ena-STUDY-Genome Institute of Singapore-08-06-2015-10:48:57:309-579
Sequencing of E. Coli UTI89 using Oxford Nanopore MinION
Genomic DNA was extracted from Escherichia coli UTI89 using the QIAamp® DNA mini kit (Qiagen). 1µg of the extracted DNA was then sheared in a total volume of 80µl using a Covaris g-TUBE according to the manufacturer’s instructions with centrifugation for 1min at 6000rpm. Sheared DNA was end repaired and A-tailed using the GeneRead™ DNA Library Prep I Kit from Qiagen according to the manufacturer’s protocol. The reaction was purified using 1X volume of Agencourt Ampure XP beads and eluted in 30µl nuclease-free water. Subsequent steps of DNA sequencing library preparation were carried out using Oxford Nanopore’s MinION Genomic DNA Sequencing Kit (SQK-MAP003) according to the manufacturer’s recommended protocol, including the addition of purified BSA (NEB) to Agencourt Ampure XP beads and Elution buffer.  Immediately prior to sequencing, 12µl of the DNA library was combined with 134µl EP buffer and 4µl Fuel Mix and mixed by inversion 10 times. The flow cell was primed by washing with two aliquots of 150µl of EP buffer, with ten minutes in between washes. 150µl of the prepared DNA Library was then loaded onto the flow cell and the Genomic DNA 48 hour sequencing run program was selected. Fresh sample was loaded onto the flow cell at 12 hour intervals throughout the run.
Genomic DNA was extracted from Escherichia coli UTI89 using the QIAamp® DNA mini kit (Qiagen). 1µg of the extracted DNA was then sheared in a total volume of 80µl using a Covaris g-TUBE according to the manufacturer’s instructions with centrifugation for 1min at 6000rpm. Sheared DNA was end repaired and A-tailed using the GeneRead™ DNA Library Prep I Kit from Qiagen according to the manufacturer’s protocol. The reaction was purified using 1X volume of Agencourt Ampure XP beads and eluted in 30µl nuclease-free water. Subsequent steps of DNA sequencing library preparation were carried out using Oxford Nanopore’s MinION Genomic DNA Sequencing Kit (SQK-MAP003) according to the manufacturer’s recommended protocol, including the addition of purified BSA (NEB) to Agencourt Ampure XP beads and Elution buffer.  Immediately prior to sequencing, 12µl of the DNA library was combined with 134µl EP buffer and 4µl Fuel Mix and mixed by inversion 10 times. The flow cell was primed by washing with two aliquots of 150µl of EP buffer, with ten minutes in between washes. 150µl of the prepared DNA Library was then loaded onto the flow cell and the Genomic DNA 48 hour sequencing run program was selected. Fresh sample was loaded onto the flow cell at 12 hour intervals throughout the run.