Evaluation of whole-genome amplification using multiple-displacement amplification of a limited number of cells

Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.

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

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2005.026 ◽

2005 ◽

Vol 43 (2) ◽

Cited By ~ 19

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

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

Journal of Microbiological Methods ◽

10.1016/j.mimet.2013.10.008 ◽

2013 ◽

Vol 95 (3) ◽

pp. 368-372 ◽

Cited By ~ 1

Author(s):

Sanjay Kumar ◽

Shefali Raj Gangoliya ◽

Mustapha Berri ◽

Annie Rodolakis ◽

Syed Imteyaz Alam

Keyword(s):

Coxiella Burnetii ◽

Whole Genome Amplification ◽

Multiple Displacement Amplification ◽

Intracellular Pathogen ◽

Whole Genome ◽

Genome Amplification ◽

Obligate Intracellular ◽

Obligate Intracellular Pathogen

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

PeerJ ◽

10.7717/peerj.5089 ◽

2018 ◽

Vol 6 ◽

pp. e5089 ◽

Cited By ~ 9

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.

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

10.1101/2021.07.09.451864 ◽

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.

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

10.7287/peerj.preprints.26939 ◽

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

10.7287/peerj.preprints.26939v1 ◽

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

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3291-3301.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3291-3301 ◽

Cited By ~ 175

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.

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