Precise quantitation and sensitive detection of copy number within genetic variations using ligation-mediated droplet digital PCR in plasma

2019 ◽  
Vol 11 (45) ◽  
pp. 5761-5767
Author(s):  
Hui Tian ◽  
Mingyue Duan ◽  
Pingping Wei ◽  
Fei Hu ◽  
Shuhao Zhao ◽  
...  
Keyword(s):  
Copy Number ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Genetic Variations ◽  
Cancer Diagnostics ◽  
Droplet Digital Pcr ◽  
Sensitive Detection

The analysis of cancer-associated genetic copy number variations (CNVs) has been employed for cancer diagnostics, treatment, and prognostic assessments.

Assessment Of Sfmbt1, Prkra, And Erlin1 Copy Number Variations In Patients With Carotid Atherosclerosis Using Droplet Digital Pcr

2019 ◽  
Vol 287 ◽  
pp. e165-e166
Author(s):  
A. Sleptcov ◽  
M. Nazarenko ◽  
A. Kazantsev ◽  
I. Lebedev ◽  
O. Barbarash ◽  
...  
Keyword(s):  
Copy Number ◽  
Carotid Atherosclerosis ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Droplet Digital Pcr

scholarly journals Droplet digital PCR for identifying copy number variations in patients with primary immunodeficiency disorders

2021 ◽  
Author(s):  
See-Tarn Woon ◽  
Julia Mayes ◽  
Alexander Quach ◽  
Hilary Longhurst ◽  
Antonio Ferrante ◽  
...  
Keyword(s):  
Primary Immunodeficiency ◽  
Copy Number ◽  
Sanger Sequencing ◽  
Cost Effective ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Droplet Digital Pcr ◽  
Inborn Errors ◽  
Primary Immunodeficiency Disorders ◽  
Large Deletions

Abstract Primary immunodeficiency disorders comprise a rare group of mostly monogenic disorders caused by inborn errors of immunity. The majority can be identified by either Sanger sequencing or Next Generation Sequencing. Some disorders result from large insertions or deletions leading to copy number variations (CNV). Sanger sequencing may not identify these mutations. Here we present droplet digital PCR as an alternative cost-effective diagnostic method to identify CNV in these genes. The data from patients with large deletions of NFKB1, SERPING1 and SH2D1A are presented.

scholarly journals Accurate measurement of transgene copy number in crop plants using droplet digital PCR

2017 ◽  
Vol 90 (5) ◽  
pp. 1014-1025 ◽  
Author(s):  
Ray Collier ◽  
Kasturi Dasgupta ◽  
Yan-Ping Xing ◽  
Bryan Tarape Hernandez ◽  
Min Shao ◽  
...  
Keyword(s):  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Crop Plants ◽  
Transgene Copy Number

scholarly journals DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

2017 ◽  
Author(s):  
Elizabeth Nacheva ◽  
Katya Mokretar ◽  
Aynur Soenmez ◽  
Alan M Pittman ◽  
Colin Grace ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Human Brain ◽  
Frontal Cortex ◽  
Genome Sequencing ◽  
Copy Number ◽  
Dna Isolation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Whole Genome ◽  
Salting Out

AbstractPotential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Akemi Katsurada ◽  
Kayoko Miyata ◽  
Andrei Derbenev ◽  
Andrea Zsombok
Keyword(s):  
Copy Number ◽  
System Analysis ◽  
Renin Angiotensin System ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Copy Numbers ◽  
Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

scholarly journals A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations

2019 ◽  
Vol 65 (9) ◽  
pp. 1153-1160 ◽  
Author(s):  
Kévin Cassinari ◽  
Olivier Quenez ◽  
Géraldine Joly-Hélas ◽  
Ludivine Beaussire ◽  
Nathalie Le Meur ◽  
...  
Keyword(s):  
Copy Number ◽  
Segregation Analysis ◽  
Genetic Diseases ◽  
Genomic Medicine ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Locked Nucleic Acid ◽  
Targeted Analysis ◽  
Cost Efficient ◽  
High Flexibility

Abstract BACKGROUND Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

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