BACKGROUND
advances in genomic technologies have enabled development of many novel genome analysis methods that may have applications in the understanding, diagnosis, and management of genetic diseases and cancer.
comprehensive high-throughput assays are available for detection of single nucleotide polymorphisms   <cit> , dna copy number variation   <cit> , microsatellite expansion or contraction  <cit>  and loss of heterozygosity   <cit> , all of which detect sometimes subtle genomic alterations associated with disease.
some of these assays require micrograms of dna which may be difficult to obtain for many clinical samples.
in addition, inadequate dna template may prevent the performance of multiple assays on a single sample  <cit> .
limited availability of dna also poses challenges for manufacturers and regulators of genetic diagnostic devices.
one of these challenges is availability of sufficient quantities of dna samples to appropriately validate a particular test under investigation, especially when uncommon mutations for a rare disease severely limit access to patient sample specimens.
since the u.s. food and drug administration ’s clearance of the first genotyping assay for cystic fibrosis  in  <dig>  the office of in vitro diagnostic device evaluation and safety within the center for devices and radiological health  has reviewed a number of other genotyping assays for inherited disorders.
some of these genotyping assays are intended to detect relatively rare heritable diseases consisting of multiple disease-causing alleles  for each disease, while others may detect more common diseases, but are intended to test very rare as well as common mutations.
to show the accuracy of these assays, manufacturers would generally use patient samples .
for rare mutations or alleles, it is often difficult to obtain sufficient quantities of such clinical samples to adequately assess test performance.
therefore, there is an interest in using whole genome amplified  dna samples created from patient samples instead of native dna samples to increase the availability of appropriate samples to query the performance of the assays.
wga is a method that amplifies small amounts of genomic dna several thousand-fold in vitro.
the wga process has the potential, however, to result in non-uniform amplification of the dna in which some regions of the genome are over-represented and others are under-represented.
such biased amplification could make the wga dna unsuitable for some of the studies designed to assess the clinical assay performance.
several wga methods based on the polymerase chain reaction  with taq polymerase were initially developed  <cit> .
these methods included the use of primers directed at highly repetitive sequences  <cit> , ligation of linkers to fragmented dna  <cit> , degenerate oligonucleotide primed pcr  <cit> , and primer extension preamplification  <cit> .
all of these methods suffer from a relatively high level of mutations in the amplified dna  and highly non-uniform amplification due to the low fidelity and low processivity of the taq polymerase, respectively.
in  <dig>  dean et al.
<cit>  described the multiple displacement amplification  technique.
this method of wga takes advantage of the high processivity and low error rate of the Φ <dig> bacteriophage dna polymerase.
this polymerase has a 3’-5’ proof-reading activity and adds an average of  <dig>  templated nucleotides to a primer  <cit> , resulting in higher fidelity and less biased amplification than with the taq polymerase methods.
the wga process using Φ <dig> polymerase is isothermal and uses random primers to target the entire genome.
the polymerase has strong strand displacement activity so that exponential amplification occurs through a branching mechanism  <cit> , resulting in a high yield of dna.
the mda process has been recently reviewed and shown to be superior to other dna amplification methods with regard to genotyping, genomic coverage, and amplification bias  <cit> .
mda-based wga has been frequently used in dna sample preparation for genotyping and sequencing in recent years  <cit> .
high call rates  and excellent concordance rates were achieved from wga samples using high-density snp arrays  <cit> .
there was a very low error rate  when high quality dna was used as template  <cit> .
mda-based wga has also been used for analyses of single cells  <cit> .
jiang et al.
successfully amplified dna from single sperm at least  <dig> fold with a single round of mda  <cit> .
dna amplified from single lymphocytes was used for multiple analyses of  <dig> different loci including the Δf <dig> deletion in exon  <dig> and two intragenic microsatellite markers in the cf gene  <cit> .
short tandem repeats  and human leucocyte antigen typing were performed using dna amplified by mda from a single cell  <cit> .
mda-wga has also been increasingly used in the field of forensic testing  <cit> .
even in the often degraded dna samples, wga showed the capability and potential to increase the quality and quantity of dna from difficult samples in forensic casework  <cit> .
although mda-based wga has been successfully applied in many studies, there are several potential problems that may affect interpretation of results.
these include the finding that large amounts of nonspecific dna amplification can be generated during mda, mostly due to primer-directed dna synthesis  <cit> , and the quantity  <cit>  and quality  <cit>  of input dna into the mda reaction can affect genotyping results.
despite the advantage of the mda-based wga method, the uniformity of the amplification process across the genome has not been extensively characterized  <cit> .
in  <dig>  paez et al.
<cit>  examined dnas amplified using a commercially available mda method, repli-g, using 10 k affymetrix snp arrays and direct sequencing of ~ <dig>  bp of dna and showed near-complete genome representation, as well as low replication error rate.
in  <dig>  pinard et al.
<cit>  assessed the bias of wga methods on bacterial genomes using massively parallel sequencing and found statistically significant amplification bias, although the mda methods produced the least bias.
arriola et al.
