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Saliva as an alternative source of high yield canine genomic DNA for genotyping studies
Katherine Mitsouras, Erica A Faulhaber
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-219
Abstract: Comparison of DNA yields from matched saliva, blood and buccal swab samples showed that yields from saliva were significantly higher than those from blood (p = 0.0198) or buccal swabs (p = 0.0008). Electrophoretic analysis revealed that blood and saliva produced higher quality DNA than buccal swabs. In addition, a 1.1-kb PCR fragment was successfully amplified using the paired DNA samples and genotyping by PCR-RFLP yielded identical results.We demonstrate that DNA yields from canine saliva are higher than those from blood or buccal swabs. The quality of DNA extracted from saliva is sufficient for successful amplification of a 1.1-kb fragment and for accurate SNP genotyping by PCR-RFLP. We conclude that saliva presents a non-invasive alternative source of high quantities of canine genomic DNA suitable for genotyping studies.The domestic dog (Canis familiaris) has emerged as a model organism to investigate the genetic basis of both normal and pathological traits. Due to controlled breeding practices within breed clubs, modern breeds are closed gene pools, with low levels of genetic variation within each breed [1]. This is in contrast to human populations where levels of genetic variation are high, rendering the identification of disease genes a challenge [1]. The genetic structure of the dog, combined with the number of genetic disorders shared among canines and humans make the dog an ideal system to study the genetic basis of disease [2]. Furthermore, sequencing of the canine genome, completion of the canine SNP map, and the development of high-throughput canine genotyping platforms, such as the Affymetrix canine SNP array (Affymetrix, Santa Clara, CA, USA) have resulted in the creation of the same technological platforms that accelerated discovery in the human genome [2]. This, in turn, has created the need for a canine sample collection method that yields sufficient quantities of high quality genomic DNA that will perform well in downstream applications.Currently,
Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development
Tao Han, Ching-Wei Chang, Joshua C Kwekel, Ying Chen, Yun Ge, Francisco Martinez-Murillo, Donna Roscoe, ?ivana Te?ak, Reena Philip, Karen Bijwaard, James C Fuscoe
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-217
Abstract: To assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA.The relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.
Genome profiling of ERBB2-amplified breast cancers
Fabrice Sircoulomb, Ismahane Bekhouche, Pascal Finetti, José Adéla?de, Azza Hamida, Julien Bonansea, Stéphane Raynaud, Charlène Innocenti, Emmanuelle Charafe-Jauffret, Carole Tarpin, Farhat Ayed, Patrice Viens, Jocelyne Jacquemier, Fran?ois Bertucci, Daniel Birnbaum, Max Chaffanet
BMC Cancer , 2010, DOI: 10.1186/1471-2407-10-539
Abstract: We defined the high resolution genome and gene expression profiles of 54 ERBB2-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions.First, we identified the ERBB2-C17orf37-GRB7 genomic segment as the minimal common 17q12-q21 amplicon, and CRKRS and IKZF3 as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA) (amplifications, gains, losses). The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in ERBB2-amplified BCs. The genomic profiles of estrogen receptor-postive (ER+) and negative (ER-) ERBB2-amplified BCs were different. The WNT/β-catenin signaling pathway was involved in ER- ERBB2-amplified BCs, and PVT1 and TRPS1 were candidate oncogenes associated with ER+ ERBB2-amplified BCs. The size of the ERBB2 amplicon was different in inflammatory (IBC) and non-inflammatory BCs. ERBB2-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i) a linear relationship between ERBB2 gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii) a potential signaling cross-talk between EGFR or IGF1R and ERBB2, which could influence response of ERBB2-positive BCs to inhibitors. FOXA1 was frequently coexpressed with ERBB2 but its expression did not impact on the outcome of patients with ERBB2-amplified tumors.We have shown that ER+ and ER- ERBB2-amplified BCs are different, distinguished ERBB2 amplicons in IBC and non-IBC, and identified genomic features that may be useful in the desig
An integrated 4249 marker FISH/RH map of the canine genome
Matthew Breen, Christophe Hitte, Travis D Lorentzen, Rachael Thomas, Edouard Cadieu, Leah Sabacan, Allyson Scott, Gwenaelle Evanno, Heidi G Parker, Ewen F Kirkness, Ruth Hudson, Richard Guyon, Gregory G Mahairas, Boris Gelfenbeyn, Claire M Fraser, Catherine André, Francis Galibert, Elaine A Ostrander
BMC Genomics , 2004, DOI: 10.1186/1471-2164-5-65
Abstract: To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS.These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps.Three major advances in the development of resources for mapping canine disease genes have been: 1) the development of a radiation hybrid (RH) map composed of large numbers of microsatellite markers and genes that link the canine and human genomes [1], 2) the development of canine specific whole chromosome paints that have allowed preliminary assignment of conserved segments between human and dog [3-5]; and 3) the publication of a 1.5x genome sequence of the dog [2]. The most recently published RH map of the dog comprises 3270 markers including 1596 microsatellite-based markers, 900 canine-specific cloned gene sequences and expressed sequence tags (ESTs), and an initial set of 668 canine-specific BAC-ends [1]. The map was constructed using the RHDF5000-2 whole genome radiation hybrid panel [6] and features markers mapped to 3009 unique positions, defining an av
Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing  [cached]
ElSharawy Abdou,Warner Jason,Olson Jeff,Forster Michael
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-500
Abstract: Background Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. Results The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. Conclusion Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.
