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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.
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,
Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0)
Katie A Clark, Patrick J Krysan
Plant Methods , 2007, DOI: 10.1186/1746-4811-3-8
Abstract: The key innovation underlying Ice-Cap 2.0 is the development of a continuous watering system. The addition of the watering system allows the seedling growth plates to be incubated without a lid for the duration of the growth period, which in turn allows for much more uniform and robust seedling growth than was observed using the original method. We also determined that inserting wooden skewers between the upper and lower plates prior to tissue harvest made it easier to separate the plates following freezing. Seedlings grown using the Ice-Cap 2.0 method remain viable in the Ice-Cap plates twice as long as seedlings grown using the original method.The continuous watering system that we have developed provides an effective solution to the problem of sub-optimal seedling growth that can be encountered when using the originally described Ice-Cap system. This novel watering system and several additional modifications to the Ice-Cap procedure have improved the robustness and utility of the method.Plant scientists increasingly need to genotype large numbers of individual plants as a part of their research programmes. For example, when performing map-based cloning to locate the genetic lesion responsible for a specific phenotype, it is often necessary to genotype thousands of individual plants from segregating populations. For crop species, the process of marker-assisted selection can involve genotyping several thousand individuals in a large population. To optimize the efficiency with which these types of experiments can be performed, it is necessary to develop a streamlined plant genotyping pipeline. We previously described a method named "Ice-Cap" to address these needs [1]. Ice-Cap is a novel high-throughput system for growing plant seedlings and collecting tissue in 96-well plate format. The key innovation in Ice-Cap is that tissue harvest for all 96 seedlings is performed in parallel, so that single plant manipulations are not necessary. This feature of Ice-Cap sets it
Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival samples
Anne Boissière, Céline Arnathau, Christophe Duperray, Laurence Berry, Laurence Lachaud, Fran?ois Renaud, Patrick Durand, Franck Prugnolle
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-163
Abstract: The method described allows isolation of Plasmodium falciparum trophozoites, genotyping and whole genome sequencing from the blood of infected patients. For trophozoite isolation, parasite and host nuclei are stained using propidium iodide (PI) followed by flow cytometry and cell sorting to separate trophozoites from host cells. Before genotyping or sequencing, whole genome amplification is used to increase the amount of DNA within sorted samples. The method has been specifically designed to deal with frozen blood samples.The results demonstrate that single trophozoite genotyping is possible and that cell sorting can be successfully applied to reduce the contaminating host DNA for subsequent whole genome sequencing of parasites extracted from infected blood samples.
Genotyping Performance between Saliva and Blood-Derived Genomic DNAs on the DMET Array: A Comparison  [PDF]
Yueshan Hu, Erik A. Ehli, Kelly Nelson, Krista Bohlen, Christophina Lynch, Patty Huizenga, Julie Kittlelsrud, Timothy J. Soundy, Gareth E. Davies
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0033968
Abstract: The Affymetrix Drug Metabolism Enzymes and Transporters (DMET) microarray is the first assay to offer a large representation of SNPs conferring genetic diversity across known pharmacokinetic markers. As a convenient and painless alternative to blood, saliva samples have been reported to work well for genotyping on the high density SNP arrays, but no reports to date have examined this application for saliva-derived DNA on the DMET platform. Genomic DNA extractions from saliva samples produced an ample quantity of genomic DNA for DMET arrays, however when human amplifiable DNA was measured, it was determined that a large percentage of this DNA was from bacteria or fungi. A mean of 37.3% human amplifiable DNA was determined for saliva-derived DNAs, which results in a significant decrease in the genotyping call rate (88.8%) when compared with blood-derived DNAs (99.1%). More interestingly, the percentage of human amplifiable DNA correlated with a higher genotyping call rate, and almost all samples with more than 31.3% human DNA produced a genotyping call rate of at least 96%. SNP genotyping results for saliva derived DNA (n = 39) illustrated a 98.7% concordance when compared with blood DNA. In conclusion, when compared with blood DNA and tested on the DMET array, saliva-derived DNA provided adequate genotyping quality with a significant lower number of SNP calls. Saliva-derived DNA does perform very well if it contains greater than 31.3% human amplifiable DNA.
