Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5′-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.     

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.     

Genotyping accuracy for whole-genome amplification of DNA from buccal epithelial cells.

We compared the accuracy of genotyping for DNA extracted from lymphocytes to that of DNA amplified from buccal epithelial cells. Amplification was via a rolling circle/phi29 DNA polymerase commercial kit. Paired buccal and lymphocyte DNA samples were available from 30 individuals. All samples were genotyped for 12 SNPs, 5 microsatellites and 2 VNTRs. The accuracy of genotyping (no-call proportions, reproducibility, and concordance) was similar for DNA from lymphocytes in comparison to amplified DNA from buccal samples. If used with caution, these data suggest that rolling-circle whole-genome amplification can be used to increase the DNA mass available for large-scale genotyping projects based on DNA from buccal cells.     

SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

Background

Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.

Results

Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.

Conclusion

Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.     

Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan

Multiple displacement amplification (MDA) has emerged as a promising new method of whole genome amplification (WGA) with the potential to generate virtually unlimited genome-equivalent DNA from only a small amount of seed DNA. To date, genome-wide high marker density assessments of MDA–DNA have focussed mainly upon suitability for single nucleotide polymorphism (SNP) genotyping applications. Suitability for short tandem repeat (STR) genotyping has not been investigated in great detail, despite their inherent instability during DNA replication, and the obvious challenge that this presents to WGA techniques. Here, we aimed to assess the applicability of MDA in STR genotyping by conducting a genome-wide scan of 768 STR markers for MDAs of 15 high quality genomic DNAs. We found that MDA genotyping call and accuracy rates were only marginally lower than for genomic DNA. Pooling of three replicate MDAs resulted in a small increase in both call rate and genotyping accuracy. We identified 34 STRs (4.4% of total markers) of which five essentially failed with MDA samples, and 29 of which showed elevated genotyping failures/discrepancies in the MDAs. We emphasise the importance of DNA and MDA quality checks, and the use of appropriate controls to identify problematic STR markers.     

Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

Background

Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods.

Results

A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point.

Conclusions

Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.     

Assessment of multiple displacement amplification in molecular epidemiology

Well-characterized epidemiological resources are generated with great effort, yet associated patient DNA samples can be limiting. The efficacy of the whole genome amplification (WGA) method, termed multiple displacement amplification (MDA), was assessed for detecting heterozygous sequence variants, mutation scanning, and PCR for challenging segments. Fifteen common polymorphisms from 10 genes located on 8 chromosomes were genotyped by direct sequencing of 300 PCR products from 115 high-quality MDA-amplified DNA samples extracted by different methods. The GC content of these analyzed segments ranges from 30% to 69%. Genotyping results demonstrate 100% accuracy. For heterozygotes, the relative intensity of peaks generated by the two alleles is highly similar for genomic and MDA-amplified genomic DNA, independent of GC content. In contrast, one of four heterozygous loci was mistyped when lower quality MDA-amplified DNA samples were used. The results of single-stranded conformation polymorphism (SSCP)-type of mutation scanningfor seven MDA-amplified DNA samples in four genes were concordant with the genomic DNA samples. PCR on MDA-amplified DNA was routinely successful for challenging 10- and 12-kb segments with GC content ranging from 30% to 80%, demonstrating that rather long segments, which are difficult to amplify with PCR, are amplified well with MDA. These results suggest that MDA is an effective method of WGA with utility in molecular epidemiology. Quality control of the MDA-amplified DNA is critical for high performance.     

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.     

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.     

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.     

Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry

A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.     

Large-scale genotyping of complex DNA

Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.     

