Targeted molecular inversion probe panel sequencing of homo sapien occipital cortex
Two hundred fifty ng of gDNA was first hybridized in a 15 ul reaction with 1.5 ul of Ampligase® 10X Reaction Buffer (VWR), 1.5 ul of the reverse blocking oligo (5'- NNNNGAAGTCGAAGGGCTATAGGCTGCCATCACANNNN-3') and the MIP pool at 63nM for 10 min at 95 °C and 24 hrs at 60 °C. Gap-fill/ligation was then performed by adding 1 unit of Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher), 4 units of Ampligase® DNA Ligase (Epicentre), 0.2 ul of Ampligase® 10X Reaction Buffer, 0.6 ul of dNTPs (10 mM) and 1ul of nuclease-free water to the MIP capture product and incubated at 60 °C for 1 hr. For exonuclease digestion, 50 units of Exonuclease III (Thermo Fisher), 10 units of Exonuclease I (Thermo Fisher), 0.2 ul of Ampligase® 10X Reaction Buffer (VWR), and 2.05 ul of nuclease free water was added to the Gap-fill/ligation product, which was incubated for 40 min at 37 °C and 5 min at 95 °C. Ten ul of the captured library is amplified in a 50 ul final reaction by adding 1 unit of Phusion Hot Start II DNA Polymerase (Thermo Fisher), 10 ul of 5X HF buffer, 1 ul of dNTPs (10mM), 1 ul of the universal MIP barcode forward primer (10 uM), 1 ul of the individual barcode reserve primer (10 uM) and 26.5 ul of nuclease-free water. MIP library amplification was then performed under the following conditions: 98 °C for 30 s; 16 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s; 72 °C for 2 min. MIP library was then purified using 2X AMPure XP Beads (Beckman Coulter,) and quantified by the Quant-iT™ dsDNA Assay HS Kit (Thermo Fisher). Ninety-six MIP libraries were pooled together and sequenced on one lane of Illumina Hiseq X.