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.

Evaluating Whole Genome Amplification via Multiply-Primed Rolling Circle Amplification for SNP Genotyping of Samples With Low DNA Yield

2005 ◽  
Vol 8 (4) ◽  
pp. 368-375 ◽  
Author(s):  
Kaisa Silander ◽  
Kati Komulainen ◽  
Pekka Ellonen ◽  
Minttu Jussila ◽  
Mervi Alanne ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Large Scale ◽  
Whole Genome Amplification ◽  
Rolling Circle Amplification ◽  
Limiting Factor ◽  
Whole Genome ◽  
Genome Amplification ◽  
Rolling Circle ◽  
Allele Dropout ◽  
Dna Yield

AbstractThe amount of available DNA is often a limiting factor in pursuing genetic analyses of large-scale population cohorts. An association between higher DNA yield from blood and several phenotypes associated with inflammatory states has recently been demonstrated, suggesting that exclusion of samples with very low DNA yield may lead to biased results in statistical analyses. Whole genome amplification (WGA) could present a solution to the DNA concentration-dependent sample selection. The aim was to thoroughly assess WGA for samples with low DNA yield, using the multiply-primed rolling circle amplification method. Fifty-nine samples were selected with the lowest DNA yield (less than 7.5µg) among 799 samples obtained for one population cohort. The genotypes obtained from two replicate WGA samples and the original genomic DNA were compared by typing 24 single nucleotide polymorphisms (SNPs). Multiple genotype discrepancies were identified for 13 of the 59 samples. The largest portion of discrepancies was due to allele dropout in heterozygous genotypes in WGA samples. Pooling the WGA DNA replicates prior to genotyping markedly improved genotyping reproducibility for the samples, with only 7 discrepancies identified in 4 samples. The nature of discrepancies was mostly homozygote genotypes in the genomic DNA and heterozygote genotypes in the WGA sample, suggesting possible allele dropout in the genomic DNA sample due to very low amounts of DNA template. Thus, WGA is applicable for low DNA yield samples, especially if using pooled WGA samples. A higher rate of genotyping errors requires that increased attention be paid to genotyping quality control, and caution when interpreting results.

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.

187 EMBRYO GENOTYPING FROM IN VIVO BIOPSIED BOVINE EMBRYOS AFTER WHOLE GENOME AMPLIFICATION

2009 ◽  
Vol 21 (1) ◽  
pp. 192 ◽  
Author(s):  
D. Le Bourhis ◽  
Y. Amigues ◽  
F. Charreaux ◽  
S. Lacaze ◽  
M. Tissier ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Detection Rate ◽  
Whole Genome Amplification ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Whole Genome ◽  
Bovine Embryos ◽  
Genome Amplification ◽  
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.

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.).

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.).

scholarly journals Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG)

2008 ◽  
Vol 2008 (2) ◽  
pp. pdb.prot4922-pdb.prot4922 ◽  
Author(s):  
N. Arneson ◽  
S. Hughes ◽  
R. Houlston ◽  
S. Done
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Genome Amplification

Whole genome amplification/hybridization following ooplasmic aspiration reveals no evidence of genomic DNA disturbance

2003 ◽  
Vol 80 ◽  
pp. 118
Author(s):  
Henry E. Malter ◽  
Dagan Wells ◽  
Mercedes Garcia Bermudez ◽  
Jacques Cohen
Keyword(s):  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Genome Amplification
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