Methylation Data Processing Protocol and Comparison of Blood and Cerebral Spinal Fluid Following Aneurysmal Subarachnoid Hemorrhage

One challenge in conducting DNA methylation-based epigenome-wide association study (EWAS) is the appropriate cleaning and quality-checking of data to minimize biases and experimental artifacts, while simultaneously retaining potential biological signals. These issues are compounded in studies that include multiple tissue types, and/or tissues for which reference data are unavailable to assist in adjusting for cell-type mixture, for example cerebral spinal fluid (CSF). For our study that evaluated blood and CSF taken from aneurysmal subarachnoid hemorrhage (aSAH) patients, we developed a protocol to clean and quality-check genome-wide methylation levels and compared the methylomic profiles of the two tissues to determine whether blood is a suitable surrogate for CSF. CSF samples were collected from 279 aSAH patients longitudinally during the first 14 days of hospitalization, and a subset of 88 of these patients also provided blood samples within the first 2 days. Quality control (QC) procedures included identification and exclusion of poor performing samples and low-quality probes, functional normalization, and correction for cell-type heterogeneity via surrogate variable analysis (SVA). Significant differences in rates of poor sample performance was observed between blood (1.1% failing QC) and CSF (9.12% failing QC;p= 0.003). Functional normalization increased the concordance of methylation values among technical replicates in both CSF and blood. SVA improved the asymptotic behavior of the test of association in a simulated EWAS under the null hypothesis. To determine the suitability of blood as a surrogate for CSF, we calculated the correlations of adjusted methylation values at each CpG between blood and CSF globally and by genomic regions. Overall, mean within-CpG correlation was low (r< 0.26), suggesting that blood is not a suitable surrogate for global methylation in CSF. However, differences in the magnitude of the correlation were observed by genomic region (CpG island, shore, shelf, open sea;p< 0.001 for all) and orientation with respect to nearby genes (3 ' UTR, transcription start site, exon, body, 5 ' UTR;p< 0.01 for all). In conclusion, the correlation analysis and QC pipelines indicated that DNA extracted from blood was not, overall, a suitable surrogate for DNA from CSF in aSAH methylomic studies.