GridION sequencing; Raw reads: 103X20190304
DNA was sequenced on an ONT GridION, with the DNA extracted from urine both with and without saponin multiplexed as a pair on a single flowcell. Urine from four of the 10 individual participants was also collected into cobas PCR Media tubes designed for diagnostic molecular N. gonorrhoeae and C. trachomatis testing. DNA was extracted from 4 ml obtained from these tubes using the QIAamp kit (without saponin treatment) and with or without a prior mechanical lysis step using bead beating as previously described,15 with the resulting DNA extracts from a single sample multiplexed as a pair on a single flowcell. Urethral swabs were obtained from nine of the participants and stored in Sigma VCM preservation medium for transport. DNA was extracted from 3ml of this medium, following vortexing for 30 seconds, using the QIAamp UCP Pathogen Mini kit without saponin treatment and sequenced as described above with a single sample per flowcell. Libraries were prepared for sequencing on an Oxford Nanopore GridION (Oxford Nanopore Technologies (ONT), Oxford, UK) using the Rapid PCR Barcoding kit (SQK-RPB004) (ONT), with modifications to the manufacturers' protocol. Briefly, up to 10 ng of input DNA and 2.5 μl of FRM (Fragmentation 170 Mix) were used in the tagmentation reaction with a final volume of 10 μl. For samples that were not able to achieve 10 ng, a total volume of 7.5 μl was used in the tagmentation reaction regardless of the quantity of DNA this represented. The PCR reaction was  performed in a double volume of 100 μl, with 25 cycles and an elongation time of 6 minutes. Post-PCR, DNA was purified with AMPure XP SPRI beads (Beckman Coulter, High Wycombe, UK) and eluted in 10 μl.
UNIVERSITY OF OXFORD
webin-reads-103X20190304
University of Oxford, Oxford, England, UK
webin-reads-103X20190304