Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development

Abstract

BackgroundGenotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized.ResultsTo 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.ConclusionThe 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.

Partition Enrichment of Nucleotide Sequences (PINS) - A Generally Applicable, Sequence Based Method for Enrichment of Complex DNA Samples

“…This observation correlates well with the low error rate previously reported for Phi29 [17], and is of high importance for the final evaluation of the sequences.…”

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations

“…Previous studies have demonstrated significant under-amplification bias in regions with high GC content and subtelomeric regions (14,22). To remove false-positive calls due to amplification bias, we evaluated each CPE-unique (490 SCNA), CAS-unique (26 SCNA) and CPE/CAS-overlapping (13 SCNAs) regions for GC content and chromosomal location.…”

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

“…Prior to the molecular analyses the genomic DNA isolated from the preserved material was randomly amplified using a DNA-amplification kit in order to ensure that we would have sufficient amounts of genomic DNA for PCR-optimization and potential future molecular analyses. This method has proven to be applicable for reproducible random amplification of genomic DNA (Han et al, 2012), while highly GC-rich regions might be amplified with slightly less efficiency. The region amplified in this study is not particularly GC-rich.…”