<cit> , using a second commercially available mda method, genomiphi, evaluated the wga dna with low density array comparative genomic hybridization , a method with higher resolution  than the traditional cgh method  <cit> .
copy number biases were found, with the extent dependent on the degree of amplification.
while these studies and others  <cit>  have consistently shown non-uniformity of amplification by the mda methods , the uniformity of amplification has not been examined with high resolution acgh at a resolution of several kb.
in addition, the consistency of the amplification process has not been evaluated in replicate amplifications of the same sample, an important parameter when such material may be used for validation of genetic diagnostic devices or tests.
in this study, we evaluated two commonly used methods of wga for their ability to produce large quantities of uniformly amplified dna with minimal introduced mutations.
biases during the amplification process were determined by measurements of dna cnvs at nearly  <dig> million positions in the genome using acgh analysis.
particular attention was given to a single gene, cftr, which encodes cystic fibrosis transmembrane conductance regulator.
mutations in cftr can cause cystic fibrosis, a rare disease manifested by thick, sticky mucus and salty sweat, which usually leads to lung transplant or early death  <cit> .
in addition to acgh, copy number variation in the cftr gene was examined by a second method, quantitative real-time pcr.
finally, the introduction of mutations during the wga process was examined by comparing the dna sequence of the resulting amplified dna to that of the unamplified dna.
the consistency of the wga process was evaluated by examining four replicate amplified dnas derived from three individuals, one healthy and two with cystic fibrosis.
RESULTS
cnv introduced by the wga process
to characterize the consistency and faithfulness of whole genome amplification methods based on Ф <dig> polymerase, three human dna samples  were amplified using two commercially available kits, repli-g and genomiphi.
the dna samples were derived from a healthy individual  and two patients with cystic fibrosis .
the experimental design is shown in figure  <dig>  each of the three dna samples was amplified  <dig> independent times by both repli-g and genomiphi kits.
dna copy number changes introduced into the amplified dna were evaluated by comparing the amplified dna to the native unamplified dna utilizing acgh.
the agilent human cgh array was used with the assumption that as a research tool it could comprehensively evaluate and reveal gross copy number changes at nearly  <dig> million loci.
in addition, unamplified normal samples were compared to unamplified normal samples four times to define the technical variability of the acgh process.
any copy number variation in such a self-self hybridization would be due to technical limits of the acgh process since the dna samples were identical.
a total of  <dig> cgh arrays were used in this study.
dna samples were purchased from precisionmed, inc. and dissolved in te buffer .
there are different mutations in the two patients with cystic fibrosis .
to better understand differences in copy number introduced by the wga process, we compared the log <dig> ratio distribution of the normal unamplified dna vs. normal unamplified dna  with the log <dig> ratio distribution of the wga normal  dna vs. normal unamplified dna by calculating the kolmogorov distances.
the pair-wise comparisons for both the repli-g and genomiphi wga methods are shown in table  <dig>  the median pair-wise kolmogorov distances between the repli-g-amplified vs. unamplified distribution and genomiphi-amplified vs. unamplified distribution are  <dig>  and  <dig> , respectively, consistent with differences between the distributions of log <dig> ratios before and after wga.
each row represents probe ratio distributions from replications of amplified dna samples; each column represents probe ratio distributions from replications of unamplified dna samples.
the median values are  <dig>  and  <dig>  for repli-g and genomiphi, respectively.
unamp ; amp-r ; amp-g .
another measure of the uniformity of the wga process is the percentage of probes with ratios below or above a certain cut-off value when the wga dna is compared to the unamplified dna.
table  <dig> shows the percentage of probes on the cgh arrays with ratios greater than  <dig> -fold, 2-fold,  <dig> -fold, 3-fold, and 4-fold for the three dna samples and the two wga methods.
the percentages are the average of the  <dig> replicates.
also shown is the percentage of probes above these cut-offs for the unamplified normal dna compared to itself; all ratios were within  <dig>  consistent with low variability of the acgh method.
both amplification methods resulted in less than 1% of probes showing cnv of greater than 2-fold, with the exception of the normal sample amplified by genomiphi .
if the fold-change criterion was relaxed to 3-fold, less than approximately  <dig> % of probes showed cnv.
table  <dig> shows regions of the genome that were consistently under-amplified by at least 3-fold across all samples by both amplification methods.
these under-amplified regions represent a small portion of total genome , and contain about  <dig> genes .
thus, while the mda technology resulted in changes in copy number, the changes were relatively small in most regions of the genome, and were concentrated in a relatively small number of chromosomal loci.
* indicates samples on the cgh array.
the averages of the  <dig> replicate wga are given.
unamp ; amp-r ; amp-g .
cytobands and chromosomal locations are based on hg  <dig> .
