Dataset Information

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

ABSTRACT:

Aims

Archived newborn bloodspots are valuable sample collections for genetic and epigenetic disease research. However, they have often been stored for long periods of time, under less than ideal circumstances, and nucleic acid yields can be low, particularly when samples become limiting. We wished to determine whether the quantity and quality of genomic DNA (gDNA) isolated from a single, surgical bloodspot punch (2?mm dia.) was adequate for accurate and reliable DNA methylome profiling on the Illumina HumanMethylation450 array.

Methods

A total of 25-750?ng of archived bloodspot or Jurkat cell gDNA were bisulfite converted and analyzed on the array without any additional DNA amplification steps. Methylation profiles were assessed for call rate, call confidence (detection p-value), and reproducibility.

Results

Using 25?ng gDNA from either Jurkat cells or dried bloodspots, array-wide call rates (?99.9%) and detection p-values (99.9% with p?-6) were excellent. There was good agreement between methylation profiles generated from 25?ng gDNA and those generated from 750?ng (? > 0.98), although a fraction of CpG sites (2-8% depending on experiment) exhibited quantitative differences. Genome-wide methylation levels were strikingly reproducible from 25?ng DNA in both replicate and interindividual samples (? > 0.98).

Conclusions

Twenty-five nanograms of gDNA, isolated from a single, surgical punch (2?mm dia.) of an archived newborn bloodspot, generate a genome-wide methylation profile on the Illumina HumanMethylation450 array that is robust, reproducible, and suitable for differential methylation studies.

SUBMITTER: Asrani K

PROVIDER: S-EPMC5568181 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Publications

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

Asrani Kripa K Shaw Gary M GM Rine Jasper J Marini Nicholas J NJ

Genetic testing and molecular biomarkers 20170613 8

<h4>Aims</h4>Archived newborn bloodspots are valuable sample collections for genetic and epigenetic disease research. However, they have often been stored for long periods of time, under less than ideal circumstances, and nucleic acid yields can be low, particularly when samples become limiting. We wished to determine whether the quantity and quality of genomic DNA (gDNA) isolated from a single, surgical bloodspot punch (2 mm dia.) was adequate for accurate and reliable DNA methylome profiling o ...[more]

PMID: 28609193

Similar Datasets

Project description:BACKGROUND: Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites. RESULTS: Probes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared. CONCLUSION: DNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.

Dataset Information

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

Aims

Methods

Results

Conclusions

Publications

DNA Methylome Profiling on the Infinium HumanMethylation450 Array from Limiting Quantities of Genomic DNA from a Single, Small Archived Bloodspot.

Similar Datasets

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