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

Han T, Chang CW, Kwekel JC, Chen Y, Ge Y, Martinez-Murillo F, Roscoe D, Težak Z, Philip R, Bijwaard K, Fuscoe JC - BMC Genomics (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Systems Biology, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA. Tao.Han@fda.hhs.gov

ABSTRACT

Background: Genotyping 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.

Results: To 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.

Conclusion: The 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.

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Quantitative real time PCR copy number assays. Taqman assays were used to examine copy number variations at 10 locations within the CFTR gene in both the unamplified and amplified DNA samples. Average calculated copy number values are plotted with bars representing minimum and maximum values from replicate measurements (n = 4). Within each amplification / sample group, the order of the probe results is I- X (see Figure 4). UnAmp (no amplification); Amp-R (REPLI-g amplified); Amp-G (GenomiPhi amplified)
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Figure 5: Quantitative real time PCR copy number assays. Taqman assays were used to examine copy number variations at 10 locations within the CFTR gene in both the unamplified and amplified DNA samples. Average calculated copy number values are plotted with bars representing minimum and maximum values from replicate measurements (n = 4). Within each amplification / sample group, the order of the probe results is I- X (see Figure 4). UnAmp (no amplification); Amp-R (REPLI-g amplified); Amp-G (GenomiPhi amplified)

Mentions: Subsequent to array analysis, a quantitative PCR method was used to further evaluate the variability between the amplified and unamplified DNA samples. Ten TaqMan Copy Number PCR probes were selected from Applied Biosystem’s pre-designed research assays across the length of the CFTR gene, a well-studied genetic disease locus, representing 9 introns and 1 exon (Table 9 and Figure 4). Relative copy number was calculated for each probe in reference to the unamplified DNA sample for each subject. Each of the ten probes gave comparable results showing less than 2-fold difference (considering both over- and under-amplification) in fold-change between amplified and unamplified DNA samples (Figure 5). This low level of variability was also evident when comparing results between the two amplification methods (REPLI-g and GenomiPhi), suggesting comparable results irrespective of the manufacturer’s protocol. Furthermore, the amount of variability across the three human samples was similar, suggesting robust amplification results regardless of possible biological differences. Thus, the TaqMan Copy Number PCR results are in agreement with the aCGH data (Figure 3) in identifying low levels (below 2-fold change) of amplification variability introduced by the WGA process in the CFTR locus.


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

Han T, Chang CW, Kwekel JC, Chen Y, Ge Y, Martinez-Murillo F, Roscoe D, Težak Z, Philip R, Bijwaard K, Fuscoe JC - BMC Genomics (2012)

Quantitative real time PCR copy number assays. Taqman assays were used to examine copy number variations at 10 locations within the CFTR gene in both the unamplified and amplified DNA samples. Average calculated copy number values are plotted with bars representing minimum and maximum values from replicate measurements (n = 4). Within each amplification / sample group, the order of the probe results is I- X (see Figure 4). UnAmp (no amplification); Amp-R (REPLI-g amplified); Amp-G (GenomiPhi amplified)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403925&req=5

Figure 5: Quantitative real time PCR copy number assays. Taqman assays were used to examine copy number variations at 10 locations within the CFTR gene in both the unamplified and amplified DNA samples. Average calculated copy number values are plotted with bars representing minimum and maximum values from replicate measurements (n = 4). Within each amplification / sample group, the order of the probe results is I- X (see Figure 4). UnAmp (no amplification); Amp-R (REPLI-g amplified); Amp-G (GenomiPhi amplified)
Mentions: Subsequent to array analysis, a quantitative PCR method was used to further evaluate the variability between the amplified and unamplified DNA samples. Ten TaqMan Copy Number PCR probes were selected from Applied Biosystem’s pre-designed research assays across the length of the CFTR gene, a well-studied genetic disease locus, representing 9 introns and 1 exon (Table 9 and Figure 4). Relative copy number was calculated for each probe in reference to the unamplified DNA sample for each subject. Each of the ten probes gave comparable results showing less than 2-fold difference (considering both over- and under-amplification) in fold-change between amplified and unamplified DNA samples (Figure 5). This low level of variability was also evident when comparing results between the two amplification methods (REPLI-g and GenomiPhi), suggesting comparable results irrespective of the manufacturer’s protocol. Furthermore, the amount of variability across the three human samples was similar, suggesting robust amplification results regardless of possible biological differences. Thus, the TaqMan Copy Number PCR results are in agreement with the aCGH data (Figure 3) in identifying low levels (below 2-fold change) of amplification variability introduced by the WGA process in the CFTR locus.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Systems Biology, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA. Tao.Han@fda.hhs.gov

ABSTRACT

Background: Genotyping 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.

Results: To 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.

Conclusion: The 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.

Show MeSH
Related in: MedlinePlus