Multiple Displacement Amplification Reaction Research Articles

Abstract 322: Ultraplex genome and exome sequencing of rare single cells

Abstract Single-cell genome analysis enables researchers to gain novel insights into tumor heterogeneity. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging since the amount of genomic DNA present in a single cell is limited. PCR-based amplification methods normally have high error rates, low coverage uniformity, and extensive allelic drop-outs making interpretation of SNVs and CNVs challenging.Here we introduce a new whole-genome amplification method which utilizes an optimized isothermal DNA amplification reaction with a Hi-fidelity, error correcting polymerase and downstream library preparation protocol to allow pooling of amplified DNA. The key DNA amplification steps use multiple displacement amplification (MDA) technology to amplify gDNA directly from single cells, nuclei or previously isolated genomic DNA. During the MDA reaction, sample-barcodes are incorporated into the newly synthetized DNA, allowing the amplified DNA to be pooled and processed in to one ultraplex library. This method saves library preparation time, reduces DNA library costs and limits plastic consumable usage.Our current workflow allows 24 samples to be multiplexed in one library and 24 libraries to be produced for a total of 576 samples per sequencing flow cell. This method can be used to investigate chromosomal instability with low pass sequencing or SNV and CNV analysis following whole genome, exome-capture workflows. Citation Format: Ioanna Andreou, Samuel Rulli, Katja Heitz, Silke Huebner, Christin Meerschiff, Eric Lader. Ultraplex genome and exome sequencing of rare single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 322.

Integrated Addressable Dynamic Droplet Array (aDDA) as Sub-Nanoliter Reactors for High-Coverage Genome Sequencing of Single Yeast Cells.

An addressable dynamic droplet array (aDDA) is presented that combines the advantages of static droplet arrays and continuous-flow droplet platforms. Modular fabrication is employed to create a self-contained integrated aDDA. All the sample preparation steps, including single-cell isolation, cell lysis, amplification, and product retrieval, are performed in sequence within a sub-nanoliter (≈300 pL) droplet. Sequencing-based validation suggests that aDDA reduces the amplification bias of multiple displacement amplification (MDA) and elevates the percentage of one-yeast-cell genome recovery to 91%, as compared to the average of 26% using conventional, 20 µL volume MDA reactions. Thus, aDDA is a valuable addition to the toolbox for high-genome-coverage sequencing of single microbial cells.

Disposable DNA Amplification Chips with Integrated Low-Cost Heaters.

Fast point-of-use detection of, for example, early-stage zoonoses, e.g., Q-fever, bovine tuberculosis, or the Covid-19 coronavirus, is beneficial for both humans and animal husbandry as it can save lives and livestock. The latter prevents farmers from going bankrupt after a zoonoses outbreak. This paper describes the development of a fabrication process and the proof-of-principle of a disposable DNA amplification chip with an integrated heater. Based on the analysis of the milling process, metal adhesion studies, and COMSOL MultiPhysics heat transfer simulations, the first batch of chips has been fabricated and successful multiple displacement amplification reactions are performed inside these chips. This research is the first step towards the development of an early-stage zoonoses detection device. Tests with real zoonoses and DNA specific amplification reactions still need to be done.

On-chip real-time monitoring of multiple displacement amplification of DNA

The integration of systems for real-time monitoring of DNA amplification reactions is rather limited, because the heating system, the temperature control and the detection system are usually realized with separate modules. In this work, a lab-on-a-chip system for real-time monitoring of the multiple displacement amplification reaction (MDA) is presented. The amplification and detection unit consists of a system-on-glass (SoG) coupled to a microfluidic chip. The SoG includes thin film metallic resistors and amorphous silicon temperature sensors to control the temperature, as well as amorphous silicon photosensors and an interference filter for fluorescence detection on a single glass plate. The amplification reaction is carried out in the microfluidic chip made of cyclic olefin copolymer (COC). By using this setup, the multiple displacement amplification reaction can be monitored in real-time using the fluorophore [Ru(phen)2(dppz)]2+.