* total number of genes located within the under-amplified regions.
consistency of the wga process
differences in the distribution of ratios within each sample group were calculated using the kolmogorov distance.
table  <dig> shows pair-wise comparisons of kolmogorov distances within each sample group, as well as the median value for each group.
the median kolmogorov value for the four self-self hybridizations  was  <dig>  and the median kolmogorov values within the other sample groups were similarly low.
thus, the wga process appears to be reproducible in terms of uniformity of amplification throughout the genome.
the phi correlation was calculated to evaluate the consistency of the position of the cnv loci .
higher values  indicate the ratios at each probe location are very similar.
results of pair-wise phi correlations  indicate that the majority of the replicates within each sample/amplification group are highly correlated .
the exception is the cf <dig> sample, which has a phi correlation just below  <dig> .
the phi correlation values are above  <dig>  if the fold cut-off is set at  <dig>  .
overall, the results from the kolmogorov distances and phi correlations show that there is high consistency in the wga process within each sample/wga method group.
* indicates samples on cgh array.
the median values for each group are shown in bold.
unamp ; amp-r ; amp-g .
* indicates samples on cgh array.
the median phi correlation values from top to bottom are shown in bold.
unamp ; amp-r ; amp-g .
comparisons between the two whole genome amplification kits
both wga kits used in this study, repli-g and genomiphi, use the same bacterial phage Φ <dig> dna polymerase.
however, the assay procedures are considerably different with regard to reaction time  and method of dna denaturation .
thus, comparison of outcomes of these procedures is important.
the median values of pair-wise kolmogorov distances for normal, cf <dig>  and cf <dig> samples between repli-g and genomiphi amplified samples are  <dig> ,  <dig> , and  <dig> , respectively  which are similar to the kolmogorov distances from samples amplified by the same method .
the median pair-wise phi correlations for normal, cf <dig>  and cf <dig> samples between repli-g and genomiphi amplified samples after cbs smoothing are  <dig> ,  <dig>  and  <dig> , respectively .
again, these values are similar to those obtained when comparing within an amplification method .
these results indicate that the two amplification methods generate similar amplified dna samples despite the procedural differences.
the median values for normal, cf <dig>  and cf <dig> groups between the wga methods are  <dig> ,  <dig> , and  <dig> , respectively.
amp-r ; amp-g .
the median values for normal, cf <dig>  and cf <dig> groups were  <dig> ,  <dig> , and  <dig> , respectively.
visual comparison of cnv generated by these amplification methods across chromosome  <dig> is illustrated in figure  <dig>  the pattern of cnvs is similar between the  <dig> amplification methods and among the  <dig> dna specimens, with much of the chromosome showing cnv < 2-fold when compared to the unamplified dna.
however, regions with consistent >2-fold cnv are evident near the chromosomal ends, as well as near 7q <dig> , 7q <dig> , and 7p <dig> , among others.
the region near the cftr gene appears to amplify uniformly and contains no cnvs greater than 2-fold.
quantitative real-time pcr copy number assay within the cftr gene region
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  <dig> introns and  <dig> exon .
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  in fold-change between amplified and unamplified dna samples .
this low level of variability was also evident when comparing results between the two amplification methods , 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  in identifying low levels  of amplification variability introduced by the wga process in the cftr locus.
chromosome locations are based on ncbi human genome build  <dig>
dna sequence analysis in the cftr gene region
bidirectional sanger dna sequencing was performed on an approximately 2 kb region of the cftr gene to examine the fidelity of the wga process using primers shown in table  <dig> .
dna sequences from the wga samples were compared with the respective unamplified dna samples.
the results are shown in table  <dig> and there were no detectable mutations introduced by either wga method in the  <dig> dna samples .
thus, the level of detectable mutations introduced by the genomiphi and repli-g wga methods in this analysis was less than  <dig> mutation per  <dig>  nucleotides examined.
these results suggest that both genomiphi and repli-g appear to be robust and accurate methods for amplifying nanogram quantities of starting dnas to microgram levels.
primer locations  are based on ncbi reference sequence ng_ <dig> .
“f” –forward primer; “r”- reverse primer.
nucleotides examined are the sum of 2-kb sequences from the four replications.
discussion
the sequencing of the human genome, coupled with advances in genomics technologies, is having a major impact on understanding basic human biology, as well as the molecular causes of diseases and toxicities  <cit> .
in fact, the leaders of the u.s. food and drug administration  and the national institutes of health  have recently outlined the opportunities in clinical medicine that are being created by advances in basic science, including genomics  <cit> .
the two agencies have announced a new collaborative effort to focus regulatory and translational sciences on bringing medical products and therapies into the age of personalized medicine.
this growing store of knowledge has enormous potential application to the development of devices and tests for use in the diagnosis, mitigation, treatment, cure, and prevention of disease and other conditions.
as of  <dig>  more than  <dig>  germline mutations in more than  <dig> genes have been associated with human inherited disease, with about  <dig> new disease genes and  <dig>  mutations being identified annually  <cit> .
in principle, dna tests could be developed for each of these conditions.
development and approval of such tests for clinical diagnostics often require that accuracy be demonstrated on patient samples.
that is, the test needs to detect the disease-causing mutation with high precision and accuracy.
in addition, measures of proficiency are required by testing laboratories to ensure continued accuracy of the results.
