Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Background

Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen^® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR^® Identifiler^® panel) and N = 49 SNP (TaqMan^®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.

Results

The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.

Conclusion

The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan^® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR^® Identifiler^® panel from wgaDNA equivalent to that of gDNA.

     

Genotyping performance assessment of whole genome amplified DNA with respect to multiplexing level of assay and its period of storage

Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at -70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.     

Evaluation of mailed pediatric buccal cytobrushes for use in a case-control study of birth defects

BACKGROUND: Buccal cell collection is a convenient DNA collection method; however, little attention has been given to the quality of DNA obtained from pediatric populations. The purpose of this study was to determine the effect of a modified cytobrush collection method on the yield and quality of infant buccal DNA collected as part of a population-based case-control study of birth defects. METHODS Cytobrushes were collected from infants, mothers, and fathers using a standard collection method in 1997 to 2003 and a modified protocol that allows air-drying of the cytobrushes after collection from 2003 to the present. Yield and quality of DNA from 1057 cytobrushes was assessed by quantitative PCR and short tandem repeat (STR) genotyping, respectively. RESULTS Air-dried cytobrushes from infants had higher median DNA yields (1300 ng) and STR completion rates (99.5%) than standard collection method cytobrushes (60 ng and 59.5%, respectively). A subset of DNA aliquots was genotyped for six single nucleotide polymorphisms (SNPs). Aliquots from both collection methods that passed the quality protocol (DNA concentration >1 ng/μl, and successful amplification of ≥1 STR) had high genotype completion rates (99-100%). The median DNA yield following whole genome amplification was more than twofold higher for air-dried than standard collection specimens (p < 0.001). CONCLUSION Yield and quality of buccal DNA collected from infants are improved by using a method that incorporates air-drying; however, DNA collected by both methods is suitable for genotyping if stringent quality control procedures are instituted. These findings may be helpful for future epidemiologic studies of birth defects and other adverse pediatric outcomes.     

Assessing the utility of whole genome amplified DNA for next‐generation molecular ecology

DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low‐quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next‐generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double‐digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de‐novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de‐novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next‐generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.     

High fidelity of whole-genome amplified DNA on high-density single nucleotide polymorphism arrays

Current microarray technology allows researchers to genotype a large number of SNPs with relatively small amounts of DNA. Nevertheless, researchers and clinicians still frequently face the problem of acquiring enough high-quality DNA for analysis. Whole-genome amplification (WGA) methods offer a solution for this problem, and earlier studies have shown that WGA samples perform reasonably well in small-scale genetic analyses (e.g. Affymetrix 10K array). To determine the performance of WGA products on a large-scale genotyping array, we compared the Affymetrix 250K array genotyping results of genomic DNA and their WGA products from four individuals. Our results indicate that WGA product performs well on the 250K array compared to genomic DNA, especially when using the BRLMM calling algorithm. WGA samples have high call rates (97.5% on average, compared to 99.4% for genomic DNA) and excellent concordance rates with their corresponding genomic DNA samples (98.7% on average). In addition, no apparent systematic genomic amplification bias can be detected. This study demonstrates that, although there is a slight decrease in the total call rates, WGA methods provide a reliable approach for increasing the amount of DNA samples for use with a common SNP genotyping array.     

High‐throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high‐throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole‐genome amplification (WGA) by primer‐extension preamplification. We used the Agena Biosciences MassARRAY^® platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high‐throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.     

A Short and Simple Improved-Primer Extension Preamplification (I-PEP) Procedure for Whole Genome Amplification (WGA) of Bovine Cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS / GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (～3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.     

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis(PGD) of patient’s embryos by next-generation sequencing(NGS) is dependent on efficient whole genome amplification(WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA(15 pg) and single cells, we showed that the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification(MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing(CNV-Seq) was applied to single cell WGA products derived by either Sure Plex or MALBAC amplification, we showed that known disease CNVs in the range of 3e15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either Sure Plex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient’s cohort for uterine transplantation.     

Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates

Background

Genome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure.

Results

Several PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001).

Conclusion

It is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-889) contains supplementary material, which is available to authorized users.     

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity—the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference—whole-blood DNA—based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy

Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500-10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10⁵ droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.     

Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.

The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.     

A short and simple improved-primer extension preamplification (I-PEP) procedure for whole genome amplification (WGA) of bovine cells

Embryo transfer is a reproductive technique that has a major impact on the dissemination of economically important genes and the rate of genetic gain in breeding schemes. In recent years, there has been increasing interest in the use of sexed and genotyped embryos in commercial embryo transfer programs. Marker/gene assisted selection (MAS/GAS) projects can be performed in the pre-implantation stage through mass production of characterized embryos. Biopsy of a few cells in the morulla stage is essential for pre-implantation genetic diagnosis (PGD), in which sex determination, evaluation of disease genes, and genotyping for candidate genes are performed. Limited quantity of cells and low amount of DNA restrict the use of multiple molecular analyses in PGD programs. Recently, whole genome amplification (WGA) techniques promise to overcome this problem by providing sufficient input DNA for analysis. Among several techniques proposed for WGA, the primer extension pre-amplification (PEP) and the improved-primer extension pre-amplification (I-PEP) methods are the most commonly used. However, these methods are time-consuming and need more than 12 h amplification cycles. Since the time is a critical parameter in the successful characterized embryo transfer, the shortening of diagnosis time is highly desirable. In this study, we developed a short and simple I-PEP procedure (~3 h) and evaluated its performance for the amplification of bovine genomic DNA. We assessed short WGA procedure by polymerase chain reaction (PCR) amplification of 7 specific loci. The results indicated that the short procedure possesses enough sensitivity for the molecular genetic analysis of 1 input cell. Although the efficiency of the method was 100%, there was an inconsistency between genomic DNA (gDNA) and whole genome amplification product (wgaDNA) genotypes for kappa-casein locus; that is, however, most likely due to allele drop-out (ADO) or false homozigocity. The results of this study indicate that with the application of reliable methods, WGA-amplified bovine DNA will be a useful source for sexing and genotyping bovine embryos in several quantitative trait locus (QTL) markers.     

Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems

As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.     

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.