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Nanobind pentagona HMW DNA Library Prep for minION Sequencing

Using the Nanobind Extracted and Short Read Eliminated pentagona HMW DNA for library prep with the Oxford Nanopore Ligation Sequencing Kit and the NEB Companion Module for Oxford Nanopore Technologies Ligation Sequencing for minION sequencing

The one extraction with the Nanobind kit is enough to make two library preps. There was about 46µl of DNA, so two PCR tubes were made with 23µl of DNA in each. PN1 and PN2. Volume was increased to 47µl with 24µl nuclease free water.

Wide bore pipette tips were used whenever possible, especially when touching sample

Some steps in this are modified from the original protocol. Modifications from J. Puritz.

DNA Repair and End-prep

  • Thawed DCS (DNA control sequence) from the nanopore kit a room temp and once it was thawed it went on ice
  • Thawed FFPE DNA Repair Buffer, FFPE DNA Repair Mix, Ultra II End-prep reaction buffer, and Ultra II End-prep enzyme mix on ice. From NEB kit
  • Made master mix for DNA repair and end prep:
    • 1µl DNA CS x 2.1 = 2.1µl
    • 3.5µl FFPE DNA repair buffer x 2.1 = 7.35µl
    • 2µl FFPE DNA repair mix x 2.1 = 4.2µl
    • 3.5µl Ultra II end prep reaction buffer x 2.1 = 7.35µl
    • 3µl Ultra II end prep enzyme mix x 2.1 = 6.3µl
  • Pipetted to mix master mix
  • Added 13µl of master mix to each sample tube in strip tubes (PN1 and PN2)
  • Flicked tubes to mix with DNA then spun down in minifuge
  • Placed in thermocycler Nanopore Incubation program: 30min at 20 degrees C then 30min at 65 degrees C

Bead Cleanup

  • Took out KAPA Pure beads, swirled to resuspend, and let come to room temp
  • Made fresh 80% ethanol
  • Took sample tubes out of the thermocyler and added 60µl beads to each tube
  • Flicked tubes to mix then spun them down on a minifuge
  • Placed tubes on shaker for 30 minutes at 300rpm
  • Placed tubes on magnet plate and waited 5 minutes
  • Removed and saved supernatant without disturbing the beads
  • Gently added 200µl 80% EtOH
  • Removed and discarded wash supernatant
  • Added 200µl 80% EtOH
  • Removed and discarded wash supernatant
  • Spun down tubes with minifuge and placed back on magnet
  • Removed any remaining supernatant
  • Let beads dry maybe 30 seconds
  • Resuspended pellet in 61µl nuclease-free water: flicked and spin down, flicked and spun down. No pipette mixing.
  • Incubated tubes room temp shaking for at least 60 minutes
  • Kept liquid with beads, placed on magnet plate and waited until clear
  • Broad Range Qubit of samples:
    • PN1: 15.02ng/µl
    • PN2: 15.01ng/µl

Adapter Ligation

  • Thawed AMX (Adapter Mix) and T4 DNA Ligase (NEB kit) on ice
  • Thawed LNB (Ligation Buffer) at room temp, spun down, pipetted to mix, then placed on ice
  • Thawed EB (Elution Buffer) at room temp, vortexed, spun down and put on ice
  • Thawed LFB (Long Fragment Buffer) at room temp, vortexed, spun down and put on ice
  • Made ligation and adapter master mix:
    • 25µl LNB x 2.1µl = 52.5µl
    • 10µl T4 DNA ligase x 2.1 = 21µl
    • 5µl AMX x 2.1 = 10.5µl
  • Flicked tube to mix (ligase!!) then spun down
  • Added 40µl ligation and adapter mix to each tube with the DNA with the beads
  • Flicked to mix the tubes then spun down
  • Incubated tubes on shaker at room temp for 30min at 300rpm

Bead Cleanup

  • Took out beads and let get to room temp
  • After incubation, spun down tubes and added 40µl beads to each sample tube
  • Flicked tubes to mix then spun down
  • Incubated tubes on shaker at room temp for 30min at 300rpm
  • Placed on magnet plate after incubation
  • Waited 5 minutes
  • Removed clear supernatant without disturbing beads and saved just in case
  • Took tubes off magnet and added 250µl LFB
  • Flicked tube to mix and spun down
  • Placed back on magnet
  • Removed and saved supernatant just in case
  • Took tubes of magnet and added 250µl LFB
  • Flicked tubes to mix and spin down
  • Placed back on magnet
  • Removed and saved supernatant
  • Spun down tubes
  • Removed any residual supernatant
  • Removed tubes from magnet and added 15µl EB
  • Flicked tubes to mix then spun down
  • Placed tubes on shaker at 300rpm at room temp overnight

Broad Range Qubit 20200122

796µl buffer, 4µl reagent
S1: 186
S2: 20742
PN1: 42.6ng/µl
PN2: 43.4ng/µl

Written on January 21, 2020