Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Pask, Rebecca; Rance, Helen E.; Barratt, Bryan J.; Nutland, Sarah; Smyth, Deborah J.; Sebastian, Meera; Twells, Rebecca C.J.; Smith, A. C. H.; Lam, Adam; Smink, Luc J.; Walker, Neil M.; Todd, John A.

doi:10.1186/1472-6750-4-15

Cited by 30 publications

(5 citation statements)

References 17 publications

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“…For example, Bergen et al [25] examined 49 TaqMan assays with regional GC% ranging from 33 to 65.8% and found a significant correlation with genotyping success. The other predictor of failure, telomere proximity, is also supported by other analyses [10,12]. While haplotypes tagging strategies [26] cannot avoid the telomeres, in studies using amplified DNA it seems advisable to avoid SNPs in G/C rich regions.…”

Section: Discussionmentioning

confidence: 91%

“…F29 Polymerase-based multiple-strand displacement wga (MDA) currently appears to be the most promising of these techniques. Multiple displacement amplification (MDA) has already been assessed using high-throughput DNA arrays on a limited number of samples [7,8,[11][12][13]. These studies report an excellent concordance of genotypes between pairs of genomic DNA (gDNA) and amplified DNA (wgaDNA) as well as good genome coverage despite the loss of a limited number of loci and regions that failed to amplify [11,13].…”

Section: Introductionmentioning

confidence: 98%

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Reliability of high‐throughput genotyping of whole genome amplified DNA in SNP genotyping studies

et al. 2007

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Whole genome amplification (wga) of DNA is being widely implemented in many laboratories to extend the life of samples only available in limited quantities for genetic analysis. We determined the reliability of wgaDNA genotypes in three sets of replicates from the same individuals: (i) 23 pairs of genomic DNA (gDNA), (ii) 43 pairs gDNA versus wgaDNA, and (iii) 29 pairs of independently amplified wgaDNA. Amplification was performed using multiple displacement amplification (MDA). Genotyping was successful for both DNA types for 1268 out of 1534 SNPs from 164 cardiovascular candidate genes assayed in a single Illumina panel. Amplified DNA failed for 77 SNPs (6%) that were genotyped successfully with genomic material. Percent of successful SNP calls, and concordance between pairs and kappa statistics (kappa) were determined. A total of 54 110 genotypes from gDNA-wgaDNA pairs were available for concordance analysis. Mean kappa for gDNA-wgaDNA pairs was 0.99. Concordance between gDNA-wgaDNA pairs was higher than amongst wgaDNA pairs (mean kappa for the 29 independently amplified pairs of wgaDNA was 0.95; interquartile range: 0.93-1.00). A statistical analysis of those SNPs which failed to genotype from amplified DNA only revealed that those loci were more likely to be closer to the telomeres and in locally GC-rich sequences. In summary, the MDA method produces wgaDNA samples that can be genotyped using high-throughput technology with a very high reproducibility to the original DNA but with slightly lower call rates. DNA amplification methodologies provide a useful solution for current and future large-scale genetic analyses especially with limited quantities of samples and DNA.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 98%

Reliability of high‐throughput genotyping of whole genome amplified DNA in SNP genotyping studies

et al. 2007

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“…Here, we used a WGA based on the MDA technology that employs the phi29 DNA polymerase and random primers to amplify gDNA without thermocycling [ 40 , 41 ]. Several studies revealed that MDA can amplify the genome with higher uniformity, longer length, and less bias compared with PCR-based WGA methods [ 41 , 42 , 43 , 44 , 45 ]. Therefore, MDA-based WGA has been shown to be the best method to obtain high quality cfDNA for different applications, such as the NGS [ 44 , 46 , 47 ].…”

Section: Discussionmentioning

confidence: 99%

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform

Vanni

Coco

Truini

et al. 2015

IJMS

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Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

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“…However, to our knowledge, this study is the first one that made use of the variable multiplexing ability of the same genotyping technology to study the relationship between genotyping performance and multiplexing level of the assays using wgaDNA, and to examine whether the storage of wgaDNA would reduce the genotyping efficiency. Indeed, existing studies [15] , [19] – [22] have successfully addressed the question of whether wgaDNA could be used in high-throughput array-based assays, e.g., Affymetrix and Illumina genotyping chips. High concordance was detected between wgaDNA and gDNA, highlighting the reliability of using wgaDNA for high-throughput genotyping with good accuracy.…”

Section: Discussionmentioning

confidence: 99%

Genotyping Performance Assessment of Whole Genome Amplified DNA with Respect to Multiplexing Level of Assay and Its Period of Storage

Yiu

Yap

et al. 2011

PLoS ONE

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Whole genome amplification can faithfully amplify genomic DNA (gDNA) with minimal bias and substantial genome coverage. Whole genome amplified DNA (wgaDNA) has been tested to be workable for high-throughput genotyping arrays. However, issues about whether wgaDNA would decrease genotyping performance at increasing multiplexing levels and whether the storage period of wgaDNA would reduce genotyping performance have not been examined. Using the Sequenom MassARRAY iPLEX Gold assays, we investigated 174 single nucleotide polymorphisms for 3 groups of matched samples: group 1 of 20 gDNA samples, group 2 of 20 freshly prepared wgaDNA samples, and group 3 of 20 stored wgaDNA samples that had been kept frozen at −70°C for 18 months. MassARRAY is a medium-throughput genotyping platform with reaction chemistry different from those of high-throughput genotyping arrays. The results showed that genotyping performance (efficiency and accuracy) of freshly prepared wgaDNA was similar to that of gDNA at various multiplexing levels (17-plex, 21-plex, 28-plex and 36-plex) of the MassARRAY assays. However, compared with gDNA or freshly prepared wgaDNA, stored wgaDNA was found to give diminished genotyping performance (efficiency and accuracy) due to potentially inferior quality. Consequently, no matter whether gDNA or wgaDNA was used, better genotyping efficiency would tend to have better genotyping accuracy.

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Investigating the utility of combining Φ29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray™ genotyping

Cited by 30 publications

References 17 publications

Reliability of high‐throughput genotyping of whole genome amplified DNA in SNP genotyping studies

Reliability of high‐throughput genotyping of whole genome amplified DNA in SNP genotyping studies

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform

Genotyping Performance Assessment of Whole Genome Amplified DNA with Respect to Multiplexing Level of Assay and Its Period of Storage

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