scholarly journals Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

2019 ◽  
Author(s):  
Marta Puig ◽  
Jon Lerga-Jaso ◽  
Carla Giner-Delgado ◽  
Sarai Pacheco ◽  
David Izquierdo ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Genomic Variation ◽  
Factors Affecting ◽  
Genome Wide ◽  
Population Information ◽  
Genetic Length ◽  
The Impact ◽  
First Time ◽  
Inversion Breakpoints

ABSTRACTDespite the interest in characterizing all genomic variation, the presence of large repeats at the breakpoints of many structural variants hinders their analysis. This is especially problematic in the case of inversions, since they are balanced changes without gain or loss of DNA. Here we tested novel linkage-based droplet digital PCR (ddPCR) assays on 20 inversions ranging from 3.1 to 742 kb and flanked by long inverted repeats (IRs) of up to 134 kb. Among these, we validated 13 inversions predicted by different genome-wide techniques. In addition, we have generated new experimental human population information across 95 African, European and East-Asian individuals for 16 of them, including four already known inversions for which there were no high-throughput methods to determine directly the orientation, like the well-characterized 17q21 inversion. Through comparison with previous data, independent replicates and both inversion breakpoints, we have demonstrated that the technique is highly accurate and reproducible. Most of the studied inversions are frequent and widespread across continents, showing a negative correlation with genetic length. Moreover, all except two show clear signs of being recurrent, and the new data allowed us to define more clearly the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, thanks to the generated genotypes, we have been able to check inversion functional effects in multiple tissues, validating gene expression differences reported before for two inversions and finding new candidate associations. Our work therefore provides a tool to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

scholarly journals Systematic evaluation of cell-type deconvolution pipelines for sequencing-based bulk DNA methylomes

2021 ◽  
Author(s):  
Yunhee Jeong ◽  
Reka Toth ◽  
Marlene Ganslmeier ◽  
Kersten Breuer ◽  
Christoph Plass ◽  
...  
Keyword(s):  
Cell Types ◽  
Systematic Evaluation ◽  
Cell Type ◽  
Factors Affecting ◽  
Genome Wide ◽  
Cell Type Composition ◽  
Type Composition ◽  
Level Information ◽  
Genomic Regions ◽  
The Impact

DNA methylation sequencing is becoming increasingly popular, yielding genome-wide methylome data at single-base pair resolution through the novel cost- and labor-optimized protocols. It has tremendous potential for cell-type heterogeneity analysis, particularly in tumors, due to intrinsic read-level information. Although diverse deconvolution methods were developed to infer cell-type composition based on bulk sequencing-based methylomes, their systematic evaluation has not been performed so far. Here, we thoroughly review and evaluate five previously published deconvolution methods: Bayesian epiallele detection (BED), PRISM, csmFinder + coMethy, ClubCpG and MethylPurify, together with two array-based methods, MeDeCom and Houseman as a comparison group. Sequencing-based deconvolution methods consist of two main steps, informative region selection and cell-type composition estimation. Accordingly, we individually assessed the performance of each step and demonstrated the impact of the former step upon the performance of the following one. In conclusion, we demonstrate the best method showing the highest accuracy in different samples, and infer factors affecting cell-type deconvolution performance according to the number of cell types in the mixture. We found that cell-type deconvolution performance is influenced by different factors according to the number of components in the mixture. Whereas selecting similar genomic regions to DMRs generally contributed to increasing the performance in bi-component mixtures, the uniformity of cell-type distribution showed a high correlation with the performance in five cell-type bulk analyses.

scholarly journals Spatial analysis of ligand-receptor interactions in skin cancer at genome-wide and single-cell resolution

2020 ◽  
Author(s):  
M Tran ◽  
S Yoon ◽  
ST Min ◽  
S Andersen ◽  
K Devitt ◽  
...  
Keyword(s):  
Cell Carcinoma ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cell Interactions ◽  
Genome Wide ◽  
Receptor Interactions ◽  
Cell Cell

