Whole genome Sequencing of Bifidobacterium sp.
DNA was quantified by QUBIT 3 Fluorometer using dS DNA HS Dye. 100 nanograms of intact DNA was enzymatically fragmented by targeting 200- 300 bp fragments size.The DNA fragments undergo end repair where in the mix converts the overhangs resulting from fragmentation into blunt ends. The 3 to 5 exonuclease activity of end repair mix removes the 3 overhangs and polymerase activity fills in the 5 overhangs. To the blunt ended fragments adenylation is performed by adding single A nucleotide to the 3 ends. To the adenylated fragments loop adapters were ligated and cleaved with uracil-specific excision reagent (USER) enzyme. The samples are further purified using AMPure beads and The DNA is then enriched by PCR with 6 cycles using NEBNext Ultra II Q5 master mix, illumina universal primer 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 uLs of 0.1X TE buffer. 1ul of the library was used to quantify by QUBIT 3 Fluorometer using dS DNA HS reagent. The fragment analysis was performed on Agilent 2100 Bioanalyzer, by loading 1 uL of the library in to Agilent DNA 7500 chip.