Evaluating an Upper Respiratory Disease Panel on the Portable MinION Sequencer

Wanda J. Lyon; Zachary K. Smith; Brian Grier; James Baldwin; Clarise R. Starr

In each case, the cDNA/DNA was end-repaired in a 100-µL reaction containing 0.5-2 µg of nucleic acid (depending on the run) in 80 µL water, 10 µL of 10X end-repair buffer (from the NEB Next End Repair Module), and 1 µL of enzyme mix. DNA and cDNA were quantified using a Qubit 3.0 fluorometer (Life Technologies) and resuspended in molecular grade water as the starting material for library preparation per the instruction of the manufacturer. End-repair was performed per the manufacturer's instructions with the exception that the incubation time was increased to 15 minutes. The resulting blunt-ended DNA was cleaned up using 1.0 × volume of AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions with the exception that 80% ethanol was used instead of 70%, and the DNA was eluted in 30 µL molecular grade water. Ten µL of adapter mix and 2 µL hairpin (HP) adapter were added to the 30 µL dA-tailed DNA, then 8 µL of molecular grade water followed by the addition of 50 µL Blunt/TA ligase master mix (NEB). The contents were briefly mixed and the reaction was left to proceed at room temperature for 15 minutes. One µL of HP tether was added, mixed briefly, and incubated 15 minutes at room temperature. The sample was cleaned up using 1.0 × by volume AMPure XP beads according to the manufacturer's instructions with the exception that the DNA was eluted into 25 µL of elution library buffer at 37°C for 10 minutes. The beads were removed by placing the tube into a 1.5-mL magnet and the supernatant was transferred to a low binding 1.5-mL Eppendorf tube and placed on ice until the sequencing flow cell was loaded. Each library All rights reserved. No reuse allowed without permission.