Whole-genome multiple displacement amplification from single cells

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.     

Nanoliter reactors improve multiple displacement amplification of genomes from single cells

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.     

Whole genome amplification from filamentous fungi using Phi29-mediated multiple displacement amplification

The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. We have used multiple displacement amplification (MDA) to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloe festucae. Up to 10 microg of high molecular weight DNA was routinely amplified from less than 10 ng of template DNA obtained using glass bead-mediated disruption of fungal spores or alkaline lysis of mycelium. PCR was possible from MDA-generated DNA and amplicons up to 10 kb were successfully amplified. RFLP analysis was successful, with bands of up to 5 kb routinely detected. Hybridization of MDA-amplified DNA to a cosmid library illustrated that the MDA product amplified from E. festucae is representative of the genome. MDA is a reliable method that could be applied to applications ranging from high-throughput screening of deletion mutants to genomic library construction.     

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.     

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.     

Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification

This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard.     

Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells

The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 microg amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).     

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.     

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.     

Genome coverage and sequence fidelity of φ29 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 29 polymerase-based genome amplification (29MDA) using direct sequencing and high density oligonucleotide arrays probing >10000 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 × 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 29MDA yields high fidelity, near-complete genome representation suitable for high resolution genetic analysis.     

Enhanced sequencing coverage with digital droplet multiple displacement amplification

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing.     

Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.     

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.     

Whole genome amplification from plant cell colonies of somatic hybrids using strand displacement amplification

The application of strand displacement amplification (SDA) is demonstrated for whole genome amplification from nanograms to micrograms for DNA isolated from small plant cell colonies. Secondary digest amplified fragment length polymorphism (SD-AFLP) analysis confirmed that the amplified genome is a representative of the entire genome. This approach allows the amplification of DNA isolated from small cell colonies of putative somatic hybrids for rapid molecular confirmation of the hybrid status of fusion products.     

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.     

Multiple displacement amplification prior to single nucleotide polymorphism genotyping in epidemiologic studies

We assessed the whole genome amplification strategy, known as multiple displacement amplification (MDA), for use with the TaqMan genotyping platform for DNA samples derived from two case-control studies nested in the Nurses' Health Study and the Physicians' Health Study. Our objectives were to (1) quantify DNA yield from samples of varying starting concentrations and (2) assess whether MDA products give an accurate representation of the original genomic sequence. Multiple displacement amplification yielded a mean 23000-fold increase in DNA quantity and genotyping results demonstrate 99.95% accuracy across six SNPs from four genes for 352 samples included in this study. These results suggest that MDA will provide a sufficiently robust amplification of limiting samples of genomic DNA that can be used for SNP genotyping in large case-control studies of complex diseases.     

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA ( 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.     

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.