Effects of electron-beam irradiation on buccal-cell DNA

Buccal cells were collected from 29 participants, by use of mouthwash rinses, and were split into equal aliquots, with one aliquot irradiated by electron-beam (E-beam) irradiation equivalent to the sterilizing dosage used by the U.S. Postal Service and the other left untreated. Aliquots were extracted and tested for DNA yields (e.g., TaqMan assay for quantifying human genomic DNA), genomic integrity, and amplification-based analysis of genetic variants (e.g., single-nucleotide polymorphisms [SNPs] and single tandem repeats [STRs]). Irradiated aliquots had lower median DNA yields (3.7 microg/aliquot) than untreated aliquots (7.6 microg/aliquot) (P<.0005) and were more likely to have smaller maximum DNA fragment size, on the basis of genomic integrity gels, than untreated aliquots (P<.0005). Irradiated aliquots showed poorer PCR amplification of a 989-bp beta-globin target (97% for weak amplification and 3% for no amplification) than untreated aliquots (7% for weak amplification and 0% for no amplification) (P<.0005), but 536-bp and 268-bp beta-globin targets were amplified from all aliquots. There was no detectable irradiation effect on SNP assays, but there was a significant trend for decreased detection of longer STRs (P=.01) in irradiated versus untreated aliquots. We conclude that E-beam irradiation reduced the yield and quality of buccal-cell specimens, and, although irradiated buccal-cell specimens may retain sufficient DNA integrity for some amplified analyses of many common genomic targets, assays that target longer DNA fragments (>989 bp) or require whole-genome amplification may be compromised.     

The effect of input DNA copy number on genotype call and characterising SNP markers in the humpback whale genome using a nanofluidic array

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.     

Detection of single nucleotide polymorphisms by PCR conformation-difference gel electrophoresis

The most common genetic variations in the human genome, single nucleotide polymorphisms (SNPs), are ideal biomarkers and are used extensively in disease research. Here we introduce a novel method of PCR-conformation-difference gel electrophoresis (PCR-CDGE) used for detecting SNPs. The principle of PCR-CDGE relies PCR products from different homozygous DNA samples showing dissimilar migration patterns upon PAGE due to their conformational differences. PCR products from heterozygous DNA samples may exhibit two or more bands in PAGE because of the existence of DNA homoduplexes and heteroduplexes. In this study, analysis of two SNPs showed that PCR-CDGE is an accurate, simple, rapid, low-cost, and high-throughput genotyping method that could be used in most laboratories.     

Large-scale SNP genotyping with canine buccal swab DNA

The dog is an attractive model for genetic studies of complex disease. With drafts of the canine genome complete, a large number of single-nucleotide polymorphisms (SNPs) that are potentially useful for gene-mapping studies and empirical estimations of canine diversity and linkage disequilibrium (LD) are now available. Unfortunately, most canine SNPs remain uncharacterized, and the amount and quality of DNA available from population-based samples are limited. We assessed how these real-world challenges influence automated SNP genotyping methods such as Illumina's GoldenGate assay. We examined 384 SNPs on canine chromosome 9 and successfully genotyped a minimum of 217 and a maximum of 275 SNPs using buccal swab samples for 181 dogs (86 beagles, 76 border collies, and 15 Australian shepherds). Call rates per SNP and sample averaged 97%, with reproducibility within and between analyses averaging 98%. The majority of these SNPs were polymorphic across all 3 breeds. We observed extensive LD, albeit less than reported for surveys using fewer dogs, consistent between breeds. Analyses of population substructure indicated that beagles are distinct from border collies and Australian shepherds. These results demonstrate the suitability of amplified canine buccal samples for high-throughput multiplex genotyping and confirm extensive LD in the dog.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

Development of new IL28B genotyping method using Invader Plus assay

IL28B polymorphism is associated with the response to pegylated interferon-α with ribavirin (PEG-IFN-α/RBV) treatment in chronic hepatitis C patients. As a genotyping assay for IL28B single nucleotide polymorphisms (SNPs) in clinical practice, the Invader Plus assay was developed. The accuracy, intra-assay, inter-assay precision, and the limit of detection of the Invader Plus assay were evaluated. Two SNPs (rs8099917 and rs12979860) associated with IL28B were genotyped by the Invader Plus and TaqMan assay in 512 Japanese patients. In comparison with direct sequencing, the Invader Plus assay showed 99% accuracy in rs8099917 and 100% accuracy in rs12979860. Intra-assay and inter-assay precision were sufficient to use in clinical practice and the detection limit was 1ngDNA/assay. Genotyping by rs8099917 showed that 361 (71%), 144 (28%) and seven (1%) of the patients were major homozygous, heterozygous and minor homozygous types, respectively. Five of the 512 cases (1%) had haplotype differences, but none showed differences between the two genotyping methods. For patients with HCV genotype 1, the prevalence of responders in the major homozygous type was 83.3%, and that of non-responders in the minor heterozygous/homozygous type was 72.5%. A convenient IL28B genotyping method using the Invader Plus assay could be useful to predict the treatment outcome in clinical practice.