WGS of Neisseria gonorrhoeae: urethral secretions
For the sample, 1 g genomic DNA was randomly fragmented to <500 bp by sonication (Covaris S220). The fragments were treated with End Prep Enzyme Mix for end repairing, 5 Phosphorylation and dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~410 bp (with the approximate insert size of 350 bp) were recovered. The sample was then amplified by PCR for 8 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, PaloAlto, CA, USA), and quantified by Qubit2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).Then libraries with different indexes were multiplexed and loaded on an Illumina HiSeq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150 paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument.
SUB4429751
L73817ZJXSH89-4A