Klebsiella pneumoniae strain SS02 genome sequencing
Genomic DNA was isolated from luxurious media-grown, stationary-phase SS02 cells using a standard kit and quantified by a QUBIT 3 fluorometer using dS DNA HS Dye. 100 nanograms of intact DNA were enzymatically fragmented by targeting 200300 bp fragment size. The DNA fragments underwent end repair, wherein the mix converted the overhangs resulting from fragmentation into blunt ends. The 3 to 5 exonuclease activity of the end repair mix removes the 3 overhangs, and polymerase activity fills in the 5 overhangs. On the blunt-ended fragments, adenylation was performed by adding single A nucleotides to the 3 ends. To the adenylated fragments, loop adapters were ligated and cleaved with the uracil-specific excision reagent (USER) enzyme. The samples were further purified using AMPure beads, and the DNA was then enriched by PCR with six cycles using NEBNext Ultra II Q5 master mix, Illumina universal primers, and sample-specific octamer primers. The amplified products are cleaned up by using AM pure beads, and the final DNA library was eluted in 15 uL of 0.1X TE buffer. 1 ul of the library was used to be quantified by a QUBIT 3 fluorometer using dS DNA HS reagent. The fragment analysis was performed on an Agilent 2100 Bioanalyzer by loading 1 uL of the library into the Agilent DNA 7500 chip. The library was sequenced in a ILLUMINA HiSeq 4000 instrument.