High-Throughput SNP Genotyping  [PDF]
Suzanne Jenkins,Neil Gibson
Comparative and Functional Genomics , 2002, DOI: 10.1002/cfg.130
Abstract: Whole genome approaches using single nucleotide polymorphism (SNP) markers have the potential to transform complex disease genetics and expedite pharmacogenetics research. This has led to a requirement for high-throughput SNP genotyping platforms. Development of a successful high-throughput genotyping platform depends on coupling reliable assay chemistry with an appropriate detection system to maximise efficiency with respect to accuracy, speed and cost. Current technology platforms are able to deliver throughputs in excess of 100 000 genotypes per day, with an accuracy of >99%, at a cost of 20–30 cents per genotype. In order to meet the demands of the coming years, however, genotyping platforms need to deliver throughputs in the order of one million genotypes per day at a cost of only a few cents per genotype. In addition, DNA template requirements must be minimised such that hundreds of thousands of SNPs can be interrogated using a relatively small amount of genomic DNA. As such, it is predicted that the next generation of high-throughput genotyping platforms will exploit large-scale multiplex reactions and solid phase assay detection systems.
Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping
B Jayashree, Praveen T Reddy, Y Leeladevi, Jonathan H Crouch, V Mahalakshmi, Hutokshi K Buhariwalla, KE Eshwar, Emma Mace, Rolf Folksterma, S Senthilvel, Rajeev K Varshney, K Seetha, R Rajalakshmi, VP Prasanth, Subhash Chandra, L Swarupa, P SriKalyani, David A Hoisington
BMC Bioinformatics , 2006, DOI: 10.1186/1471-2105-7-383
Abstract: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics.A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/gt-bt/lims/lims.asp webcite.Plant genotyping is a technology that has widespread applications in the fields of breeding, research and commerce. With the increasing availability of sequence information, more and more molecular markers are becoming available for use in plant genotyping; for example SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms) [1]. These markers are widely used in screening genebank collections of cultivated and wild germplasm, genome mapping for traits of interest and marker assisted selectio
Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples
Zsofia Nemoda, Maria Horvat-Gordon, Christine K Fortunato, Emilie K Beltzer, Jessica L Scholl, Douglas A Granger
BMC Medical Research Methodology , 2011, DOI: 10.1186/1471-2288-11-170
Abstract: Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses.The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible.Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.In the wake of the Human Genome Project, information from Genome Wide Association (GWA) studies is accumulating at a rapid rate. GWA studies include large numbers of well-characterized cases and several hundred thousand polymorphisms in an attempt to identify candidate genes with plausible linkag
Detection of hepatitis C virus RNA in saliva samples from patients with seric anti-HCV antibodies
Gon?alves, Patrícia L.;Cunha, Carla B.;Busek, Solange C. U.;Oliveira, Guilherme C.;Ribeiro-Rodrigues, Rodrigo;Pereira, Fausto EL;
Brazilian Journal of Infectious Diseases , 2005, DOI: 10.1590/S1413-86702005000100006
Abstract: we examined the frequency of hcv-rna in saliva samples from anti-hcv positive patients. both plasma and saliva samples from 39 hcv patients (13 with normal liver enzymes, 19 with abnormal liver enzymes and 13 with cirrhosis) were investigated. stimulated saliva and fresh plasma were centrifuged (900 x g,10 min) and stored at -70oc, after the addition of guanidine isothiocyanate rna extraction buffer. hcv-rna was detected by rt- nested-pcr (amplification of hcv-cdna for two rounds, using hcv primers 939/209 and 940/211). hcv genotyping was carried out by rflp (using mva i and hinf 1 or hae iii and rsa i restriction enzymes). thirty-two out of 39 (82%; 95% ci=70-94%) anti-hcv-positive patients had hcv-rna in plasma samples. eight out of 39 (20.5%; 95% ci=6.6-34.4%) had hcv-rna in the saliva. the hcv genotype in saliva samples from these patients matched the genotype found for plasma hcv-rna. no significant correlation between the presence of hcv and either age, gender, hcv genotype or any risk factor for hcv infection was found. the observed prevalence (20.5% of anti hcv positive patients or 25% of the patients with hcv-rna in plasma) was lower than that previously reported from other countries. the low frequency of hcv-rna in saliva samples observed in our study may be due to the use of cell-free saliva. other authors reporting higher frequencies of hcv-rna in saliva used whole saliva, without centrifugation.
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