the clinical laboratory improvement advisory committee has recently issued a good practices report for molecular genetic testing and has stressed the need for performance assessments  <cit> .
such recommendations are supportive of the  <dig> report of the secretary’s advisory committee on genetics, health, and society   <cit> .
additionally, the sacghs has identified the development of genetic reference materials as one of five critical gaps in the oversight system of genetic testing  <cit> .
the device approval process, use of reference and control materials, and ongoing proficiency assessments require relatively large quantities of clinical samples in order to assure test performance is adequate and maintained over time.
the availability of sufficient samples with appropriate mutations has been recognized as a critical issue in genetic testing, given the paucity of validated clinical inherited disease gene samples  <cit> .
therefore, methods that can expand the limited supply of validated clinical samples would have major impacts on both the test and device performance evaluation and approval process, and the on-going proficiency assessment of the test providers.
the importance of the amount of template dna for mda-based wga has been thoroughly discussed for snp assays and str genotyping in previous studies  <cit> .
for example, large number of snps could be accurately detected from as low as  <dig>  ng of dna template  <cit>  and even degraded dna samples can be used for forensic snp typing  <cit> , while over 100 ng of dna template was needed for optimal str genotyping  <cit> .
in this study, we mainly focused on evaluating the uniformity and fidelity of wga dnas using cgh arrays, taqman copy number assays, and dna sequencing.
the results indicated the amplified dna and its native unamplified dna that we examined were similar, although not identical, in terms of dna copy number variation.
importantly, no detectable introduced mutations were found under our experimental conditions.
the agilent human cgh array used in this study includes approximately  <dig>  probes at  <dig> to 2 kb intervals throughout the human genome.
to ensure the quality of this study, four independent replications for both the wga and acgh processes were used.
the self-self hybridization of unamplified normal dna samples provided us the base-line measurement of noise for acgh technology.
the box plot of unamplified normal samples indicated less than  <dig> % of the approximately  <dig>  probes on the cgh array have fold changes greater than  <dig>  and no cnvs can be detected by acgh .
the low kolmogorov distances  and high phi correlations  between the four replicates of unamplified samples further showed the acgh technology was reproducible and could be a reliable tool to access variability of the wga process.
the wga technologies have evolved over the years from taq dna polymerase-based  to bacteriophage Ф <dig> dna polymerase-based methods .
we focused on mda methods in this study because of the processivity and reportedly low replication error rate of the Ф <dig> dna polymerase.
in this study, we compared two mda-based commercial wga kits: the repli-g and genomiphi kits.
the comparison between the unamplified and amplified dna samples showed that relatively few probes  were over- or under-amplified by more than 2-fold .
genomiphi-amplified dna, however, showed consistently more cnvs than the repli-g-amplified dna.
kolmogorov distances also indicated that differences in the distribution of dna copy number existed between the unamplified and amplified samples , and that these differences were greater than the differences between the repli-g and genomiphi amplified samples .
thus, even though the kits differ in method of dna denaturation , buffer composition, and reaction time , they produce similar amplified dna, likely the result of the common Ф <dig> dna polymerase.
the data in this report indicate that the two wga methods examined can consistently amplify small amounts of dna  to large quantities  with relatively small changes in dna copy number along the chromosomes.
changes in dna copy number of greater than 3-fold are evident as under-amplified regions at the ends of chromosomes, as illustrated in figure  <dig>  and in discrete regions on many of the chromosomes .
because of the consistency of the dna amplification, knowledge of the positions of likely under-amplification can prevent the inappropriate use of wga dna.
results presented in table  <dig> may serve as a reference guide to avoid target genes in these regions.
inspection of the gc content within these consistently under-amplified regions showed an average gc content of nearly 53% which was higher than the overall gc content of the human genome of 41%.
further investigations will be needed to evaluate the significance of this observation.
cystic fibrosis  is caused by severe dysfunction of cystic fibrosis transmembrane conductance regulator , which commonly leads to progressive lung disease and a shortened life  <cit> .
currently, there is no cure available for cf, even though multiple interventions have been developed to slow its progression.
since the fda cleared the first genotyping assay for cf in  <dig>  manufacturers have developed a number of genotyping tests for rare heritable diseases caused by multiple mutant alleles, using patient samples to assess performance.
there is an increased interest in using wga dna samples created from patient samples to support the performance of the assays.
this study shows that within certain limits, the wga process produces large quantities of dna that may be useful for this purpose.
to address the concerns of mutations introduced by wga, taqman copy number assays were used to analyze cnvs at  <dig> locations within cftr gene region and a random selected 2 kb region in cftr gene was also sequenced.
results from the taqman copy number assay are in excellent agreement with the acgh results.
the dna sequencing analysis showed there were no mutations induced by wga in this approximately 2 kb region of the cftr gene , which indicates that wga dnas can be used for enrichment of dna samples for cystic fibrosis genotyping assays.
the objective of the study presented here was to determine whether wga amplified samples may be a reliable alternative to native clinical specimens for assessing the performance of a test under investigation.
the conclusions from this study provide scientific input that may serve to support regulatory decisions in the ascertainment of safety and effectiveness of diagnostic products that use whole genome amplified samples in clinical studies.
this study may serve as a guide to the technical qualification of wga dna for assessing the performance of genotyping assays.
