NEXTERA XT library preparation

Libraries were prepared using the NEXTERA XT kit (Illumina, Cat. #: FC-131-1024), as described by Masser et al [7]. Briefly, dual-indexed libraries were generated according to the manufacturer’s protocol. Initial PCRs were performed on bisulfite converted DNA using a ProFlex thermocycler, under the following conditions: 95 °C for 3 min, then 45 cycles of 98 °C for 20s, 58 to 62°c (depending on the primer pair used) for 15 s, and 72 °C for 15 s, and finished with 72 °C for 1 min. Purified PCR products were quantified by Quant-IT Picogreen (Thermo Scientific, cat. #: P7589) and pooled to a final 0.2 ng/μl concentration. A total of 1 ng was used for library generation in a 96-well plate format. Transposome-mediated simultaneous DNA fragmentation and adapter ligation, that is, tagmentation, was performed at 55 °C for 5 min. After the tagmentation reaction, indexing specific PCR primers were added two per well for unique dual indexing. A 12-cycle PCR was performed to amplify the libraries and incorporate the index sequences to the libraries using the following reaction conditions: 72 °C for 3 min and 95 °C for 30s, then 11 cycles of 95 °C for 10s, 55 °C for 30s, and 72 °C for 30s, and finished with 72 °C for 5 min and hold at 10 °C. Amplified libraries were purified using 30 to 50 μl AMPure XP beads (Beckman-Coulter, Brea, CA, USA), and eluted off the beads in 52.5 μl resuspension buffer (provided with Nextera XT). The quality of double-stranded libraries was checked using a High Sensitivity DNA chip run on the Agilent 2100 Bioanalyzer (Agilent Technologies, cat. #: 5067-4626) for size and molarity determination. Based on these metrics, libraries were diluted to 2nM in 10 mM Tris with 0.5% Tween. Equimolar libraries were pooled in equal volumes for denaturation and dilution in HT1 (Illumina, San Diego, CA, USA) buffer. Briefly, 10 μl of pooled NGS library was mixed with 10 μl 0.2 N NaOH for 5 min, then the library was diluted to 20 pM in HT1 buffer. PhiX control libraries were used to increase diversity of base calling during sequencing. 10 nM stock of PhiX library was denatured in 0.2 N NaOH, then diluted to 20 pM in HT1 buffer. Diluted, multiplexed libraries were mixed with diluted PhiX at 4:1 volume ratios, followed by a final dilution to 11pM with HT1 buffer (1 ml final volume). 600 μl was loaded onto the reagent cartridge for MiSeq sequencing. In contrast with the aforementioned Targeted-BS sequencing approach, here classical Illumina sequencing primers were used.