Multiple Displacement Amplification Method Research Articles

Pushing the limits of whole genome amplification: successful sequencing of RADseq library from a single microhymenopteran (Chalcidoidea, Trichogramma).

A major obstacle to high-throughput genotyping of microhymenoptera is their small size. As species are difficult to discriminate, and because complexes may exist, the sequencing of a pool of specimens is hazardous. Thus, one should be able to sequence pangenomic markers (e.g., RADtags) from a single specimen. To date, whole genome amplification (WGA) prior to library construction is still a necessity as at most 10 ng of DNA can be obtained from single specimens (sometimes less). However, this amount of DNA is not compatible with manufacturer’s requirements for commercial kits. Here we test the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (F0 and F1 generation, about1 ng input DNA for the WGA (0.17–2.9 ng)) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that 99% of the examined loci (up to 48,189 for one of the crosses, 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) and Mendelian inheritance of alleles. The remaining 1% (0.01% of the analysed nucleotides) could represent WGA bias or other experimental/analytical bias. This study shows that the multiple displacement amplification method on which the GenomiPhi kit relies, could also be of great help for the high-throughput genotyping of microhymenoptera used for biological control, or other organisms from which only a very small amount of DNA can be extracted, such as human disease vectors (e.g., sandflies, fleas, ticks etc.).

Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing

BackgroundThe current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations.ResultsBy analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells.ConclusionsIn conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.

Testing the Reproducibility of Multiple Displacement Amplification on Genomes of Clonal Endosymbiont Populations

The multiple displacement amplification method has revolutionized genomic studies of uncultured bacteria, where the extraction of pure DNA in sufficient quantity for next-generation sequencing is challenging. However, the method is problematic in that it amplifies the target DNA unevenly, induces the formation of chimeric reads and also amplifies contaminating DNA. Here, we have tested the reproducibility of the multiple displacement amplification method using serial dilutions of extracted genomic DNA and intact cells from the cultured endosymbiont Bartonella australis. The amplified DNA was sequenced with the Illumina sequencing technology, and the results were compared to sequence data obtained from unamplified DNA in this study as well as from a previously published genome project. We show that artifacts such as the extent of the amplification bias, the percentage of chimeric reads and the relative fraction of contaminating DNA increase dramatically for the smallest amounts of template DNA. The pattern of read coverage was reproducibly obtained for samples with higher amounts of template DNA, suggesting that the bias is non-random and genome-specific. A re-analysis of previously published sequence data obtained after amplification from clonal endosymbiont populations confirmed these predictions. We conclude that many of the artifacts associated with the use of the multiple displacement amplification method can be alleviated or much reduced by using multiple cells as the template for the amplification. These findings should be particularly useful for researchers studying the genomes of endosymbionts and other uncultured bacteria, for which a small clonal population of cells can be isolated.

Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms

Highly accurate and high-throughput SNP genotyping platforms are increasingly popular but the performance of suboptimal DNA samples remains unclear. The aim of our study was to determine the best platform, amplification technique, and loading concentration to maximize genotype accuracy and call rate using degraded samples. We amplified high-molecular weight genomic DNA samples recently extracted from whole blood and degraded DNA samples extracted from 50-year-old patient sera. Two whole-genome amplification (WGA) methodologies were used: an isothermal multiple displacement amplification method (MDA) and a fragmentation-PCR-based method (GenomePlex [GPLEX]; Sigma-Aldrich, St. Louis, MO). Duplicate runs were performed on genome-wide dense SNP arrays (Nsp-Mendel; Affymetrix) and custom SNP platforms based on molecular inversion probes (Targeted Genotyping [TG]; Affymetrix) and BeadArray technology (Golden Gate [GG]; Illumina). Miscalls and no-calls on Mendel arrays were correlated with each other, with confidence scores from the Bayesian calling algorithm, and with average probe intensity. Degraded DNA amplified with MDA gave low call rates and concordance across all platforms at standard loading concentrations. The call rate with MDA on GG was improved when a 5 x concentration of amplified DNA was used. The GPLEX amplification gave high call rate and concordance for degraded DNA at standard and higher loading concentrations on both TG and GG platforms. Based on these analyses, after standard filtering for SNP and sample performance, we were able to achieve a mean call rate of 99.7% and concordance 99.7% using degraded samples amplified by GPLEX on GG technology at 2 x loading concentration. These findings may be useful for investigators planning case-control association studies with patient samples of suboptimal quality.

Ancient DNA forces reconsideration of evolutionary history of Mediterranean pygmy elephantids

During the Pleistocene pygmy elephantids, some only a quarter of their ancestors' size, were present on Mediterranean islands until about 10,000 years ago (y.a.). Using a new methodology for ancient DNA (aDNA) studies, the whole genomic multiple displacement amplification method, we were able to retrieve cytochrome b (cytb) DNA fragments from 4200 to 800,000 y.a. specimens from island and mainland samples, including pygmy and normal-sized forms. The short DNA sequence (43 bp) retrieved from the 800,000 y.a. sample is one of the oldest DNA fragment ever retrieved. Duplication of the experiments in two laboratories, the occurrence of three diagnostic sites and the results of the phylogenetic analyses strongly support its authenticity. Our results challenge the prevailing view that pygmy elephantids of the eastern Mediterranean originated exclusively from Elephas, suggesting independent histories of dwarfism and the presence of both pygmy mammoths and elephant-like taxa on these islands. Based on our molecular data, the origin of the Tilos and Cyprus elephantids from a lineage within the genus Elephas is confirmed, while the DNA sequence from the Cretan sample falls clearly within the mammoth clade. Thus, the name Mammuthus creticus rather than Elephas creticus, seems to be justified for this form. Our findings also suggest a need to re-evaluate the evolutionary history of the Sicilian/Maltese species, traditionally included in the genus Elephas.

Effects of Electron-Beam Irradiation on Whole Genome Amplification

Electron-beam (E-beam) irradiation, currently being used to sterilize mail addressed to selected ZIP codes in the United States, has significant negative effects on the genomic integrity of DNA extracted from buccal-cell washes. We investigated the yield, composition, and genotyping performance of whole genome amplified DNA (wgaDNA) derived from 24 matched samples of E-beam-irradiated and nonirradiated genomic DNA (gDNA) as a model for the effects of degraded gDNA on the performance of whole genome amplification. gDNA was amplified using the Multiple Displacement Amplification method. Three methods of DNA quantification analysis were used to estimate the yield and composition of wgaDNA, and 65 short tandem repeat and single nucleotide polymorphism genotyping assays were used to evaluate the genotyping performance of irradiated and nonirradiated gDNA and wgaDNA. Compared with wgaDNA derived from nonirradiated gDNA, wgaDNA derived from irradiated gDNA exhibited a significantly reduced yield of wgaDNA and significantly reduced short tandem repeat and single nucleotide polymorphism genotyping completion and concordance rates (P < 0.0001). Increasing the amount of irradiated gDNA input into whole genome amplification improved genotyping performance of wgaDNA but not to the level of wgaDNA derived from nonirradiated gDNA. Multiple Displacement Amplification wgaDNA derived from E-beam-irradiated gDNA is not suitable for genotyping analysis.