NEST is an ongoing prospective birth cohort study with 2,681 pregnant women recruited in two waves between 2005 and 2011; enrollment of participants is described in detail elsewhere. Briefly, pregnant women were recruited from prenatal clinics serving Duke University Hospital and Durham Regional Hospital obstetrics facilities in Durham, NC. Eligible participants were: (1) pregnant, (2) at least 18 years of age, (3) English-speaking, and (4) intending to deliver at one of two obstetric facilities. Women with HIV or intending to give up custody of their offspring were excluded. At delivery, umbilical cord blood was obtained. For the current study, we used 8 umbilical cord blood samples, and processed them twice with the Human Imprintome Array to assess the reliability of the array.
Extracted molecule
genomic DNA
Extraction protocol
For the preparation of samples, 200 ng of DNA were bisulfite converted using the EZ DNA Methylation kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions.
Label
Cy3, Cy5
Label protocol
Bisulfite-converted DNA samples were randomly assigned to a chip well on the Human Imprintome array BeadChip (Illumina, Inc., San Diego, CA), amplified, hybridized onto the array, stained, washed, and imaged with the Illumina iScan SQ instrument (Illumina, Inc., San Diego, CA) to obtain raw image intensities.
Hybridization protocol
Bisulfite-converted DNA samples were randomly assigned to a chip well on the Human Imprintome array BeadChip (Illumina, Inc., San Diego, CA), amplified, hybridized onto the array, stained, washed, and imaged with the Illumina iScan SQ instrument (Illumina, Inc., San Diego, CA) to obtain raw image intensities.
Scan protocol
Bisulfite-converted DNA samples were randomly assigned to a chip well on the Human Imprintome array BeadChip (Illumina, Inc., San Diego, CA), amplified, hybridized onto the array, stained, washed, and imaged with the Illumina iScan SQ instrument (Illumina, Inc., San Diego, CA) to obtain raw image intensities.
Data processing
To preprocess the DNA methylation values, we utilized the sesame package due to its compatibility with custom arrays. Specifically, we employed the readIDATpair followed by the getBetas functions, which require the IDAT file locations and the custom manifest to generate a beta value matrix. To visualize the distribution of beta values we employed the densityPlot function from the minfi package
