1D-ligation-Trypsin-resin_2
Total genomic DNA was extracted from blood using an enzymatic based DNA extraction, yielding 131.4 ng/ul after resuspending in TE buffer. Sequencing was undertaken using the Oxford Nanopore MinIon (machine ID No. MN26033; flow-cell version SQK-LSK109) with R9.4 chemistry and conducted using Minion protocol version 1d-gDNA-sqk-lsk109-GDE_9063_v109_revC_23May2018 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 was eluted to 37 ul in TE buffer, (b) NEBNext FFPE Repair Buffer of 3.5 ul, (c) NEBNext FFPE Repair Mix of 2 ul (c) NEBNext Ultra II End Repair/dA-Tailing Mix of 3 ul, (d) NEBNext Ultra II End Repair/dA-Tailing Buffer of 2 ul, (e) Oxford Nanopore DNA CS 1 ul for a total volume of 50 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. Immobilized Trypsin-Resin in Place of SPRI Cleanup For this run a deviated protocol to increase library yield from wash cycles and avoid unnecessary DNA shearing from agitation, applied an enzymatic digest of end-repair enzymes in step 1 above replacing the use of SPRI beads. This was done using an activated trypsin. 2.1. To activate trypsin resin, resin was gently mixed adding 20 ul of resin to 120 ul of 10 mM Tris HCl pH 8.5 buffer in a 1.5 ml tube. This was inverted for mixing flowed by a 6000 rpm spin, with the supernatant discarded; this was repeated two additional times, with 20 ul of 10 mM Tris HCl pH 8.5 buffer added to the end product.2.2. The (a) end-repaired DNA from step 1 of 50 ul, and (b) activated trypsin resin of 20 ul from step 2.1 were (c) incubate at 37 C for 30 min. Only 60 ul of the total volume of 70 ul was retained leaving 10 ul of pelleted resin behind.3. Adaptor Ligation Add to (a) eluted End-prepped DNA above of 60 ul, (b) Oxford Nanopore Adapter Mix (AMX) of 5 ul, and (c) NEB T4 Ligase of 10 ul, (d) Ligation Buffer (LNB) 25.0 ul, for a total volume of 100.0 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 50 ul and let stand for 2 min followed by discarding supernatant while on magnet, (b) wash with Long Fragment Buffer of 100 ul X 2, and (c) elute in Oxford Nanopore Elution Buffer of 15 ul for DNA sequencing.Library was run on a SQK-LSK109 flow-cell.