Quantitative Analysis of Copy Number Variants Based on Real‐Time LightCycler PCR

Ma, Lijiang; Chung, Wendy K.

doi:10.1002/0471142905.hg0721s80

Cited by 47 publications

(46 citation statements)

References 19 publications

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“…a F v /F m , b winter survival, c ranking of genotypes according to HvCBF2 CNRQ in the two VRN-H1 groups fall between integers (e.g. copy number measuring 1.3) making interpretation more difficult (Ma and Chung 2014).…”

Section: Discussionmentioning

confidence: 99%

Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley

Francia

Morcia²,

Pasquariello

et al. 2016

Plant Mol Biol

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A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat

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Section: Discussionmentioning

confidence: 99%

Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley

Francia

Morcia²,

Pasquariello

et al. 2016

Plant Mol Biol

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“…Filtering discordant sequence reads to identify paired ends that span SV breakpoints is one successful strategy to pinpoint breakpoints; however, it is difficult to uniquely map short reads to repetitive DNA. One way to increase the likelihood of capturing breakpoints in repeats is by creating large-insert 'jumping' libraries, where mate pairs span both sides of interspersed repeats [110,111]. This is especially useful for inversions and balanced translocations that are not detected by copy-number methods.…”

Section: Consequences Of Svmentioning

confidence: 99%

Human Structural Variation: Mechanisms of Chromosome Rearrangements

2015

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Chromosome structural variation (SV) is a normal part of variation in the human genome, but some classes of SV can cause neurodevelopmental disorders. Analysis of the DNA sequence at SV breakpoints can reveal mutational mechanisms and risk factors for chromosome rearrangement. Large-scale SV breakpoint studies have become possible recently owing to advances in next-generation sequencing (NGS) including whole-genome sequencing (WGS). These findings have shed light on complex forms of SV such as triplications, inverted duplications, insertional translocations, and chromothripsis. Sequence-level breakpoint data resolve SV structure and determine how genes are disrupted, fused, and/or misregulated by breakpoints. Recent improvements in breakpoint sequencing have also revealed non-allelic homologous recombination (NAHR) between paralogous long interspersed nuclear element (LINE) or human endogenous retrovirus (HERV) repeats as a cause of deletions, duplications, and translocations. This review covers the genomic organization of simple and complex constitutional SVs, as well as the molecular mechanisms of their formation.

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“…Assessment of copy number variation was determined using relative quantification for qPCR and the comparative CT method (the 2 −ΔΔCT method; Livak and Schmittgen, 2001). This method determined gene copy number using a known single-copy gene as the comparator (Ma and Chung, 2014;D'haene et al, 2010). Recombination activating gene 1 (RAG1) is known to be single-copy across amphibian species (Chiari et al, 2009;Evans et al, 2005) and was used as the comparator for assessment of EBF3N copy number.…”

Section: Assessment Of Ebf3n Copy Number Using the 2 −δδCt Methodsmentioning

confidence: 99%

A quantitative-PCR based method to estimate ranavirus viral load following normalisation by reference to an ultraconserved vertebrate target

Leung

Thomas‐Walters

Garner

et al. 2017

Journal of Virological Methods

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A B S T R A C TRanaviruses are important pathogens of amphibians, reptiles and fish. To meet the need for an analytical method for generating normalised and comparable infection data for these diverse host species, two standard-curve based quantitative-PCR (qPCR) assays were developed enabling viral load estimation across these host groups. A viral qPCR targeting the major capsid protein (MCP) gene was developed which was specific to amphibianassociated ranaviruses with high analytical sensitivity (lower limit of detection: 4.23 plasmid standard copies per reaction) and high reproducibility across a wide dynamic range (coefficient of variation below 3.82% from 3 to 3 × 10 8 standard copies per reaction). The comparative sensitivity of the viral qPCR was 100% (n = 78) based on agreement with an established end-point PCR. Comparative specificity with the end-point PCR was also 100% (n = 94) using samples from sites with no history of ranavirus infection. To normalise viral quantities, a host qPCR was developed which targeted a single-copy, ultra-conserved non-coding element (UCNE) of vertebrates. Viral and host qPCRs were applied to track ranavirus growth in culture. The two assays offer a robust approach to viral load estimation and the host qPCR can be paired with assays targeting other pathogens to study infection burdens.

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Quantitative Analysis of Copy Number Variants Based on Real‐Time LightCycler PCR

Cited by 47 publications

References 19 publications

Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley

Copy number variation at the HvCBF4–HvCBF2 genomic segment is a major component of frost resistance in barley

Human Structural Variation: Mechanisms of Chromosome Rearrangements

A quantitative-PCR based method to estimate ranavirus viral load following normalisation by reference to an ultraconserved vertebrate target

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