Array-Based Whole-Genome Survey of Dog Saliva DNA Yields High Quality SNP Data  [PDF]
Jennifer S. Yokoyama,Carolyn A. Erdman,Steven P. Hamilton
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010809
Abstract: Genome-wide association scans for genetic loci underlying both Mendelian and complex traits are increasingly common in canine genetics research. However, the demand for high-quality DNA for use on such platforms creates challenges for traditional blood sample ascertainment. Though the use of saliva as a means of collecting DNA is common in human studies, alternate means of DNA collection for canine research have instead been limited to buccal swabs, from which dog DNA is of insufficient quality and yield for use on most high-throughput array-based systems. We thus investigated an animal-based saliva collection method for ease of use and quality of DNA obtained and tested the performance of saliva-extracted canine DNA on genome-wide genotyping arrays.
Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples
Stephanie J. Loomis, Lana M. Olson, Louis R. Pasquale, Janey Wiggs, Daniel Mirel, Andrew Crenshaw, Melissa Parkin, Brandon Rahhal, Stephanie Tetreault, Peter Kraft, Shelley S. Tworoger, Jonathan L. Haines and Jae H. Kang
Biomarker Insights , 2012, DOI: 10.4137/BMI.S5062
Abstract: It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses’ Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (.97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (.95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform.
Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples
Stephanie J. Loomis,Lana M. Olson,Louis R. Pasquale,Janey Wiggs
Biomarker Insights , 2010,
Abstract: It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses’ Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (.97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (.95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform.
Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping
Kristine L Bucasas, Gagan A Pandya, Sonal Pradhan, Robert D Fleischmann, Scott N Peterson, John W Belmont
BMC Genetics , 2009, DOI: 10.1186/1471-2156-10-85
Abstract: Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold.We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.Array technologies are designed to rapidly genotype hundreds of thousands of single nucleotide polymorphisms (SNPs) across the genome using a relatively small amount of DNA. Advances in genotyping platforms have allowed cost-effective, whole-genome scans of multiple individuals in large-scale association studies. These studies are aimed at identifying genetic factors affecting many important complex human diseases. DNA samples from carefully characterized populations that are necessary to carry out adequately powered genome-wide association studies (GWAS), however, are often limiting. Collecting a sufficient number of appropriate samples for GWAS can be a complex and expensive collaborative challenge. There are potential alternative sources of genomic DNA for which important medical phenotypes have been recorded but their utility for GWAS has
Influence of anatomic reference on the buccal contour of prosthetic crowns
Vasconcelos, Flávia Sabrina Queirós;Neves, Ana Christina Claro;Silva-Concílio, Laís Regiane da;Cunha, Leonardo Gon?alves;Rode, Sigmar de Mello;
Brazilian Oral Research , 2009, DOI: 10.1590/S1806-83242009000300002
Abstract: during clinical practice, when performing prosthetic rehabilitation with single crowns, improper reproduction of the dental contour by the dental laboratory is a common occurrence. therefore, the present study evaluated the fidelity of the reproduction of the buccal contour in an upper left canine performed by three dental prosthesis technicians (dpt) using the indirect laminate veneer technique. first, the dpts confected the veneers based on a model obtained from the upper arch of a dental dummy, containing a replica of an upper left canine with a prosthetic preparation for a laminate veneer. then, the same dpts received other identical models, now with the replica of the upper left canine with no preparation, to be used as an anatomical reference for confecting the laminate veneers. the laminate veneers were then bonded to the plaster models and had their buccal contour individually measured. measurements were also made of the buccal contour of the reference canine. the data were analyzed by anova and the t-test (p = 0.05). results showed 100% of buccal overcontour when the laminate veneers were compared to the reference canine, regardless of which dpt confected the veneer and regardless of using or not the anatomical reference. the dpts who participated in the present study were unable to acomplish a faithful anatomical reproduction of the buccal contour, creating an overcontour in all samples. this situation may be responsible for increasing the probability of periodontal and esthetic harm in clinical practice.
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