Seqcap epi accessory kit

One microgram of the amplified Bisulfite-converted Sample Library was added to a new 1.5 ml centrifuge tube, with a hole pierced in the tube lid, containing 1 μl of SeqCap HE Universal Oligo (1000 μM), 1 μl of the appropriate SeqCap HE Index Oligo (1000 μM) and 10 μl of Bisulfite Capture Enhancer (SeqCap HE Oligo Kits A and B and SeqCap Epi Accessory Kit, Roche NimbleGen). With the lid closed, the tubes containing the Sample Libraries, Bisulfite Capture Enhancer and Oligos were dried down using a DNA vacuum concentrator on high heat. To the dried-down amplified Bisulfite-converted Sample Library plus Oligos and Bisulfite Capture Enhancer, 7.5 μl of 2× Hybridization Buffer and 3 μl of Hybridization Component A (SeqCap EZ Hybridization and Wash Kit, Roche NimbleGen) were added. The hole at the top of the 1.5 ml tubes was covered with lab tape and the samples were vortexed and briefly centrifuged. The samples were then heated in a 95°C heat block for 10 min. After denaturation, the samples were vortexed, allowed to return to room temperature and briefly centrifuged. The entire volume was then transferred to a 0.2 ml tube containing the SeqCap Epi Choice probe pool (Roche NimbleGen) that had been previously aliquoted in 4.5 μl amounts in 0.2 ml PCR strip tubes. The samples were mixed briefly and then incubated in a thermal cycler for 68 h at 47°C (with a heated lid).

Sequencing libraries consisted of 16–18 samples and were prepared according to the SeqCap Epi protocol (Roche, Basel, Switzerland) with KAPA HyperPrep Kit (Roche). Diagnostic whole-blood DNA from AML patients (800–1200 ng) was first mixed with the Bisulfite-conversion Control (unmethylated DNA from phage lambda) provided in the SeqCap Epi Accessory kit (Roche) and then fragmented either via E220 Focused ultrasonicator (Covaris, Woburn, MA, USA) or Bioruptor Pico instrument (Diagenode, Liège, Belgium) to get an average size of 200 bp. EZ DNA Methylation Lightning Kit (Zymo Research, Irvine, CA, USA) was used for the bisulfite conversion. Pooled samples from each library were hybridized for about 68 h with a custom set of probes (made by Roche Company). The final concentration of the libraries was measured using KAPA Library Quantification Kit (Roche), and the average size of the libraries’ fragments was assessed on 4200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) using the MiSeq Reagent Kit v2 (300-cycles) (Illumina).