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Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays.

ABSTRACT: BACKGROUND:The recommended genomic DNA input requirements for whole genome single nucleotide polymorphism microarrays can limit the scope of molecular epidemiological studies. We performed a large-scale evaluation of whole genome amplified DNA as input into high-density, whole-genome Illumina® Infinium® SNP microarray. RESULTS:Overall, 6622 DNA samples from 5970 individuals were obtained from three distinct biospecimen sources and genotyped using gDNA and/or wgaDNA inputs. When genotypes from the same individual were compared with standard, native gDNA input amount, we observed 99.94% mean concordance with wgaDNA input. CONCLUSIONS:Our results demonstrate that carefully conducted studies with wgaDNA inputs can yield high-quality genotyping results. These findings should enable investigators to consider expansion of ongoing studies using high-density SNP microarrays, currently challenged by small amounts of available DNA.

SUBMITTER: Dagnall CL

PROVIDER: S-EPMC5838969 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays.

Dagnall Casey L CL Morton Lindsay M LM Hicks Belynda D BD Li Shengchao S Zhou Weiyin W Karlins Eric E Teshome Kedest K Chowdhury Salma S Lashley Kerrie S KS Sampson Joshua N JN Robison Leslie L LL Armstrong Gregory T GT Bhatia Smita S Radloff Gretchen A GA Davies Stella M SM Tucker Margaret A MA Yeager Meredith M Chanock Stephen J SJ

BMC genomics 20180306 1

<h4>Background</h4>The recommended genomic DNA input requirements for whole genome single nucleotide polymorphism microarrays can limit the scope of molecular epidemiological studies. We performed a large-scale evaluation of whole genome amplified DNA as input into high-density, whole-genome Illumina® Infinium® SNP microarray.<h4>Results</h4>Overall, 6622 DNA samples from 5970 individuals were obtained from three distinct biospecimen sources and genotyped using gDNA and/or wgaDNA inputs. When ge ...[more]

PMID: 29510662

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Project description:BACKGROUND: Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. RESULTS: Here we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA). Overnight amplification in a 20-microlitre reaction produced 3.7 - 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA) for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H - 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H. CONCLUSION: The results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which considerably expands the potential of chromosome flow sorting in plant genomics.

Dataset Information

Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays.

Publications

Successful use of whole genome amplified DNA from multiple source types for high-density Illumina SNP microarrays.

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