title: Standard_Protocol_Barcode115_SQK-108
design: Total genomic DNA was extracted from blood using the Qiagen Blood & Cell Culture DNA Midi Kit (Hilden, Germany, cat# 13343), yielding 242 ng/ul provided in TE buffer. Sequencing was undertaken using the Oxford Nanopore MinIon (machine ID No. MN19030; flow-cell version SQK-108)  with R9.4 chemistry and conducted using Minion protocol version GDE_9002_v108_revT_18Oct2016 for library preparation with modification. A nick-repair enzyme (NEBNext FFPE Repair Mix) to repair breaks in one side of the DNA double helix, which can prematurely shorten DNA reads was added. To increase library yield, NEBNext Ultra II End Repair/dA-Tailing Module was used to eliminate an additional SPRI (Solid Phase Reversible Immobilization) cleanup step, here applying Agencourt AMPure XP beads. To increase library yield, 2 ul of concentrated NEB T4 DNA ligase was added during adaptor ligation.1. DNA Nick-Repair, Blunt-Ending, and End Repair Add in a clean low bind PCR tube, (a) ~ 2 ug genomic Tuatara DNA as described above of 8.27 ul, (b) NEBNext FFPE Repair Mix of 3 ul, (c) NEBNext Ultra II End Repair/dA-Tailing Mix of 3 ul, (d) NEBNext Ultra II End Repair/dA-Tailing Buffer of 7 ul, (e) 100X NAD+ of 0.6 ul, and (f) 10 mM Tris HCl pH 8.5 buffer of 38.13 ul for a total volume of 60 ul. This is mixed gently via inversion and incubated at 20 C for 60 min and 65 C for 30 min in a thermocycler.2. SPRI Cleanup Add to the above (a) Agencourt AMPure XP beads of 60 ul and let stand for 2 min followed by discarding supernatant while on magnet, (b) wash with 70% EtOH of 140 ul X 2, and (c) elute in Nuclease-free water of 31 ul.3. Adaptor Ligation Add to (a) eluted End-prepped DNA above of 30 ul, (b) 1D Adapter Mix of 20 ul, and (c) NEB Instant Sticky End Ligase of 50 ul, for a total volume of 100 ul. This is mixed gently via inversion and incubated at 20 C for 10 min in a thermocycler.4. Library Purification, with additional SPRI Cleanup Add to the above (a) Agencourt AMPure XP beads of 40 ul and let stand for 2 min followed by discarding supernatant while on magnet, (b) wash with Adaptor Bead Binding Buffer of 140 ul X 2, and (c) elute in Oxford Nanopore Elution Buffer of 25 ul for DNA sequencing.Library was run on a SQK-108 flow-cell.