Iscan sq scanner

Samples were processed as previously described [30 (link)]. Briefly, DNA was extracted from umbilical cord blood samples using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Mettmann, Germany), according to the manufacturer’s instructions. DNA was bisulfite-converted using the EZ DNA Methylation kit (Zymo Research, Irvine, California, USA), according to manufacturer’s instructions and hybridized in the Illumina Infinium HumanMethylation450 BeadChip array (Illumina Inc., San Diego, California, USA). Raw data was extracted by iScan SQ scanner (Illumina) with GenomeStudio software (v.2011.1), using the methylation module v.1.9.0 (Illumina), into IDAT files used for further analyses. Raw data files were normalized using Quantile and corrected for cell composition using a cord blood dataset as implemented in minfi. Multidimensionality graphs (MDS) were also used to obtain the biological variability of the data. The X and Y chromosome probes were excluded to avoid sexual identity bias. Non-specific probes were excluded due to the high probability of co-hybridization. Single Value Decomposition method (SVD) identified technical bias (slide), which was corrected using ComBat. Probes from HM450K were annotated according to their nearest genes using FDb.InfiniumMethylation.hg19 package. Our main interest here with DNAm data was obtaining the epigenetic clock (EC).

Genome-wide DNA methylation profiling of Gallus gallus skeletal muscle from four developmental stages was established using an Illumina Hiseq 2500 platform, and 150 bp paired-end reads were generated according to the manufacturer’s standard procedure. In brief, genomic DNA was fragmented by sonication to 200–300 bp with Covaris S220, followed by end repair and adenylation. Then, these DNA fragments were treated with bisulfite using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, United States), before PCR amplification using KAPA HiFi HotStart Uracil + ReadyMix (2X). The resulting DNA products were used for library preparation and subsequent sequencing, and more than 140 million raw reads were produced in each sample. Raw image intensities were scanned by the iScan SQ scanner (iScan System, Illumina, United States) and processed by the Genome Studio software (Illumina, United States). The methylated rate of cytosine (Mc) was calculated as β value in designed windows bins (bin size is 10 kb), and the β value varied from 0 (completely unmethylated) to 1 (completely methylated). Overall, more than 4,100 Mb cytosine sites were measured in all of the samples. The β value was corrected according to Lister et al. (2013) (link) studies to mitigate false positives from bisulfite non-conversion rate.

Following bisulfite treatment of 1 μg genomic DNA using the EZ DNA Methylation kit (Zymo Research, Irvine, CA), the bisulfite-converted DNA was hybridized onto the Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA), following the Illumina Infinium HD Methylation protocol in the Genomics Core Facility at UAMS. The Methylation EPIC BeadChip covers over 850,000 CpG sites, and has increased genome coverage of regulatory regions and higher reproducibility and reliability compared to previous versions [3 (link)]. Whole genome amplification, hybridization, staining and scanning steps for all samples were performed, the Illumina iScan SQ scanner was used to create images of the single arrays, and the intensities of the images were extracted using the Methylation module (v.1.9.0) of the GenomeStudio (v.2011.1) software (Illumina). Raw intensity data as IDAT files were imported into GenomeStudio for the computation of detection p-value of the probes, and all further steps including data import, normalization, filtering and analyses were performed using the methylation pipeline in Partek Genomics Suite^™ 6.6 (Partek Inc., St. Louis, MO).

Following bisulfite treatment of 1 μg genomic DNA using the EZ DNA Methylation kit (Zymo Research, Irvine, CA), the bisulfite-converted DNA was hybridized onto the Infinium Methylation EPIC BeadChip (Illumina, San Diego, CA), following the Illumina Infinium HD Methylation protocol in the Genomics Core Facility at UAMS. The Methylation EPIC BeadChip covers >850000 CpG sites, and has increased genome coverage of regulatory regions and higher reproducibility and reliability compared to previous versions^{15 (link)}. Whole genome amplification, hybridization, staining and scanning steps for all samples were performed, the Illumina iScan SQ scanner was used to create images of the single arrays, and the intensities of the images were extracted using the Methylation module (v.1.9.0) of the GenomeStudio (v.2011.1) software (Illumina). Raw intensity data as IDAT files were imported into the ChAMP R package^{11 (link)} for the processing and analysis of the methylation arrays. The BMIQ algorithm was used in the normalization of the data. Probes on a blacklist of probes that are known to be cross-reactive were removed.