CONCLUSIONS
in summary, wga generates large quantities of dna with relatively high uniformity and low replication error rate when compared to unamplified dna.
this suggests that wga dna may be suitable for accurate genotyping.
however, because there are 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.

 advances in genomic technologies have enabled development of many novel genome analysis methods that may have applications in the understanding, diagnosis, and management of genetic diseases and cancer. comprehensive high-throughput assays are available for detection of single nucleotide polymorphisms, dna copy number variation, microsatellite expansion or contraction and loss of heterozygosity, all of which detect sometimes subtle genomic alterations associated with disease. some of these assays require micrograms of dna which may be difficult to obtain for many clinical samples. in addition, inadequate dna template may prevent the performance of multiple assays on a single sample. limited availability of dna also poses challenges for manufacturers and regulators of genetic diagnostic devices. one of these challenges is availability of sufficient quantities of dna samples to appropriately validate a particular test under investigation, especially when uncommon mutations for a rare disease severely limit access to patient sample specimens. since the u.s. food and drug administration ’s clearance of the first genotyping assay for cystic fibrosis in the office of in vitro diagnostic device evaluation and safety within the center for devices and radiological health has reviewed a number of other genotyping assays for inherited disorders. some of these genotyping assays are intended to detect relatively rare heritable diseases consisting of multiple disease-causing alleles for each disease, while others may detect more common diseases, but are intended to test very rare as well as common mutations. to show the accuracy of these assays, manufacturers would generally use patient samples. for rare mutations or alleles, it is often difficult to obtain sufficient quantities of such clinical samples to adequately assess test performance. therefore, there is an interest in using whole genome amplified dna samples created from patient samples instead of native dna samples to increase the availability of appropriate samples to query the performance of the assays. wga is a method that amplifies small amounts of genomic dna several thousand-fold in vitro. the wga process has the potential, however, to result in non-uniform amplification of the dna in which some regions of the genome are over-represented and others are under-represented. such biased amplification could make the wga dna unsuitable for some of the studies designed to assess the clinical assay performance. several wga methods based on the polymerase chain reaction with taq polymerase were initially developed. these methods included the use of primers directed at highly repetitive sequences, ligation of linkers to fragmented dna, degenerate oligonucleotide primed pcr, and primer extension preamplification. all of these methods suffer from a relatively high level of mutations in the amplified dna and highly non-uniform amplification due to the low fidelity and low processivity of the taq polymerase, respectively. in dean et al. described the multiple displacement amplification technique. this method of wga takes advantage of the high processivity and low error rate of the Φ bacteriophage dna polymerase. this polymerase has a 3’-5’ proof-reading activity and adds an average of templated nucleotides to a primer, resulting in higher fidelity and less biased amplification than with the taq polymerase methods. the wga process using Φ polymerase is isothermal and uses random primers to target the entire genome. the polymerase has strong strand displacement activity so that exponential amplification occurs through a branching mechanism, resulting in a high yield of dna. the mda process has been recently reviewed and shown to be superior to other dna amplification methods with regard to genotyping, genomic coverage, and amplification bias. mda-based wga has been frequently used in dna sample preparation for genotyping and sequencing in recent years. high call rates and excellent concordance rates were achieved from wga samples using high-density snp arrays. there was a very low error rate when high quality dna was used as template. mda-based wga has also been used for analyses of single cells. jiang et al. successfully amplified dna from single sperm at least fold with a single round of mda. dna amplified from single lymphocytes was used for multiple analyses of different loci including the Δf deletion in exon and two intragenic microsatellite markers in the cf gene. short tandem repeats and human leucocyte antigen typing were performed using dna amplified by mda from a single cell. mda-wga has also been increasingly used in the field of forensic testing. even in the often degraded dna samples, wga showed the capability and potential to increase the quality and quantity of dna from difficult samples in forensic casework. although mda-based wga has been successfully applied in many studies, there are several potential problems that may affect interpretation of results. these include the finding that large amounts of nonspecific dna amplification can be generated during mda, mostly due to primer-directed dna synthesis, and the quantity and quality of input dna into the mda reaction can affect genotyping results. despite the advantage of the mda-based wga method, the uniformity of the amplification process across the genome has not been extensively characterized. in paez et al. examined dnas amplified using a commercially available mda method, repli-g, using 10 k affymetrix snp arrays and direct sequencing of ~  bp of dna and showed near-complete genome representation, as well as low replication error rate. in pinard et al. assessed the bias of wga methods on bacterial genomes using massively parallel sequencing and found statistically significant amplification bias, although the mda methods produced the least bias. arriola et al., using a second commercially available mda method, genomiphi, evaluated the wga dna with low density array comparative genomic hybridization, a method with higher resolution than the traditional cgh method. copy number biases were found, with the extent dependent on the degree of amplification. while these studies and others have consistently shown non-uniformity of amplification by the mda methods, the uniformity of amplification has not been examined with high resolution acgh at a resolution of several kb. in addition, the consistency of the amplification process has not been evaluated in replicate