AbstractThe ability to study cancer-immune cell communication across the whole tumor section without tissue dissociation is important to understand molecular mechanisms of cancer immunotherapy and drug targets. Current experimental methods such as immunohistochemistry allow researchers to investigate a small number of cells or a limited number of ligand-receptor pairs at tissue scale with limited cellular resolution. In this work, we developed a powerful experimental and analytical pipeline that allows for the genome-wide discovery and targeted validation of cellular communication. By profiling thousands of genes, spatial transcriptomic and single-cell RNA sequencing data show genes that are possibly involved in interactions. The expression of the candidate genes could be visualized by single-molecule in situ hybridization and droplet digital PCR. We developed a computational pipeline called STRISH that enables us to quantitatively model cell-cell interactions by automatically scanning for local expression of RNAscope data to recapitulate an interaction landscape across the whole tissue. Furthermore, we showed the strong correlation of microscopic RNAscope imaging data analyzed by STRISH with the gene expression values measured by droplet digital PCR. We validated the unique ability of this approach to discover new cell-cell interactions in situ through analysis of two types of cancer, basal cell carcinoma and squamous cell carcinoma. We expect that the approach described here will help to discover and validate ligand receptor interactions in different biological contexts such as immune-cancer cell interactions within a tumor.

The Impact of Constitutional Copy Number Variants in Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 496-496
Author(s):  
Matthew W Jenner ◽  
David C Johnson ◽  
Paola E Leone ◽  
Brian A Walker ◽  
David Gonzalez ◽  
...  
Keyword(s):  
Copy Number ◽  
Copy Number Change ◽  
Copy Number Variations ◽  
Genomic Variation ◽  
Chromosome 21 ◽  
Nucleotide Polymorphisms ◽  
Copy Number Changes ◽  
The Impact ◽  
First Time ◽  
Tumor Dna

Abstract Single nucleotide polymorphisms (SNPs) have been long regarded as being important in determining variation and disease predisposition. Recently, chromosomal structural variation in the form of deletions, insertions and duplifications have been identified frequently in the genome of the general population. Such copy number variations (CNVs) have been shown to contribute to a range of human diseases. In recent studies we have utilized Affymetrix 50K and 500K arrays to identify acquired copy number change in myeloma tumor samples. In those studies we had access to paired constitutional DNA and in the present study have been able to report for the first time a CNV map of the constitutional genome of myeloma patients. Affymetrix 500K mapping arrays were used to identify copy number changes in 63 paired samples using DNA from peripheral blood and CD138 selected plasma cells. Tumor samples were analyzed in CNAG using both a paired and unpaired analysis to distinguish between inherited and acquired copy number change. Constitutional DNA was analyzed by both CNAG and GEMCA using 90 Caucasian samples from the Hapmap database as a reference set. For maximum calling accuracy, only those regions identified by both algorithms were called as CNVs. As with similar studies, overlapping CNVs identified using this approach were merged to generate a list of CNV regions (CNVRs) characteristic of the constitutional DNA of these myeloma cases. Using this approach, we identified 292 CNVs across 63 cases, with a median of 4 regions per sample. There were 155 discrete CNVRs, of which 46 were recurrent. The recurrent CNVRs were found most frequently in the pericentric regions of chromosome 14 and 15 in keeping with other studies. We then compared these recurrent CNVRs with a comparable dataset of normal individuals generated using Affymetrix 500K arrays. In this analysis, 25/46 recurrent CNVRs in the myeloma cases were novel. The two most frequent novel CNVRs in the myeloma cases were gains on chromosome 21 and 15. We also compared the characteristics of the constitutional CNVs with the acquired copy number changes in the corresponding tumor samples and identified that the constitutional CNVs were generally considerably smaller. However, using unpaired analysis it was possible to determine the presence of the constitutional CNV in the tumor sample, providing validation of the CNVs. We were also able to demonstrate that acquired copy number change in the tumor cells can either exaggerate or ameliorate the effect of the inherited CNV in the tumor genome, such as cases with acquired trisomy 15 and deletion or gain of regions of 15q in the constitutional DNA. These findings also reinforce the need for paired non-tumor DNA when undertaking copy number analysis of tumor DNA using SNP arrays. In this study we have been able to identify for the first time the presence of CNVs in the constitutional genome of individuals with myeloma. We have been able to systematically catalogue these CNVRs. These results provide the basis for future studies aimed at identifying how this type of genomic variation may influence the development of and outcome of myeloma and a broad range of other hematological conditions.

