Whole genome sequence of Mycobacterium orygis
DNA quantification and purity ratios were checked with NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA), Qubit, and Agarose Gel Electrophoresis (1% Agarose Gel) respectively. DNA size distribution is analysed by FEMTO pulse (Agilent Technologies, California, USA).The first step in the generation of a SMRTbell library is the production of appropriately sized double-stranded DNA fragments which was achieved by shearing DNA to 7-10kb for bacterial samples. DNA shearing was performed on the Megaruptor 3 (Diagenode, Belgium) system at a speed setting of 40 with the disposable shearing syringe. Sheared DNA was purified using AMPure PB beads (Pacific Biosciences, California, USA). The sheared and purified DNA was evaluated for size distribution using the Agilent FEMTO pulse analyzer (Agilent Technologies, California, USA) and quantified using DNA HS assay kit (Thermofisher #Q32851, Thermofisher Scientific, Massachusetts, USA) on Qubit 3.0 fluorometer (Thermofisher #Q33238, Thermofisher Scientific, Massachusetts, USA).SMRTbell libraries were prepared using Template Prep Kit 2.0. Next,  to  avoid shearing at the ends single-strand overhangs were created, and hence,  single-strand overhangs were removed, DNA damage repair and End-Repair/A-tailing were carried out before SMRTbell library preparation. SMRTbell library was prepared by ligating bell shape adapters to the End-Repaired DNA fragments. The SMRTbell library itself is produced by ligating A-tailed/Barcoded adapters onto double-stranded DNA fragments. The hairpin dimers formed during this process are removed using Ampure PB beads and prepared adapter ligated SMRT libraries were evaluated using Agilent FEMTO pulse.Purified SMRTbell library was subjected to primer annealing and polymerase binding using Sequel II binding kit 2.2 to prepare bound complex. About 90 pM of the library was loaded onto one SMRTcells containing 8M ZMW and sequenced on PacBio Sequel II system in CCS/HiFi mode (Nucleome Informatics Pvt. Ltd.)