amplifications of the same sample, an important parameter when such material may be used for validation of genetic diagnostic devices or tests. in this study, we evaluated two commonly used methods of wga for their ability to produce large quantities of uniformly amplified dna with minimal introduced mutations. biases during the amplification process were determined by measurements of dna cnvs at nearly million positions in the genome using acgh analysis. particular attention was given to a single gene, cftr, which encodes cystic fibrosis transmembrane conductance regulator. mutations in cftr can cause cystic fibrosis, a rare disease manifested by thick, sticky mucus and salty sweat, which usually leads to lung transplant or early death. in addition to acgh, copy number variation in the cftr gene was examined by a second method, quantitative real-time pcr. finally, the introduction of mutations during the wga process was examined by comparing the dna sequence of the resulting amplified dna to that of the unamplified dna. the consistency of the wga process was evaluated by examining four replicate amplified dnas derived from three individuals, one healthy and two with cystic fibrosis.  cnv introduced by the wga process to characterize the consistency and faithfulness of whole genome amplification methods based on Ф polymerase, three human dna samples were amplified using two commercially available kits, repli-g and genomiphi. the dna samples were derived from a healthy individual and two patients with cystic fibrosis. the experimental design is shown in figure each of the three dna samples was amplified independent times by both repli-g and genomiphi kits. dna copy number changes introduced into the amplified dna were evaluated by comparing the amplified dna to the native unamplified dna utilizing acgh. the agilent human cgh array was used with the assumption that as a research tool it could comprehensively evaluate and reveal gross copy number changes at nearly million loci. in addition, unamplified normal samples were compared to unamplified normal samples four times to define the technical variability of the acgh process. any copy number variation in such a self-self hybridization would be due to technical limits of the acgh process since the dna samples were identical. a total of cgh arrays were used in this study. dna samples were purchased from precisionmed, inc. and dissolved in te buffer. there are different mutations in the two patients with cystic fibrosis. to better understand differences in copy number introduced by the wga process, we compared the log ratio distribution of the normal unamplified dna vs. normal unamplified dna with the log ratio distribution of the wga normal dna vs. normal unamplified dna by calculating the kolmogorov distances. the pair-wise comparisons for both the repli-g and genomiphi wga methods are shown in table  the median pair-wise kolmogorov distances between the repli-g-amplified vs. unamplified distribution and genomiphi-amplified vs. unamplified distribution are and, respectively, consistent with differences between the distributions of log ratios before and after wga. each row represents probe ratio distributions from replications of amplified dna samples; each column represents probe ratio distributions from replications of unamplified dna samples. the median values are and for repli-g and genomiphi, respectively. unamp; amp-r; amp-g. another measure of the uniformity of the wga process is the percentage of probes with ratios below or above a certain cut-off value when the wga dna is compared to the unamplified dna. table  shows the percentage of probes on the cgh arrays with ratios greater than -fold, 2-fold, -fold, 3-fold, and 4-fold for the three dna samples and the two wga methods. the percentages are the average of the replicates. also shown is the percentage of probes above these cut-offs for the unamplified normal dna compared to itself; all ratios were within consistent with low variability of the acgh method. both amplification methods resulted in less than 1% of probes showing cnv of greater than 2-fold, with the exception of the normal sample amplified by genomiphi. if the fold-change criterion was relaxed to 3-fold, less than approximately % of probes showed cnv. table  shows regions of the genome that were consistently under-amplified by at least 3-fold across all samples by both amplification methods. these under-amplified regions represent a small portion of total genome, and contain about genes. thus, while the mda technology resulted in changes in copy number, the changes were relatively small in most regions of the genome, and were concentrated in a relatively small number of chromosomal loci. * indicates samples on the cgh array. the averages of the replicate wga are given. unamp; amp-r; amp-g. cytobands and chromosomal locations are based on hg. * total number of genes located within the under-amplified regions. consistency of the wga process differences in the distribution of ratios within each sample group were calculated using the kolmogorov distance. table  shows pair-wise comparisons of kolmogorov distances within each sample group, as well as the median value for each group. the median kolmogorov value for the four self-self hybridizations was and the median kolmogorov values within the other sample groups were similarly low. thus, the wga process appears to be reproducible in terms of uniformity of amplification throughout the genome. the phi correlation was calculated to evaluate the consistency of the position of the cnv loci. higher values indicate the ratios at each probe location are very similar. results of pair-wise phi correlations indicate that the majority of the replicates within each sample/amplification group are highly correlated. the exception is the cf sample, which has a phi correlation just below. the phi correlation values are above if the fold cut-off is set at. overall, the results from the kolmogorov distances and phi correlations show that there is high consistency in the wga process within each sample/wga method group. * indicates samples on cgh array. the median values for each group are shown in bold. unamp; amp-r; amp-g. * indicates samples on cgh array. the median phi correlation values from top to bottom are shown in bold. unamp; amp-r; amp-g. comparisons between the two whole genome amplification kits both wga kits used in this study, repli-g and genomiphi, use the same bacterial phage Φ dna polymerase. however, the assay procedures are considerably different with regard to reaction time and method of dna denaturation. thus, comparison of outcomes of these procedures is important. the median values of pair-wise kolmogorov distances for normal, cf and cf samples between