WGS for Bf12P1-resistant culture sample B. fragilis NCTC 9343 RP_Bf_22
Overnight culture was pelleted via centrifugation, supernatant removed, and DNA extracted using the NEB Monarch Spin gDNA Extraction Kit per the manufacturers instructions. Concentration and purity were measured using NanoDrop and Qubit, and DNA integrity was confirmed on an Agilent TapeStation. For library preparation, 100 ng gDNA was fragmented using the WGS Fragmentation Mix (Qiagen), and custom sequencing adapters (annealed TCTTTCCCTACACGACGCTCTTCCGATCTT and AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) were ligated using the T4 ligase (NEB). Indexed PCR amplification was performed using KAPA polymerase, 10M custom P5 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG) and uniquely barcoded P7 (CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTG) extension primers. Thermocycling conditions included initial denaturation at 95C for 3 min, followed by 8-12 cycles of 98C for 20 sec, 55C for 20 sec, and 72C for 3 min. Indexed libraries were purified using a double-sided size selection with SPRI beads at a 0.6-0.8x ratio.
SUB15670933
RP_Bf_22_S351