scholarly journals Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina

2018 ◽  
Vol 115 (4) ◽  
pp. 816-821 ◽  
Author(s):  
Benjamin Laenen ◽  
Andrew Tedder ◽  
Michael D. Nowak ◽  
Per Toräng ◽  
Jörg Wunder ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Mating System ◽  
Purifying Selection ◽  
Mating Strategies ◽  
Genomic Variation ◽  
The Arctic ◽  
Mixed Mating ◽  
Genome Wide ◽  
Arabis Alpina ◽  
The Impact

Plant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina. We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina. These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.

scholarly journals A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2229
Author(s):  
Ke Duan ◽  
Ying-Jie Zhao ◽  
Zi-Yi Li ◽  
Xiao-Hua Zou ◽  
Jing Yang ◽  
...  
Keyword(s):  
Agronomic Traits ◽  
Crop Improvement ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Sugar Transporter ◽  
Transporter Gene ◽  
Engineering Technology ◽  
Selectable Markers ◽  
Vector Backbone ◽  
First Time

Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

The impact of the difficult vascular access, fear, and anxiety level in children on the success of first-time phlebotomy

2018 ◽  
Vol 19 (6) ◽  
pp. 620-625 ◽  
Author(s):  
Gülçin Özalp Gerçeker ◽  
Dijle Ayar ◽  
Emine Zahide Özdemir ◽  
Murat Bektaş
Keyword(s):  
Vascular Access ◽  
Cross Sectional Study ◽  
Affecting Factors ◽  
Cross Sectional ◽  
Factors Affecting ◽  
Intravenous Access ◽  
Children’S Anxiety ◽  
Fear And Anxiety ◽  
The Impact ◽  
First Time

Purpose: This study aimed to investigate the success of first-time phlebotomy and the affecting factors in children between 4 and 10 years of age. Methods: This descriptive, comparative, and cross-sectional study was conducted on 155 children who underwent phlebotomy. The Sociodemographic Data Form, the Children’s Anxiety Meter–State, the Children’s Fear Scale, and the Difficult Intravenous Access score were used to collect the data for the study. The relationship between the success of first-time phlebotomy, mean pre-phlebotomy fear and anxiety score, and Difficult Intravenous Access score were examined. The variables affecting the success of first-time phlebotomy were assessed by regression analysis. Results: Phlebotomies failed in 18.1% of children. A statistically significant relationship was found between the success of first-time phlebotomy, Children’s Anxiety Meter–State, Children’s Fear Scale mean scores assessed by the researchers, and Difficult Intravenous Access score. Factors affecting the success of first-time phlebotomy include difficult vascular access, age, mean Children’s Anxiety Meter–State score, mean Difficult Intravenous Access score, and duration of the last phlebotomy performed. These factors explain 42% of the total factors affecting the success of first-time phlebotomy. Conclusion: Child’s fear, anxiety before phlebotomy, and difficult vascular access affects the first-time phlebotomy success.

scholarly journals Phenotypic factors associated with lamb live weight and carcass composition measurements in an Irish multi-breed sheep population1

2020 ◽  
Vol 4 (4) ◽  
Author(s):  
Fiona Mary McGovern ◽  
Noirin McHugh ◽  
Shauna Fitzmaurice ◽  
Thierry Pabiou ◽  
Kevin McDermott ◽  
...  
Keyword(s):  
Management Strategies ◽  
Live Weight ◽  
Carcass Composition ◽  
Sheep Population ◽  
Factors Affecting ◽  
Factors Associated ◽  
Ewe Lambs ◽  
Genetic Evaluations ◽  
The Impact ◽  
First Time

