scholarly journals Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

2006 ◽  
Vol 72 (5) ◽  
pp. 3291-3301 ◽  
Author(s):  
Carl B. Abulencia ◽  
Denise L. Wyborski ◽  
Joseph A. Garcia ◽  
Mircea Podar ◽  
Wenqiong Chen ◽  
...  
Keyword(s):  
Contaminated Soils ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Department Of Energy ◽  
Contaminated Site ◽  
Small Subset ◽  
Whole Genome ◽  
Genome Coverage ◽  
Genome Amplification ◽  
Amplification Bias

ABSTRACT Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using φ29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and “clusters of orthologous groups” (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.

scholarly journals Quantifying genome DNA during whole-genome amplification via quantitative real-time multiple displacement amplification

RSC Advances ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 4617-4621
Author(s):  
Jing Tu ◽  
Yi Qiao ◽  
Yuhan Luo ◽  
Naiyun Long ◽  
Zuhong Lu
Keyword(s):  
Real Time ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Genome Amplification

Monitoring multiple displacement amplification by fluorescence signals.

Whole genome amplification of buccal cell DNA: genotyping concordance before and after multiple displacement amplification

2005 ◽  
Vol 43 (2) ◽  
Author(s):  
Miles D. Thompson ◽  
Raffick A. R. Bowen ◽  
Betty Y. L. Wong ◽  
Joan Antal ◽  
Zhanqin Liu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Buccal Cell ◽  
Whole Genome ◽  
Buccal Cells ◽  
Genome Amplification ◽  
Dna Yield ◽  
Epidemiological Surveys ◽  
Before And After

AbstractWhile buccal cells provide an easily accessible source of genomic DNA, the amount extracted may be insufficient for many studies. Whole genome amplification (WGA) using multiple displacement amplification (MDA) may optimize buccal cell genomic DNA yield. We compared the usefulness, in epidemiological surveys, of DNA derived from buccal cells collected by alcohol mouthwash and amplified by WGA protocol and standard protocols. Buccal cell collection kits were mailed to 300 randomly selected members of a large cohort study, and 189 subjects returned buccal cell samples. We determined: (i) which QIAamp

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

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5089 ◽  
Author(s):  
Bruno A. S. de Medeiros ◽  
Brian D. Farrell
Keyword(s):  
Population Genetics ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Whole Genome ◽  
Promising Technique ◽  
Genome Amplification ◽  
Template Dna

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.

scholarly journals Comparison of single-cell whole-genome amplification strategies

2018 ◽  
Author(s):  
Nuria Estévez-Gómez ◽  
Tamara Prieto ◽  
Amy Guillaumet-Adkins ◽  
Holger Heyn ◽  
Sonia Prado-López ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Sequencing Data ◽  
Genome Amplification ◽  
Dna Yield ◽  
Amplification Bias ◽  
Random Amplification ◽  
Low Pass ◽  
Amplicon Size

Single-cell genomics is an alluring area that holds the potential to change the way we understand cell populations. Due to the small amount of DNA within a single cell, whole-genome amplification becomes a mandatory step in many single-cell applications. Unfortunately, single-cell whole-genome amplification (scWGA) strategies suffer from several technical biases that complicate the posterior interpretation of the data. Here we compared the performance of six different scWGA methods (GenomiPhi, REPLIg, TruePrime, Ampli1, MALBAC, and PicoPLEX) after amplifying and low-pass sequencing the complete genome of 230 healthy/tumoral human cells. Overall, REPLIg outperformed competing methods regarding DNA yield, amplicon size, amplification breadth, amplification uniformity –being the only method with a random amplification bias–, and false single-nucleotide variant calls. On the other hand, non-MDA methods, and in particular Ampli1, showed less allelic imbalance and ADO, more reliable copy-number profiles and less chimeric amplicons. While no single scWGA method showed optimal performance for every aspect, they clearly have distinct advantages. Our results provide a convenient guide for selecting a scWGA method depending on the question of interest while revealing relevant weaknesses that should be considered during the analysis and interpretation of single-cell sequencing data.

scholarly journals Comparison of seven single cell Whole Genome Amplification commercial kits using targeted sequencing

2017 ◽  
Author(s):  
Tamir Biezuner ◽  
Ofir Raz ◽  
Shiran Amir ◽  
Lilach Milo ◽  
Rivka Adar ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Targeted Sequencing ◽  
Whole Genome ◽  
Genome Coverage ◽  
Genome Amplification ◽  
Cell Experiment ◽  
Commercial Kits ◽  
Genomic Regions

AbstractAdvances in biochemical technologies have led to a boost in the field of single cell genomics. Observation of the genome at a single cell resolution is currently achieved by pre-amplification using whole genome amplification (WGA) techniques that differ by their biochemical aspects and as a result by biased amplification of the original molecule. Several comparisons between commercially available single cell dedicated WGA kits (scWGA) were performed, however, these comparisons are costly, were only performed on selected scWGA kit and more notably, are limited by the number of analyzed cells, making them limited for reproducibility analysis. We benchmarked an economical assay to compare all commercially available scWGA kits that is based on targeted sequencing of thousands of genomic regions, including highly mutable genomic regions (microsatellites), from a large cohort of human single cells (125 cells in total). Using this approach, we could analyze the genome coverage, the reproducibility of genome coverage and the error rate of each kit. Our experimental design provides an affordable and reliable comparative assay that simulates a real single cell experiment. Results demonstrate the needfor a dedicated kit selection depending on the desired single cell assay.

scholarly journals Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

2021 ◽  
Author(s):  
Charles Michael Thurlow ◽  
Sandeep J Joseph ◽  
Lilia M Ganova-Raeva ◽  
Samantha Katz ◽  
Lara Pereira ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Treponema Pallidum ◽  
The United States ◽  
Whole Genome ◽  
Genome Amplification ◽  
Clinical Specimens ◽  
Enrichment Methods

Downstream next generation sequencing of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing Multiple Displacement Amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA followed by MDA as enrichment methods to improve the yields of T. pallidum DNA in rabbit propagated isolates and lesion specimens from patients with primary and secondary syphilis. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/μl input for SWGA and >129 genomic copies/μl for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.

scholarly journals Pushing the limits of whole genome amplification: Successful sequencing of RADseq libraries from single micro-hymenoptera (Chalcidoidea, Trichogramma)

2018 ◽  
Author(s):  
Astrid Cruaud ◽  
Géraldine Groussier ◽  
Guenaëlle Genson ◽  
Laure Sauné ◽  
Jean-Yves Rasplus
Keyword(s):  
High Throughput ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Mendelian Inheritance ◽  
Whole Genome ◽  
Genome Amplification ◽  
Amplification Method ◽  
As Species ◽  
F1 Generation ◽  
Genomic Material

A major obstacle to high-throughput genotyping of micro-hymenoptera 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 only ca 10ng of DNA can be obtained from single specimens. However this amount of DNA is not compatible with manufacturer’s requirements for commercialised kits. Here we tested the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (generation F0 and F1, ca 1 ng input DNA for the WGA) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that ca 99% of the examined loci (up to 48,189; 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) or a Mendelian inheritance of alleles. The remaining 1% (ca 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 micro-hymenoptera used for biological control or other organisms from which only a very low amount of DNA can be extracted such as human disease vectors (e.g. sand flies, fleas, ticks etc.).

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