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 < 5 × 10-6) were excellent. There was good agreement between methylation profiles generated from 25 ng gDNA and those generated from 750 ng (ρ > 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 (ρ > 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.