Abstract Understanding the phenotypic factors that affect lamb live weight and carcass composition is imperative to generating accurate genetic evaluations and further enables implementation of functional management strategies. This study investigated phenotypic factors affecting live weight across the growing season and traits associated with carcass composition in lambs from a multibreed sheep population. Four live weight traits and two carcass composition traits were considered for analysis namely; birth, preweaning, weaning, and postweaning weight, and ultrasound muscle depth and fat depth. A total of 427,927 records from 159,492 lambs collected from 775 flocks between the years 2016 and 2019, inclusive were available from the Irish national sheep database. Factors associated with live weight and carcass composition were determined using linear mixed models. The heaviest birth, preweaning, and weaning weights were associated with single born lambs (P < 0.001), however by postweaning, there was no difference observed in the weights of single and twin born lambs (P > 0.01). Breed class affected lamb live weight and carcass composition with terminal lambs weighing heaviest and having greater muscle depth than all other breed classes investigated (P < 0.001). Lambs born to first parity dams were consistently lighter, regardless of time of weighing (P < 0.001), while dams lambing for the first time as ewe lambs produced lighter lambs than those lambing for the first time as hoggets (P < 0.001). Greater heterosis coefficients (i.e., >90% and ≤100%) resulted in heavier lambs at weaning compared with lambs with lower levels of heterosis coefficients (P < 0.001). A heterosis coefficient class <10% resulted in lambs with greater muscle depth while recombination loss of <10% increased ultrasound fat depth (P < 0.001). Results from this study highlight the impact of multiple animal level factors on lamb live weight and carcass composition which will enable more accurate bio-economic models and genetic evaluations going forward.

scholarly journals Epigenetic age-predictions in mice using pyrosequencing, droplet digital PCR or barcoded bisulfite amplicon sequencing

2020 ◽  
Author(s):  
Yang Han ◽  
Miloš Nikolić ◽  
Michael Gobs ◽  
Julia Franzen ◽  
Gerald de Haan ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Model Organism ◽  
Amplicon Sequencing ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Biological Aging ◽  
Epigenetic Aging ◽  
Epigenetic Age ◽  
Trait Locus ◽  
The Impact

AbstractAge-associated DNA methylation reflects aspects of biological aging - therefore epigenetic clocks for mice can help to elucidate the impact of treatments or genetic background on the aging process in this model organism. Initially, age-predictors for mice were trained on genome-wide DNA methylation profiles, whereas we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three CG dinucleotides (CpGs). Here, we have re-evaluated pyrosequencing approaches in comparison to droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CpGs the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified in murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the epigenetic age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe further optimized and alternative targeted methods to determine epigenetic clocks in mice.

scholarly journals The Impact of Diversification of Production Activities by Major Public Oil Companies on the Value of Their Shares

2019 ◽  
Vol 13 (4) ◽  
pp. 7-18
Author(s):  
Evgeny Busygin
Keyword(s):  
Positive Impact ◽  
Internal Factors ◽  
Factors Affecting ◽  
Oil Companies ◽  
Market Participants ◽  
Investment Companies ◽  
Financial Indicator ◽  
The Impact ◽  
First Time ◽  
Profitability Ratios

The studies devoted to the analysis of the diversification of production activities of the largest public oil companies andits impact on their cost do not consider production or financial factors, which are important indicators for assessing thedevelopment prospects of companies.In this article, an econometric analysis will be carried out to identify the external and internal factors affecting thecapitalization of the largest vertically integrated oil companies, and for the first time, profitability ratios for each ofthem will be used to test the hypothesis about the positive impact of diversification of activities on the upstream anddownstream segments.As a result of the study, it was found that an increase in profitability in the upstream segment leads to an increase in thevalue of oil companies shares, while profitability in the downstream segment turned out to be an insignificant factor thatnegatively affects the dependent variable.The obtained results indicate that investors are more oriented to the financial indicator related to the production sector,ignoring the refining segment, which may lead to underestimation of oil companies and subsequent adjustments of stockprices.The final conclusions can be used by investment companies and other stock market participants as part of investmentdecision making process regarding the acquisition/sale of shares of large vertically integrated oil companies.As part of the development of a study on the valuation of oil companies, it could be analyzed the influence of the factorsconsidered in the work on firms that conduct production activities separately in upstream and downstream segments.

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