Hybridization capture and sequencing
Hybridization capture of the FLc-Capture method is similar to that of previously published capture protocols (Li et al., 2013) with some modifications. For each capture reaction, 500 ng of DNA libraries and 200 ng of cDNA probes are used. In order to increase the capture efficiency, we used a touch-down hybridization program: after denaturation at 94°C for five minutes, the hybridization starts from 65°C decreased by 5°C every 6 hr and ended at 45°C, for a total duration of 30 hours. The hybridized DNA fragments are captured with streptavidin magnetic beads (Dynabeads MyOne bead, Life Technologies). The beads are washed to remove unhybridized DNAs and eluted in 30 μl of 1× TE to release the captured DNA fragments. The captured libraries are amplified with Illumina P5 and P7 universal primers. Finally, the captured libraries of different capture experiments are pooled in equal concentrations and sequenced on three lanes of Illumina HiSeq X-ten with paired-end 150-bp mode (~400 G of total data). The workflow of the hybridization capture experiment is shown in Figure 1c.