Dataset Information

Quantitative criteria for improving performance of buccal DNA for high-throughput genetic analysis.

ABSTRACT: DNA from buccal brush samples is being used for high-throughput analyses in a variety of applications, but the impact of sample type on genotyping success and downstream statistical analysis remains unclear. The objective of the current study was to determine laboratory predictors of genotyping failure among buccal DNA samples, and to evaluate the successfully genotyped results with respect to analytic quality control metrics. Sample and genotyping characteristics were compared between buccal and blood samples collected in the population-based Genetic and Environmental Risk Factors for Hemorrhagic Stroke (GERFHS) study (https://gerfhs.phs.wfubmc.edu/public/index.cfm).Seven-hundred eight (708) buccal and 142 blood DNA samples were analyzed for laboratory-based and analysis metrics. Overall genotyping failure rates were not statistically different between buccal (11.3%) and blood (7.0%, p?=?0.18) samples; however, both the Contrast Quality Control (cQC) rate and the dynamic model (DM) call rates were lower among buccal DNA samples (p?

SUBMITTER: Woo JG

PROVIDER: S-EPMC3447646 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Quantitative criteria for improving performance of buccal DNA for high-throughput genetic analysis.

Woo Jessica G JG Martin Lisa J LJ Ding Lili L Brown W Mark WM Howard Timothy D TD Langefeld Carl D CD Moomaw Charles J CJ Haverbusch Mary M Sun Guangyun G Indugula Subba R SR Cheng Hong H Deka Ranjan R Woo Daniel D

BMC genetics 20120825

<h4>Background</h4>DNA from buccal brush samples is being used for high-throughput analyses in a variety of applications, but the impact of sample type on genotyping success and downstream statistical analysis remains unclear. The objective of the current study was to determine laboratory predictors of genotyping failure among buccal DNA samples, and to evaluate the successfully genotyped results with respect to analytic quality control metrics. Sample and genotyping characteristics were compare ...[more]

PMID: 22920755

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Project description:Filter aided sample preparation (FASP) is becoming a central method for proteomic sample cleanup and peptide generation prior to LC-MS analysis. We previously adapted this method to a 96-well filter plate, and applied to prepare protein digests from cell lysate and body fluid samples in a high throughput quantitative manner. While the 96FASP approach is scalable and can handle multiple samples simultaneously, two key advantages compared to single FASP, it is also time-consuming. The centrifugation-based liquid transfer on the filter plate takes 3-5 times longer than single filter. To address this limitation, we now present a quick 96FASP (named q96FASP) approach that, relying on the use of filter membranes with a large MWCO size (~30kDa), significantly reduces centrifugal times. We show that q96FASP allows the generation of protein digests derived from whole cell lysates and body fluids in a quality similar to that of the single FASP method. Processing a sample in multiple wells in parallel, we observed excellent experimental repeatability by label-free quantitation approach. We conclude that the q96FASP approach promises to be a promising cost- and time-effective method for shotgun proteomics and will be particularly useful in large scale biomarker discovery studies. SIGNIFICANCE:High throughput sample processing is of particular interests for quantitative proteomics. The previously developed 96FASP is high throughput and appealing, however it is time-consuming in the context of centrifugation-based liquid transfer (~1.5h per spin). This study presents a truly high throughput sample preparation method based on large cut-off 96-well filter plate, which shortens the spin time to ~20min. To our knowledge, this is the first multi-well method that is entirely comparable with conventional FASP. This study thoroughly examined two types of filter plates and performed side-by-side comparisons with single FASP. Two types of samples, whole cell lysate of a UTI (urinary tract infection)-associated Klebsiella pneumoniae cell and human urine, were tested which demonstrated its capability for quantitative proteomics. The q96FSAP approach makes the filter plate-based approach more appealing for protein biomarker discovery projects, and could be broadly applied to large scale proteomics analysis.

Dataset Information

Quantitative criteria for improving performance of buccal DNA for high-throughput genetic analysis.

Publications

Quantitative criteria for improving performance of buccal DNA for high-throughput genetic analysis.

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