title: Illumina NovaSeq 6000 paired end sequencing; Targeted MIP sequencing for healthy samples
design: Targeted MIP sequencing for healthy samples
library_construction_protocol: 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.