SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification

Whole genome amplification by multiple displacement amplification (MDA) offers investigators using precious genomic DNA samples a high fidelity method for amplifying nanogram quantities of DNA several thousandfold. This becomes especially important for the modemrn day genomics researcher who more and more commonly is applying today's genome scanning technologies to patient cohort samples collected years ago that are irrecoverable and invariably in short supply. We present evidence here that MDA-prepared genomic DNA includes artifacts of chromosomal copy number that resemble copy number polymorphisms (CNPs) upon analysis of the DNA on the Affymetrix 10K GeneChip. The study of CNPs in both health and disease is a rapidly growing area of research, however our current understanding of the relevance of CNPs is incomplete. Our data indicate that utilization of whole genome-amplified samples for analysis heavily reliant on accurate copy number retention could be confounded if the genomic DNA sample was subjected to MDA. We recommend that small amounts of patient cohort DNA stocks be set aside and not subjected to whole genome amplification in order to facilitate the unbiased determination of chromosomal copy numbers when desired.     

SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1)

The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand-Myanmar (Burma) border (Thai-Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n = 45), Mae Hongson (n = 18), Ranong (n = 11), and Tak (n = 22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube? automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.     

Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification

Major efforts are underway to systematically define the somatic and germline genetic variations causally associated with disease. Genome-wide genetic analysis of actual clinical samples is, however, limited by the paucity of genomic DNA available. Here we have tested the fidelity and genome representation of phi29 polymerase-based genome amplification (phi29MDA) using direct sequencing and high density oligonucleotide arrays probing >10,000 SNP alleles. Genome representation was comprehensive and estimated to be 99.82% complete, although six regions encompassing a maximum of 5.62 Mb failed to amplify. There was no degradation in the accuracy of SNP genotyping and, in direct sequencing experiments sampling 500,000 bp, the estimated error rate (9.5 x 10(-6)) was the same as in paired unamplified samples. The detection of cancer-associated loss of heterozygosity and copy number changes, including homozygous deletion and gene amplification, were similarly robust. These results suggest that phi29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.     

Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads

Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10 ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.     

Sequence quality is maintained after multiple displacement amplification of non-invasively obtained macaque semen DNA

Whole genome amplification protocols are revolutionizing the fields of molecular and conservation biology as they open the possibility of obtaining a large number of copies of a complete genome from minute amounts of sample. Multiple displacement amplification (MDA) is a whole genome amplification technique based on the properties of the phi29 DNA polymerase, which leads to a uniform representation of the genome with very low error rates. In this study we performed MDA on 28 macaque DNA samples extracted from blood or non-invasively collected semen from which we obtained mitochondrial control region sequences both before and after MDA. The length of the readable sequences was longer for the original samples than for the MDA products, but the number of unresolved positions was comparable both before and after MDA. We conclude that the MDA technique is useful for increasing the amount of DNA for sequencing mitochondrial regions in the case of non-invasively collected semen samples.     

Detection of plant genes using a rapid, nonorganic DNA purification method

We have developed a simple procedure for the preparation of plant genomic DNA using FTA paper. Plant leaves were crushed against FTA paper, and the genomic DNA was purified using simple, nonorganic reagents. The 18S rRNA gene and the gene encoding the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from the chloroplast genome were detected by PCR amplification of DNA on FTA paper. DNA amplification was successful using extracts from 16 dicot and monocot plants. Studies of specific plant extracts revealed that extracts of leaf samples could be collected and stored at room temperature on FTA paper without a decrease in the DNA amplification success rate for more than a month. Both the 18S RNA gene and the rbcL gene were detected in the genomic DNA isolated from various soybean cultivars stored in this manner. Furthermore, by modestly increasing the number of cycles of DNA amplification, we were able to detect the uidA gene in transgenic tobacco and rice leaves as well as a single copy gene linked to the resistance gene of cyst nematode race 3 using genomic DNA isolated on FTA paper. These results demonstrate that genomic DNA isolated using FTA paper can be used for the detection of plant genes, from a wide range of plants with either high or low gene copy number and of either nuclear or cytoplasmic origin.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments

Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and phi 29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations >or= 10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples.     

Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125

Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms.     

Status of potential PfATP6 molecular markers for artemisinin resistance in Suriname

ABSTRACT: BACKGROUND: Polymorphisms within the PfATP6 gene have been indicated as potential molecular markers for artemisinin efficacy. Since 2004, the use of artemisinin combination therapy (ACT) was introduced as first-line treatment of the uncomplicated malaria cases in Suriname. The aim of this research was to determine changes in Suriname in the status of the polymorphic markers in the PfATP6 gene before and after the adoption of the ACT-regimen, particularly of the S769N mutation, which was reported to be associated with in vitro Artemether resistance in the neighboring country French Guiana. METHODS: The PfATP6 gene from Plasmodium falciparum parasites in Suriname was investigated in 28 samples using PCR amplification and restriction enzyme analysis, to assess and determine the prevalence of potentially interesting single nucleotide polymorphisms. The polymorphisms [L263E; A623E; S769N], which may be associated with the artemisinin resistant phenotype were characterized in parasites from three endemic regions before and after the adoption of the ACT-regimen. In addition, the status of these molecular markers was compared in paired P. falciparum isolates from patients with recurring malaria after controlled ACT. RESULTS: All the investigated samples exhibit the wild-type genotype at all three positions; L263, A623, S769. CONCLUSION: All investigated isolates before and after the adoption of the ACT-regimen and independent of endemic region harbored the wild-type genotype for the three investigated polymorphisms. The study revealed that decreased artemisinin susceptibility could occur independent from PfATP6 mutations, challenging the assumption that artemisinin resistance is associated with these mutations in the PfATP6 gene.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.     

