title: Validation of birth weight-associated DNA methylation variable positions with deep bisulfite sequencing on GS FLX Titanium
design: As long as the stocks lasted, the bisulfite DNA used for the genome-wide scan was used for validation analysis or a new bisulfite treatment was performed using standard protocols. In brief, 2 M sodium bisulfite and 0.6 M NaOH was added to 300 ng genomic DNA, which was then incubated for 15 min at 99°C and 30 min at 50°C, followed by 2 cycles of 5 min at 99°C and 90 min at 50°C. Afterwards, bisulfite-treated DNA was sequentially desulfonated (with 0.3 M NaOH), washed with 1xTE and recovered in 50 µl 0.5xTE using centrifugal filter units YM-30 (Millipore, Schwalbach, Germany).  Amplicons were generated using region-specific primers having on their 5´-ends the recommended GS-FLX A and B adaptors sequences and multiplex identifiers (MID) (Roche, Mannheim, Germany). Bisulfite PCRs were carried out in 30 µl mixes, including 1-3 µl bisulfite-treated DNA, 0.2 mM of each dNTP, 3 U HOT FIREPol DNA polymerase (Solis BioDyne, Tartu, Estonia), 1x reaction buffer B (Solis BioDyne), 2.5 mM MgCl2, or 1.5 U HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) and 1x PCR buffer (Qiagen). Primer sequences, concentrations and PCR conditions are summarized in Table S5 in additional file 1.  PCR products were visualized on 1.2% agarose gels, purified using the Gel/PCR DNA Fragments extraction kit (AVEGENE, Taipei, Taiwan) and measured by intercalating fluorescence dye using the Qubit Fluorometer (Qubit HS-Kit, Invitrogen, Darmstadt, Germany). After equimolar amplicon pooling, emulsion PCR was performed using Lib-L emPCR protocols. DNA containing beads were recovered, enriched and sequenced on a XLR70 Titanium PicoTiterPlate (Roche) separated into 8 regions, according to the manufacturer’s protocols. Reads were extracted from primary sff-files and assigned to the reference sequence. In total, 340 amplicons were sequenced and 331,768 high quality sequences were obtained with an average conversion rate >99%.