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Saliva DNA quality and genotyping efficiency in a predominantly elderly population.

ABSTRACT: The question of whether DNA obtained from saliva is an acceptable alternative to DNA from blood is a topic of considerable interest for large genetics studies. We compared the yields, quality and performance of DNAs from saliva and blood from a mostly elderly study population.Two thousand nine hundred ten DNAs from primarily elderly subjects (mean age ± standard deviation (SD): 65 ± 12 years), collected for the Primary Open-Angle African-American Glaucoma Genetics (POAAGG) study, were evaluated by fluorometry and/or spectroscopy. These included 566 DNAs from blood and 2344 from saliva. Subsets of these were evaluated by Sanger sequencing (n = 1555), and by microarray SNP genotyping (n = 94) on an Illumina OmniExpress bead chip platform.The mean age of subjects was 65, and 68 % were female in both the blood and saliva groups. The mean ± SD of DNA yield per ml of requested specimen was significantly higher for saliva (17.6 ± 17.8 ?g/ml) than blood (13.2 ± 8.5 ?g/ml), but the mean ± SD of total DNA yield obtained per saliva specimen (35 ± 36 ?g from 2 ml maximum specimen volume) was approximately three-fold lower than from blood (106 ± 68 ?g from 8 ml maximum specimen volume). The average genotyping call rates were >99 % for 43 of 44 saliva DNAs and >99 % for 50 of 50 for blood DNAs. For 22 of 23 paired blood and saliva samples from the same individuals, the average genotyping concordance rate was 99.996 %. High quality PCR Sanger sequencing was obtained from ? 98 % of blood (n = 297) and saliva (n = 1258) DNAs. DNA concentrations ?10 ng/?l, corresponding to total yields ? 2 ?g, were obtained for 94 % of the saliva specimens (n = 2344).In spite of inferior purity, the performance of saliva DNAs for microarray genotyping was excellent. Our results agree with other studies concluding that saliva collection is a viable alternative to blood. The potential to boost study enrollments and reduce subject discomfort is not necessarily offset by a reduction in genotyping efficiency. Saliva DNAs performed comparably to blood DNAs for PCR Sanger sequencing.

SUBMITTER: Gudiseva HV

PROVIDER: S-EPMC4823890 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Saliva DNA quality and genotyping efficiency in a predominantly elderly population.

Gudiseva Harini V HV Hansen Mark M Gutierrez Linda L Collins David W DW He Jie J Verkuil Lana D LD Danford Ian D ID Sagaser Anna A Bowman Anita S AS Salowe Rebecca R Sankar Prithvi S PS Miller-Ellis Eydie E Lehman Amanda A O'Brien Joan M JM

BMC medical genomics 20160407

<h4>Background</h4>The question of whether DNA obtained from saliva is an acceptable alternative to DNA from blood is a topic of considerable interest for large genetics studies. We compared the yields, quality and performance of DNAs from saliva and blood from a mostly elderly study population.<h4>Methods</h4>Two thousand nine hundred ten DNAs from primarily elderly subjects (mean age ± standard deviation (SD): 65 ± 12 years), collected for the Primary Open-Angle African-American Glaucoma Genet ...[more]

PMID: 27052975

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Project description:BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman™ and Illumina Beadchip™ genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek®) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by Gen-Probe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ?g, range 0.2-52 ?g) than from blood (mean 210 ?g, range 58-577 ?g) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.

Project description:BackgroundWhole-genome sequencing (WGS) is becoming an increasingly important tool for detecting genomic variation. Blood derived DNA is the current standard for WGS for research or clinical purposes but may not always be feasible to acquire. The usability of DNA from saliva for WGS is not known. We compared the quality of WGS between blood versus saliva derived DNA.MethodsWGS was performed in DNA from 531 blood and 502 saliva samples (including 5 paired samples) from participants enrolled in a heart disease biorepository. We compared the proportion of sequencing reads that mapped to non-human sources (microbiome), the sequencing coverage, and the yield and concordance of single nucleotide variant (SNV) and copy number variant (CNV) calls between blood and saliva genomes.ResultsOf 531 blood and 502 saliva samples, 46% saliva DNA failed quality control (QC) requirements for WGS compared to 6% QC failure for blood DNA. An average of 10.7% WGS reads in the saliva samples mapped to the human oral microbiome compared to 0.09% WGS reads in blood samples. However, these reads were readily excluded by excluding reads that did not map to the human reference genome. Sequencing coverage met or exceeded the target sequencing depth of 30x in all the blood samples and 4 of the 5 saliva samples; the fifth saliva sample had an average sequencing depth of 22.6x. Over 95% of SNVs identified in saliva were concordant with those identified in blood across the genome, within all gene coding regions, and within cardiovascular disease-related gene coding regions. Rare SNVs, defined as those with a minor allele frequency of less than 1% in the Genome Aggregation Database, had a lower concordance of 90% between blood and saliva genomes. CNVs had only 76% concordance between blood and saliva samples.ConclusionsHigh quality saliva samples that meet stringent QC criteria can be used for WGS when blood-derived DNA is not available or is not suitable. Saliva DNA provides an acceptable yield of SNV calls but has a lower yield for CNV calls compared to blood DNA.

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Saliva DNA quality and genotyping efficiency in a predominantly elderly population.

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Saliva DNA quality and genotyping efficiency in a predominantly elderly population.

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