DNA Methylation Analysis from Blood Spots: Increasing Yield and Quality for Genome-Wide and Locus-Specific Methylation Analysis

Abstract

Blood represents an easily accessible human tissue for numerous research and clinical applications, including surrogate roles for biomarkers of other tissues. Dried blood spots (DBS) are space- and cost-efficient storage forms of blood while stably retaining many of its chemical constituents. Consequently, neonatal DBS are routinely collected in many countries, and their biobanks represent gold mines for research. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome-wide profiling. In particular, DNA methylome analysis in DBS has proven to be technically challenging, mainly due to the requirement for stringent preprocessing, such as bisulfite conversion. Moreover, DNA amplification, required to increase its yield, often leads to a bias in the analysis, particularly in methylome profiles. Thus, it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Using a combination of in-house-derived and modified commercial extraction methods, we developed two robust protocols that produced increased DNA yield and quality from DBS. Though both protocols are more efficient relative to other published methods, one protocol yields less DNA compared to the other, but shows improved 260/280 spectrophotometric ratios, which are useful for sample quality assessment. Both protocols consist of two sequential phases, each involving several critical steps. Phase I comprises blood extraction off the filter papers, cell lysis, and protein digestion. Phase II involves DNA precipitation, purification, and elution. Results from subsequent locus-specific and genome-wide DNA methylation analyses demonstrate the high quality, reproducibility, and consistency of the data. This work may prove useful to meet the increased demand for research on DBS, particularly with a focus on the epigenetic origins of human diseases and newborn screening programs.

Keywords: Blood spot; DNA extraction; DNA methylation; Epigenetics; Illumina HM450 arrays; Pyrosequencing; Whole bisulfitome amplification.