repli-g and genomiphi amplified samples are,, and, respectively which are similar to the kolmogorov distances from samples amplified by the same method. the median pair-wise phi correlations for normal, cf and cf samples between repli-g and genomiphi amplified samples after cbs smoothing are, and, respectively. again, these values are similar to those obtained when comparing within an amplification method. these results indicate that the two amplification methods generate similar amplified dna samples despite the procedural differences. the median values for normal, cf and cf groups between the wga methods are,, and, respectively. amp-r; amp-g. the median values for normal, cf and cf groups were,, and, respectively. visual comparison of cnv generated by these amplification methods across chromosome is illustrated in figure the pattern of cnvs is similar between the amplification methods and among the dna specimens, with much of the chromosome showing cnv < 2-fold when compared to the unamplified dna. however, regions with consistent >2-fold cnv are evident near the chromosomal ends, as well as near 7q, 7q, and 7p, among others. the region near the cftr gene appears to amplify uniformly and contains no cnvs greater than 2-fold. quantitative real-time pcr copy number assay within the cftr gene region 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 introns and exon. 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 in fold-change between amplified and unamplified dna samples. this low level of variability was also evident when comparing results between the two amplification methods, 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 in identifying low levels of amplification variability introduced by the wga process in the cftr locus. chromosome locations are based on ncbi human genome build dna sequence analysis in the cftr gene region bidirectional sanger dna sequencing was performed on an approximately 2 kb region of the cftr gene to examine the fidelity of the wga process using primers shown in table . dna sequences from the wga samples were compared with the respective unamplified dna samples. the results are shown in table  and there were no detectable mutations introduced by either wga method in the dna samples. thus, the level of detectable mutations introduced by the genomiphi and repli-g wga methods in this analysis was less than mutation per nucleotides examined. these results suggest that both genomiphi and repli-g appear to be robust and accurate methods for amplifying nanogram quantities of starting dnas to microgram levels. primer locations are based on ncbi reference sequence ng_. “f” –forward primer; “r”- reverse primer. nucleotides examined are the sum of 2-kb sequences from the four replications. discussion the sequencing of the human genome, coupled with advances in genomics technologies, is having a major impact on understanding basic human biology, as well as the molecular causes of diseases and toxicities. in fact, the leaders of the u.s. food and drug administration and the national institutes of health have recently outlined the opportunities in clinical medicine that are being created by advances in basic science, including genomics. the two agencies have announced a new collaborative effort to focus regulatory and translational sciences on bringing medical products and therapies into the age of personalized medicine. this growing store of knowledge has enormous potential application to the development of devices and tests for use in the diagnosis, mitigation, treatment, cure, and prevention of disease and other conditions. as of more than germline mutations in more than genes have been associated with human inherited disease, with about new disease genes and mutations being identified annually. in principle, dna tests could be developed for each of these conditions. development and approval of such tests for clinical diagnostics often require that accuracy be demonstrated on patient samples. that is, the test needs to detect the disease-causing mutation with high precision and accuracy. in addition, measures of proficiency are required by testing laboratories to ensure continued accuracy of the results. the clinical laboratory improvement advisory committee has recently issued a good practices report for molecular genetic testing and has stressed the need for performance assessments. such recommendations are supportive of the report of the secretary’s advisory committee on genetics, health, and society. additionally, the sacghs has identified the development of genetic reference materials as one of five critical gaps in the oversight system of genetic testing. the device approval process, use of reference and control materials, and ongoing proficiency assessments require relatively large quantities of clinical samples in order to assure test performance is adequate and maintained over time. the availability of sufficient samples with appropriate mutations has been recognized as a critical issue in genetic testing, given the paucity of validated clinical inherited disease gene samples. therefore, methods that can expand the limited supply of validated clinical samples would have major impacts on both the test and device performance evaluation and approval process, and the on-going proficiency assessment of the test providers. the importance of the amount of template dna for mda-based wga has been thoroughly discussed for snp assays and str genotyping in previous studies. for example, large number of snps could be accurately detected from as low as  ng of dna template and even degraded dna samples can be used for forensic snp typing, while over 100 ng of dna template was needed for optimal str genotyping. in this study, we mainly focused on evaluating the uniformity and fidelity of wga dnas using cgh arrays, taqman copy number assays, and dna sequencing. the results indicated the amplified dna and its native unamplified dna that we examined were similar, although not identical, in terms of dna copy number variation. importantly, no detectable introduced mutations were found under our experimental conditions. the agilent human cgh array used in this study includes approximately probes at to 2 kb intervals throughout the human genome. to ensure the quality of this study, four independent replications for both the wga and acgh processes were used. the self-self hybridization of unamplified normal dna samples provided us the base-line measurement of noise for acgh technology. the box plot of unamplified normal samples indicated less than % of the approximately probes on the cgh array have fold changes greater than and no cnvs can be detected by acgh. the low kolmogorov distances and high phi correlations between the four replicates of