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.