DNA quantification of whole genome amplified samples for genotyping on a multiplexed bead array platform

Area of Science:

• Genomics and Molecular Biology
• Genetic Epidemiology
• Biotechnology

Background:

• Whole genome amplification (WGA) is crucial for genotyping scarce DNA samples in genetic epidemiology.
• WGA can generate nucleic acid byproducts, potentially compromising DNA quantification and downstream analyses.
• Effective quality control of whole genome amplified DNA (wgaDNA) is essential but lacks standardized quantification methods.

Purpose of the Study:

• To evaluate and compare four distinct quantification methods for wgaDNA.
• To determine the best method for assessing wgaDNA suitability prior to high-throughput genotyping.
• To establish a reliable quality control measure for wgaDNA used in genetic studies.

Main Methods:

• Quantified wgaDNA from 54 buccal/blood samples using UV absorbance, PicoGreen fluorescence (calibrated with lambda bacteriophage or calf thymus DNA), and real-time PCR (Alu Yd6).
• Genotyped wgaDNA and paired high-quality genomic DNA samples using a 384-plex Illumina Golden Gate Panel.
• Correlated quantification results with genotyping concordance rates.

Main Results:

• Genotyping concordance varied: 39 samples showed high (>99%), 7 moderate (90-99%), and 8 poor (≤90%) agreement between wgaDNA and genomic DNA.
• Absolute quantification via human-specific real-time PCR of Alu Yd6 successfully differentiated wgaDNA samples based on concordance levels.
• wgaDNA quantities correlated with concordance: high concordance (4.14-118.32 ng/µL), moderate (0.29-2.19 ng/µL), and poor (0.01-0.27 ng/µL).

Conclusions:

• Human-specific quantitative PCR targeting Alu sequences is a superior method for wgaDNA quantification.
• This qPCR method effectively predicts wgaDNA suitability for high-throughput SNP genotyping.
• Implementing this qPCR-based quality control enhances the reliability of genetic epidemiology studies using WGA.