be ruled out that under significant environmental changes or respectively. The lower Alu correlation is in part due to the small pathological conditions, DNA methylation of different ReDS range of methylation values measured for the Alu assay. There may be more strongly associated and there may be greater intra- was an average difference of only 1.6% methylation between Alu individual correlation. Conversely, DNA methylation of one type technical replicates. of dispersed sequence may be affected by a given condition while Illumina Infinium HumanMethylation27 beadchip array. All others are spared. Thus, the DNA methylation status of mul- samples were run on the Illumina Infinium HumanMethylation27 tiple ReDS are valuable measures to consider. We propose that BeadChip array. Data for villi, fetal tissues and blood samples LINE-1 and Alu DNA methylation not be extrapolated to repre- were previously published in an analysis of gestational-age sent trends at other ReDS; they should be reported as a measure changes in DNA methylation35 and for identification of novel of methylation at CpG sites in the consensus sequence of specific differentially methylated regions between fetal tissues and REs. Additionally, we recommend that DNA methylation results blood.51 Briefly, genomic DNA was bisulfite converted using the only be compared when elements are measured by the same tech- EZ DNA Methylation Kit (Zymo Research), digested and then nique. The findings of our study reveal that the interpretation of hybridized to the Inifinium HumanMethylation27 BeadChip dispersed DNA methylation data collected via a diverse range of array (Illumina). The Illumina Infinium HumanMethylation27 assays is complex and the true power of these individual measure- BeadChip array is a robust assay and technical replicates were ments and their relationship to each other should be scrutinized. highly correlated (r = 0.99). Using GenomeStudio 2008 1.0.5 software (Illumina), sex chromosome probes were removed Methods from analysis to reduce sex-bias (n = 1,092). Poor quality probes (n = 32), defined as probes with detection p value of > 0.05, in Sample collection. Samples were collected through the BC >10% of samples were removed. Additionally we removed probes Women’s Hospital pathology department and previously that were polymorphic at the target CpG (n = 263). Signal inten- described in Yuen et al.50,51 The study was approved by the eth- sity output from genome studio was read in R (www.R-project. ics committees of the University of British Columbia and the org) with the Bioconductor methylumi package to calculate an Children’s and Women’s Health Centre of British Columbia M value based on the ratio of the intensity of the methylated (H04-70488, H06-70085). First ©trime2ster0pla1cen2ta (Lmaale nn= d8, etsouBnmietohylsatecdipreobnes.5c2Tehe.lumi Bioconductor package was female n = 2) were obtained anonymously from elective termina- used to correct color biases from chip to chip and then a β value tions between 8–12 weeks gestation. Second trimester placenta was calculated for each probe. (male n = 5, female n = 6) and fetal tissues (Dkidoney, nmaole nt= d5, istGrpiGbIEu2.0t53ewa.s used to annotate the genome with the loca- female n = 6; brain, male n = 4, female n = 4; muscle, male tion, size and density of CpG islands based on Weber’s defini- n = 4, female n = 6) were obtained from anonymous chromosom- tion.9 This annotation was intersected with the genomic location ally normal pregnancies terminated for medical reasons such as of array probes in Galaxy (galaxyproject.org) to group probes premature rupture of membranes or placental abruption between into three categories based on the density of the CpG island in 17–24 weeks gestation. Term placenta (38–41 weeks gestation, which they fell. Strong island probes (n = 14,391) were defined male n = 5, female n = 5) and adult female blood (n = 10) were as those that mapped to CpG islands with >55% GC content, samples used for previous studies of DNA methylation in the pla- >0.75 observed/expected GC ratio and >500 bps in length. Weak centa.34 After collection, a 1 cm3 piece was sampled from the fetal island probes (n = 2,786) were defined as those that mapped to side of each placenta to avoid maternal decidual contamination. CpG islands with >50% GC content, >0.48 observed/expected The amniotic membrane and chorion were removed and then GC ratio and >200 bps in length. Probes in weak islands that DNA was extracted from one quarter of the piece of chorionic bordered strong islands (n = 2,229) were not included since villi. A similar sized piece of each fetal tissue was also taken for they may act like CpG island shores and thus confound analy- DNA extraction. The remainder of each sample was stored at ses. Non-island probes (n = 5,805) were all remaining probes. -80°C for future use. Extraction was performed by standard salt Probes that mapped to Alu (n = 623) and LINE-1 (n = 222) method for all samples and DNA was stored at -20°C. were removed from CpG island groups and used for evolutionary Pyrosequencing. Methylation of LINE-1 and Alu elements age comparison of DNA methylation of REs. An estimate of the was measured by Pyrosequencing. Three hundred nanograms representation of all Alu and LINE-1 sequences was obtained by of genomic DNA was bisulfite converted using the EZ DNA dividing the number of probes on the array by the total number of Methylation Gold Kit (Zymo Research). LINE-1 and Alu ele- sequences in each subfamily. The total number of Alu sequences ments were amplified using published primer sets designed to was estimated based on supporting data in Price et al.54 whereas complement the LINE-1H and AluSx consensus sequences and the total number of LINE-1 sequences was estimated base on cycling conditions used were as previously published in reference counts from the UCSC RepeatMasker track (hg18). 28. PCR products were subsequently sequenced by a PyroMark Binning of probes into distance to nearest TSS was accom- MD system (Biotage). The DNA methylation status for each plished using distances provided in the Illumina Infinium CpG dinucleotide was evaluated using PyroQ-CpG software HumanMethylation27 BeadChip array annotation file. Strong, (Biotage) and the average for all CpG sites was calculated as a weak and non-island probes were initially grouped separately percentage for each sample. Correlation of distinct bisulfite con- into each of six non-overlapping bins: (1) -1,500 to -1,001 bps, versions for the LINE-1 and Alu assays were r = 0.99 and r = 0.51 (2) -1,000 to -501 bps, (3) -500 to 0 bps, (4) 1 to 500 bps, www.landesbioscience.com Epigenetics 661