Whole-genome amplification in double-digest RADseq results in adequate libraries but fewer sequenced loci.

Whole-genome amplification by multiple displacement amplification (MDA) is a promising technique to enable the use of samples with only limited amount of DNA for the construction of RAD-seq libraries. Previous work has shown that, when the amount of DNA used in the MDA reaction is large, double-digest RAD-seq (ddRAD) libraries prepared with amplified genomic DNA result in data that are indistinguishable from libraries prepared directly from genomic DNA. Based on this observation, here we evaluate the quality of ddRAD libraries prepared from MDA-amplified genomic DNA when the amount of input genomic DNA and the coverage obtained for samples is variable. By simultaneously preparing libraries for five species of weevils (Coleoptera, Curculionidae), we also evaluate the likelihood that potential contaminants will be encountered in the assembled dataset. Overall, our results indicate that MDA may not be able to rescue all samples with small amounts of DNA, but it does produce ddRAD libraries adequate for studies of phylogeography and population genetics even when conditions are not optimal. We find that MDA makes it harder to predict the number of loci that will be obtained for a given sequencing effort, with some samples behaving like traditional libraries and others yielding fewer loci than expected. This seems to be caused both by stochastic and deterministic effects during amplification. Further, the reduction in loci is stronger in libraries with lower amounts of template DNA for the MDA reaction. Even though a few samples exhibit substantial levels of contamination in raw reads, the effect is very small in the final dataset, suggesting that filters imposed during dataset assembly are important in removing contamination. Importantly, samples with strong signs of contamination and biases in heterozygosity were also those with fewer loci shared in the final dataset, suggesting that stringent filtering of samples with significant amounts of missing data is important when assembling data derived from MDA-amplified genomic DNA. Overall, we find that the combination of MDA and ddRAD results in high-quality datasets for population genetics as long as the sequence data is properly filtered during assembly.

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL) volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.

Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis.

BackgroundWhole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts of DNA, such as samples from low biomass environments. It is well known that multiple displacement amplification (MDA), the most commonly used WGA method for microbial samples, skews the genomic representation in the sample. We have combined MDA with droplet microfluidics to perform the reaction in a homogeneous emulsion. Each droplet in this emulsion can be considered an individual reaction chamber, allowing partitioning of the MDA reaction into millions of parallel reactions with only one or very few template molecules per droplet.ResultsAs a proof-of-concept, we amplified genomic DNA from a synthetic metagenome by MDA either in one bulk reaction or in emulsion and found that after sequencing, the species distribution was better preserved and the coverage depth was more evenly distributed across the genomes when the MDA reaction had been performed in emulsion.ConclusionsPartitioning MDA reactions into millions of reactions by droplet microfluidics is a straightforward way to improve the uniformity of MDA reactions for amplifying complex samples with limited amounts of DNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0197-7) contains supplementary material, which is available to authorized users.

DNA Nanoparticles for Improved Protein Synthesis In Vitro

AbstractThe amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

DNA Nanoparticles for Improved Protein Synthesis In Vitro.

The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

BackgroundShotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested.ResultsUsing mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes.ConclusionsMDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Random‐Primed, Phi29 DNA Polymerase‐Based Whole Genome Amplification

Whole-genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and random hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The random-primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits available.

Genome of the pathogen Porphyromonas gingivalis recovered from a biofilm in a hospital sink using a high-throughput single-cell genomics platform

