60 publications

References 8 publications

DNA Barcoding and Pharmacovigilance of Herbal Medicines

DNA Barcoding and Pharmacovigilance of Herbal Medicines

Differential Effect of Three Base Modifications on DNA Thermostability Revealed by High Resolution Melting

Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

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