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.     

Single-nucleotide polymorphism array coupled with multiple displacement amplification: accuracy and spatial resolution for analysis of chromosome copy numbers in few cells

When coupled with multiple displacement amplification (MDA), microarray-based comparative genomic intensity allows detection of chromosome copy number aberrations even in single or few cells, but the actual performance of the system and their influencing factors have not been well defined. Here, using single-nucleotide polymorphism (SNP) array, we analyzed copy number profiles from DNA amplified by MDA in 1-10 cells and estimated the accuracy and spatial resolution of the analysis. Based on the concordance of SNP copy numbers for DNA with and without MDA, the accuracy of the system can be significantly enhanced by using MDA-amplified DNA as reference and also by increasing the cell numbers. Analyses under different smoothing treatments revealed a practical resolution of 2?Mb for 10 cells and 10?Mb for a single cell. When both cells with known chromosomal duplication and deletion were analyzed, this platform detected a copy number "loss" more accurately than a "gain" (P < 0.01), particularly in single-cell MDA products. Together, we demonstrated that SNP array coupled with MDA is reliable and efficient for detection of copy number aberrations in a small number of cells, and its accuracy and resolution can both be significantly enhanced with increasing the number of cells as MDA template.     

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.     

Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.     

Multiple Displacement Amplification for Generating an Unlimited Source of DNA for Genotyping in Nonhuman Primate Species

We evaluated a whole genome amplification method—multiple displacement amplification (MDA)—as a means to conserve valuable nonhuman primate samples. We tested 148 samples from a variety of species and sample sources, including blood, tissue, cell-lines, plucked hair and noninvasively collected semen. To evaluate genotyping success and accuracy of MDA, we used routine genotyping methods, including short tandem repeat (STR) analysis, denaturing gradient gel electrophoresis (DGGE), Alu repeat analysis, direct sequencing, and nucleotide detection by tag-array minisequencing. We compared genotyping results from MDA products to genotypes generated from the original (non-MD amplified) DNA samples. All genotyping methods showed good results with the MDA products as a DNA template, and for some samples MDA improved genotyping success. We show that the MDA procedure has the potential to provide a long-lasting source of DNA for genetic studies, which would be highly valuable for the primate research field, in which genetic resources are limited and for other species in which similar sampling constraints apply.     

Allele-specific disparity in breast cancer

Background

Molecular alterations critical to development of cancer include mutations, copy number alterations (amplifications and deletions) as well as genomic rearrangements resulting in gene fusions. Massively parallel next generation sequencing, which enables the discovery of such changes, uses considerable quantities of genomic DNA (> 5 ug), a serious limitation in ever smaller clinical samples. However, a commonly available microarray platforms such as array comparative genomic hybridization (array CGH) allows the characterization of gene copy number at a single gene resolution using much smaller amounts of genomic DNA. In this study we evaluate the sensitivity of ultra-dense array CGH platforms developed by Agilent, especially that of the 1 million probe array (1 M array), and their application when whole genome amplification is required because of limited sample quantities.

Methods

We performed array CGH on whole genome amplified and not amplified genomic DNA from MCF-7 breast cancer cells, using 244 K and 1 M Agilent arrays. The ADM-2 algorithm was used to identify micro-copy number alterations that measured less than 1 Mb in genomic length.

Results

DNA from MCF-7 breast cancer cells was analyzed for micro-copy number alterations, defined as measuring less than 1 Mb in genomic length. The 4-fold extra resolution of the 1 M array platform relative to the less dense 244 K array platform, led to the improved detection of copy number variations (CNVs) and micro-CNAs. The identification of intra-genic breakpoints in areas of DNA copy number gain signaled the possible presence of gene fusion events. However, the ultra-dense platforms, especially the densest 1 M array, detect artifacts inherent to whole genome amplification and should be used only with non-amplified DNA samples.

Conclusions

This is a first report using 1 M array CGH for the discovery of cancer genes and biomarkers. We show the remarkable capacity of this technology to discover CNVs, micro-copy number alterations and even gene fusions. However, these platforms require excellent genomic DNA quality and do not tolerate relatively small imperfections related to the whole genome amplification.     

Impact of whole genome amplification on analysis of copy number variants

Large-scale copy number variants (CNVs) have recently been recognized to play a role in human genome variation and disease. Approaches for analysis of CNVs in small samples such as microdissected tissues can be confounded by limited amounts of material. To facilitate analyses of such samples, whole genome amplification (WGA) techniques were developed. In this study, we explored the impact of Phi29 multiple-strand displacement amplification on detection of CNVs using oligonucleotide arrays. We extracted DNA from fresh frozen lymph node samples and used this for amplification and analysis on the Affymetrix Mapping 500k SNP array platform. We demonstrated that the WGA procedure introduces hundreds of potentially confounding CNV artifacts that can obscure detection of bona fide variants. Our analysis indicates that many artifacts are reproducible, and may correlate with proximity to chromosome ends and GC content. Pair-wise comparison of amplified products considerably reduced the number of apparent artifacts and partially restored the ability to detect real CNVs. Our results suggest WGA material may be appropriate for copy number analysis when amplified samples are compared to similarly amplified samples and that only the CNVs with the greatest significance values detected by such comparisons are likely to be representative of the unamplified samples.     

Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase

Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Data Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.