MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Yu, Ming; Carter, Kelly; Makar, Karen W.; Vickers, Kathy; Ulrich, Cornelia M.; Schoen, Robert E.; Brenner, Dean E.; Markowitz, Sanford D.; Grady, William M.

doi:10.1080/15592294.2015.1068490

Cited by 65 publications

(46 citation statements)

References 25 publications

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“…The clinical significance of cryptic FM alleles requires further epigenotype-phenotype studies. If MS-QMA were to become a standard first-line test, other more analytically sensitive methods, such as pyrosequencing and droplet digital PCR with reported lower limit of detection Ͻ5% in other applications (41,42 ), would need to be developed for second-line testing. Testing of multiple tissues and more extensive CGG triplet repeat size testing of other family members, especially mothers, could also be used for confirmation of MS-QMA positive results.…”

Section: Discussionmentioning

confidence: 99%

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

et al. 2016

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Section: Discussionmentioning

confidence: 99%

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

et al. 2016

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“…Whale et al [66], Morisset et al [74], and Yu et al [75]). As discussed in the Design subsection of this review, various factors, such as sample preparation and pipetting errors, may violate the assumptions necessary to allow a pooling of technical replicates.…”

Section: Sample-to-sample Variationmentioning

confidence: 98%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

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“…Single Base Primer Extension Assay (SNaPshot) was also used by many laboratories for epigenetic age prediction, but the accuracy is apparently lower (Lee et al 2015;Hong et al 2017). Recently, droplet digital PCR (ddPCR) was reported to enable precise DNAm measurements (Yu et al 2015;Zemmour et al 2018), and hence it might facilitate epigenetic age predictions without PCR bias. Barcoded bisulfite amplicon sequencing (BBA-seq), which is based on next generation sequencing, enables multiplexed analysis of PCR amplicons (Bernstein et al 2015;Theophilou et al 2015).…”

Section: Introductionmentioning

confidence: 99%

New Targeted Approaches for Epigenetic Age Predictions

Han

Franzen

Stiehl

et al. 2019

Preprint

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Aging causes epigenetic modifications, which are utilized as a biomarker for the aging process.While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective highthroughput analysis. In this study, we utilized 4,650 Illumina BeadChip datasets of blood to select the best suited CpG sites for targeted analysis. DNA methylation analysis at these sites with either pyrosequencing or droplet digital PCR (ddPCR) revealed a high correlation with chronological age.In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower precision at individual CpGs. However, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often accompanied by a CTCF binding site at the peak. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified but reveals a stochastic pattern. Based on this, we have developed an alternative model for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. Thus, the stochastic evolution of age-associated DNA methylation patterns, which seems to resemble epigenetic drift, enables epigenetic clocks for individual DNA strands.

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MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles

Cited by 65 publications

References 25 publications

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

Identification of Males with Cryptic Fragile X Alleles by Methylation-Specific Quantitative Melt Analysis

The Future of Digital Polymerase Chain Reaction in Virology

New Targeted Approaches for Epigenetic Age Predictions

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