Pyrosequencing vacuum prep tool

DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1: IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing after sodium bisulfite DNA treatment. Genomic DNA (500 ng) was modified by sodium bisulfite treatment using the EpiTect kit (Qiagen). Bisulfite-treated DNA (25 ng) was subsequently used as the template for PCR amplification prior to pyrosequencing as previously described in Bruno et al., 2015 [63 (link)]. Primers are available in Additional file 5: Table S4. Pyrosequencing reactions were performed in the PyroMark Q24 System (Qiagen) with the PyroGold SQA reagent kit according to the manufacturer’s instructions (Pyrosequencing AB, Uppsala, Sweden). The biotinylated PCR products were purified and denatured using the Pyrosequencing Vacuum Prep Tool (Qiagen). Pyrosequencing was performed on a Pyrosequencer Q24 (Qiagen). The DNA methylation level was calculated as the ratio of the C to T peaks at a given CpG site in pyrograms using Pyromark Q24 Software v.2.0.6 (Qiagen). Considering the presence of SNPs and high variability on one CpG of H19/IGF2-CTCF6 (no. 5) and two CpGs of IGF2-DRMR2 (no. 8 and 9), these CpGs were not considered for quantitative methylation analysis.

Bisulfite pyrosequencing was performed by EpigenDx laboratories LLC using their established protocol^{59 (link), 77 –79 (link)}. Briefly, 500ng of extracted DNA was bisulfite treated by EpigenDx using a proprietary bisulfite salt solution. Bisulfite treated DNA was purified using Zymogen DNA columns. For SNP mutation analysis, 5 ng of genomic DNA was used for PCR. The PCR was performed with 0.2 μM of each primer and one of the PCR primers is biotinylated to purify the final PCR product using Sepharose beads. The PCR product was bound to Streptavidin Sepharose HP (GE Healthcare Life Sciences), and purified, washed and denatured using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen). PCR products were sequenced by Pyrosequencing PSQ96 HS System (Pyrosequencing, Qiagen) following manufacturer’s instructions (Pyrosequencing, Qiagen). The methylation status of each locus was analyzed individually as a T/C SNP using QCpG software (Pyrosequencing, Qiagen).

The genomic DNA was bisulfite converted from unmethylated cytosine to uracil by using the Epitect Bisulfite kit (Qiagen). Primers were designed by the PyroMark Assay Design 2.0. PCR was performed with the following cycling conditions: initial denaturation step: 95°C for 3 min; 40 cycles of PCR in denaturation step: 94°C for 30 s; annealing step: Tm of primer; extension step: 72°C for 1 min and final extension: 72°C for 7 min. Subsequently, streptavidin coated sepharose beads, PyroMark Gold reagents (Qiagen), Pyrosequencing Vacuum Prep Tool and PyroMark Q96 software (Qiagen) were used for the determination and analysis of DNA methylation according to the manufacturers' instructions.

The methylation status of LINE-1 was measured by pyrosequencing. The primer sequences and PCR conditions have been previously described in detail [30 (link), 31 (link)]. Briefly, PCR was carried out in a 25 μL reaction mix containing 50ng bisulfite-converted DNA, 1x Pyromark PCR Master Mix (Qiagen, Valencia, CA), 1x Coral Load Concentrate (Qiagen) and 0.2 uM forward and reverse primers, using the following PCR program: 95°C for 15 minutes, then 44 cycles of 95°C for 30 seconds followed by 56°C for 30 seconds and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. The biotinylated PCR products were purified and converted into single-strands to act as a template in the pyrosequencing reaction as recommended by the manufacturer of the Pyrosequencing Vacuum Prep Tool (Qiagen). Then, 0.3 nmol/L of pyrosequencing primer was annealed to the purified single-stranded PCR product and pyrosequencing was conducted on a PyroMark Q96 MD (Qiagen). We used non-CpG cytosine residues as internal controls to verify efficient sodium bisulfite DNA conversion and universal unmethylated and methylated DNAs (Zymo Research) were used as experimental controls. The intra- and inter-assay coefficients of variation were 0.7% and 1.4%, respectively.

CpG site-targeted bisulfite pyrosequencing was used to confirm the Infinium HumanMethylation450 BeadChip Array results. Forward, biotinylated- reverse and sequencing primers were designed using the PyroMark assay design 2.0 software. PCR reaction (25μL) was performed according to manufacturer’s instructions (PyroMark PCR kit, Qiagen, Hilden, Germany); containing 50 ng bisulfite-treated DNA and 400nM of forward primer and biotin-labeled reverse primer. The primer sequences and PCR conditions are summarized in S1 Table. Amplification was carried out as follows: 95°C 15 min, then 45 cycles of 95°C 30 sec, annealing temperature for 30 sec (S1 Table), 72°C for 30 sec, followed by 72°C for 10 min. Biotin-labeled PCR products were captured with Streptavidin Sepharose beads (GE Healthcare, UK), and made single stranded using a Pyrosequencing Vacuum Prep Tool (Qiagen, Hilden, Germany). Sequencing primer (S1 Table) was annealed to the single-stranded PCR product by heating to 80°C, followed by slow cooling. Pyrosequencing was performed on a PyroMark Q24 system (Qiagen, Hilden, Germany) and cytosine methylation was quantified using PyroMark Q24 1.010 software.