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.

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

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.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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.

scholarly journals Multiplex ligation-dependent probe amplification – a short overview

2020 ◽  
Vol 28 (2) ◽  
pp. 123-131
Author(s):  
Valeriu Moldovan ◽  
Elena Moldovan
Keyword(s):  
Copy Number ◽  
Genetic Disorders ◽  
Cost Effective ◽  
Copy Number Variations ◽  
Comparative Genomic ◽  
Gene Deletions ◽  
Conventional Cytogenetic Analysis ◽  
Main Technique ◽  
Cost Effective Technique ◽  
Dependent Probe

AbstractMultiplex Ligation-dependent Probe Amplification is a technique proposed for the detection of deletions or duplications that may lead to copy number variations in genomic DNA, mainly due to its higher resolution, and shorter overall diagnosis time, when compared with techniques traditionally used, namely karyotyping, fluorescence in situ hybridization, and array comparative genomic hybridization. Multiplex Ligation-dependent Probe Amplification is a fast (about 2 days), useful and cost-effective technique, being suitable for the diagnosis of hereditary conditions caused by complete or partial gene deletions or duplications, as these conditions are either more difficult or impossible to be diagnosed by other techniques, such as PCR, Real-Time PCR, or sequencing (Sanger or Next Generation). Due to its numerous advantages over conventional cytogenetic analysis techniques, Multiplex Ligation-dependent Probe Amplification could be used in the near future as the main technique for the molecular investigation of genetic conditions caused by copy number variations, in both rare and complex genetic disorders.

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