Although biofilms have been shown to be reservoirs of pathogens, our knowledge of the microbial diversity in biofilms within critical areas, such as health care facilities, is limited. Available methods for pathogen identification and strain typing have some inherent restrictions. In particular, culturing will yield only a fraction of the species present, PCR of virulence or marker genes is mainly focused on a handful of known species, and shotgun metagenomics is limited in the ability to detect strain variations. In this study, we present a single-cell genome sequencing approach to address these limitations and demonstrate it by specifically targeting bacterial cells within a complex biofilm from a hospital bathroom sink drain. A newly developed, automated platform was used to generate genomic DNA by the multiple displacement amplification (MDA) technique from hundreds of single cells in parallel. MDA reactions were screened and classified by 16S rRNA gene PCR sequence, which revealed a broad range of bacteria covering 25 different genera representing environmental species, human commensals, and opportunistic human pathogens. Here we focus on the recovery of a nearly complete genome representing a novel strain of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis JCVI SC001) using the single-cell assembly tool SPAdes. Single-cell genomics is becoming an accepted method to capture novel genomes, primarily in the marine and soil environments. Here we show for the first time that it also enables comparative genomic analysis of strain variation in a pathogen captured from complex biofilm samples in a healthcare facility.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Whole‐community genome amplification (WCGA) leads to compositional bias in methane‐oxidizing communities as assessed by pmoA‐based microarray analyses and QPCR

SummaryWhole‐genome amplification (WGA) using multiple displacement amplification (MDA) has recently been introduced to the field of environmental microbiology. The amplification of single‐cell genomes or whole‐community metagenomes decreases the minimum amount of DNA needed for subsequent molecular community analyses. The resolution of profiling methods of environmental microbial communities will increase substantially by the use of the whole‐community genome amplification (WCGA) procedure, assuming that the original community composition is not affected qualitatively as well as quantitatively. The present study aims to test if WCGA introduces a bias when applied to aerobic proteobacterial methanotrophic communities. For this, first, we subjected samples from freshwater lake sediment to WCGA, and amplified using primers targeting the pmoA gene coding for the α‐subunit of the methane monooxygenase enzyme. Second, we analysed community composition using a diagnostic microarray and quantitative PCR (QPCR) assays. These methods clearly demonstrated that the WCGA amplification introduced a bias. Thus, numbers of γ‐proteobacterial methanotrophs (‘type Ia’) increased significantly while the α‐proteobacterial methanotrophs (‘type II’) were not amplified by the WCGA procedure. It is hypothesized that this bias is caused by the differences in GC content, which may compromise the efficiency of the MDA reaction.

Genomic DNA amplification by the multiple displacement amplification (MDA) method

Large amounts of DNA are frequently required for use in detection assays and genomic analysis. The limited availability of DNA can be a critical obstacle to meeting research and clinical needs. DNA amplification methods are often required to generate sufficient material from small specimens or environmental samples with low DNA content. The MDA (multiple displacement amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with older whole genome amplification methods and the high DNA yields, even from exceedingly low amounts of starting material. Remarkably, MDA enables genomic sequencing even from single microbial cells. Some of the uses of MDA and its strengths and limitations will be discussed.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil

Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with phi29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

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.

USE OF MULTIPLE DISPLACEMENT AMPLIFICATION TO INCREASE THE DETECTION AND GENOTYPING OF TRYPANOSOMA SPECIES SAMPLES IMMOBILIZED ON FTA FILTERS

Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterized by low parasitemias. Multiple displacement amplification (MDA) amplifies DNA with a simple in vitro step and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a 20-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multicopy ribosomal ITS region or 177-bp repeats, and a 20-fold increase in sensitivity over nested PCR against a single-copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.

Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping

Although multiple displacement amplification (MDA) is being used increasingly to amplify genomes, the amplification bias generated by the ϕ29 polymerase can be a concern with genotyping applications. It has been noted that the bias is pronounced with small template amounts, particularly with single nucleotide polymorphism (SNP) and short tandem repeat (STR) genotyping. Bias may occur between loci, or between alleles within a locus, and may differ between sample donors at the same loci. Previous research has suggested that omitting denaturation of the template prior to amplification can reduce the observed bias significantly. Comparison of the two methods (with and without denaturation) has found that nondenaturation of template reverses the direction of bias observed between allelic pairs following MDA. By combining two MDA reactions, one denatured and one nondenatured, the bias was found to be reduced significantly, aiding copy number analysis and subsequent genotyping.