Targeted MIP sequencing for healthy samples
Targeted MIP sequencing for healthy samples
DNA samples were received from the Princess Margaret cancer center, University Health Network (UHN), Canada) (under UHN IRB protocol 01-0573). Per reaction a total DNA of 50–500 ng/ul was used. DNA samples were received from the Princess Margaret cancer center, University Health Network (UHN), Canada) (under UHN IRB protocol 01-0573).Per reaction a total DNA of 50–500 ng/ul was used. One microliter DNA template was added to a hybridization mix together with a MIP pool (final concentration of 0.04 pM per probe) in 0.85× Ampligase buffer. Mix was incubated in a thermal cycler at 98°C for 3 min, followed by 85°C for 30 min, 60°C for 60 min and 56°C for 60 min. Product was mixed with (final concentration in brackets): dNTPs (14 pM), Betaine (375 mM), NAD+ (1 mM), additional Ampligase buffer (0.5×), Ampligase (total of 1.25U) and Q5 High-Fidelity DNA Polymerase (0.4 U, New England Biolabs). All the product of the hybridization was incubated at 56°C for 5 min followed by 72°C for 5 min. Enzymatic digestion of linear probes was performed by adding Exonuclease I (8U) and Exonuclease III (50 U). Mixture was incubated at 37°C for 10 min, followed by 80°C for 20 min. Final product was amplified using NEBNext Ultra II Q5 Master Mix (New England Biolabs). Samples were pooled and concentrated using AMPure XP beads at 0.75× volumetric concentration and sequenced as abovementioned described.