unamplified samples further showed the acgh technology was reproducible and could be a reliable tool to access variability of the wga process. the wga technologies have evolved over the years from taq dna polymerase-based to bacteriophage Ф dna polymerase-based methods. we focused on mda methods in this study because of the processivity and reportedly low replication error rate of the Ф dna polymerase. in this study, we compared two mda-based commercial wga kits: the repli-g and genomiphi kits. the comparison between the unamplified and amplified dna samples showed that relatively few probes were over- or under-amplified by more than 2-fold. genomiphi-amplified dna, however, showed consistently more cnvs than the repli-g-amplified dna. kolmogorov distances also indicated that differences in the distribution of dna copy number existed between the unamplified and amplified samples, and that these differences were greater than the differences between the repli-g and genomiphi amplified samples. thus, even though the kits differ in method of dna denaturation, buffer composition, and reaction time, they produce similar amplified dna, likely the result of the common Ф dna polymerase. the data in this report indicate that the two wga methods examined can consistently amplify small amounts of dna to large quantities with relatively small changes in dna copy number along the chromosomes. changes in dna copy number of greater than 3-fold are evident as under-amplified regions at the ends of chromosomes, as illustrated in figure and in discrete regions on many of the chromosomes. because of the consistency of the dna amplification, knowledge of the positions of likely under-amplification can prevent the inappropriate use of wga dna. results presented in table  may serve as a reference guide to avoid target genes in these regions. inspection of the gc content within these consistently under-amplified regions showed an average gc content of nearly 53% which was higher than the overall gc content of the human genome of 41%. further investigations will be needed to evaluate the significance of this observation. cystic fibrosis is caused by severe dysfunction of cystic fibrosis transmembrane conductance regulator, which commonly leads to progressive lung disease and a shortened life. currently, there is no cure available for cf, even though multiple interventions have been developed to slow its progression. since the fda cleared the first genotyping assay for cf in manufacturers have developed a number of genotyping tests for rare heritable diseases caused by multiple mutant alleles, using patient samples to assess performance. there is an increased interest in using wga dna samples created from patient samples to support the performance of the assays. this study shows that within certain limits, the wga process produces large quantities of dna that may be useful for this purpose. to address the concerns of mutations introduced by wga, taqman copy number assays were used to analyze cnvs at locations within cftr gene region and a random selected 2 kb region in cftr gene was also sequenced. results from the taqman copy number assay are in excellent agreement with the acgh results. the dna sequencing analysis showed there were no mutations induced by wga in this approximately 2 kb region of the cftr gene, which indicates that wga dnas can be used for enrichment of dna samples for cystic fibrosis genotyping assays. the objective of the study presented here was to determine whether wga amplified samples may be a reliable alternative to native clinical specimens for assessing the performance of a test under investigation. the conclusions from this study provide scientific input that may serve to support regulatory decisions in the ascertainment of safety and effectiveness of diagnostic products that use whole genome amplified samples in clinical studies. this study may serve as a guide to the technical qualification of wga dna for assessing the performance of genotyping assays.  in summary, wga generates large quantities of dna with relatively high uniformity and low replication error rate when compared to unamplified dna. this suggests that wga dna may be suitable for accurate genotyping. however, because there are 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. 
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. 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. to assess amplification uniformity, we used array-based comparative genomic hybridization to evaluate dna copy number variations 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 cystic fibrosis patients. 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. each dna sample was amplified independent times and compared to its native unamplified dna. the two amplification kits, repli-g and genomiphi, generate very similar amplified dna samples despite the differences between the unamplified and amplified dna samples. however, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized. less than 2% of the probes showed more than 2-fold cnv introduced by the amplification process. to overcome the problem of limiting amounts of available dna, whole genome amplification methods have been developed. whole genome amplificationarray-based comparative genomic hybridizationtaqman copy number assaydna sequencing the multiple displacement amplification method using Φ polymerase has become the preferred choice due to its high processivity and low error rate. this was confirmed by quantitative real-time pcr copy number assays at locations within the cftr gene. dna sequencing analysis of a 2-kb region within the cftr gene showed no mutations introduced by wga. komogorov distances and phi correlations showed a high consistency within each sample group. regions of the genome that were consistently under-amplified were found to contain higher than average gc content.
 genotyping assays often require substantial amounts of dna. to overcome the problem of limiting amounts of available dna, whole genome amplification methods have been developed. the multiple displacement amplification method using Φ 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.  to assess amplification uniformity, we used array-based comparative genomic hybridization to evaluate dna copy number variations 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 cystic fibrosis patients. each dna sample was amplified 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 locations within the cftr gene. dna sequencing analysis of a 2-kb region within the cftr gene showed no mutations introduced by wga.  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. whole genome amplificationarray-based comparative genomic hybridizationtaqman copy number assaydna sequencing 