WGS sequencing of T1RM-null Enterobacter asburiae
Briefly, 8 g of extracted total genomic DNA was sheared mechanically using g-TUBE (Covaris, USA) into smaller DNA fragment, ranging from 10 kb to 20 kb. After shearing, the sample was purified and concentrated using 0.45-fold volume of Agencourt AMPure XP beads (Beckman Coulter, USA). Subsequently, the concentration of the purified DNA sample was quantified using Qubit dsDNA Broad Range (BR) Assay Kit (Thermo Fisher Scientific, USA). The integrity of the purified sample was qualitatively investigated using Pippin Pulse Electrophoresis System (Sage Science Inc., USA). The preparation of SMRTbell template was prepared using SMRTbell Template Preparation Reagent Kits (Pacific Biosciences, USA), in which the sheared DNA sample was subjected to end repair, adapter ligation and exonucleases digestion of incompletely ligated products. Size selection using BluePippin DNA Size Selection Electrophoresis System (Sage Science Inc., USA) was performed on the SMRTbell template, in which SMRTbell template with the size of 7,000 bp to 50,000 bp was selected for sequencing. The size-selected SMRTbell template were purified using AMPure PB Kit (Pacific Biosciences, USA) followed by quantification using Qubit dsDNA High Sensitivity (HS) Assay Kit (Thermo Fisher Scientific, USA). Calculator (version 2.1.1) (Pacific Biosciences, USA) was used to calculate theloading concentration of SMRTbell template-DNA polymerase complex for annealing and binding. Library sequencing primers and P6 DNA polymerase, from DNA Polymerase Binding Reagent Kit (Pacific Biosciences, USA), were annealed to the SMRTbell template. Prior to sequencing, the SMRTbell template-DNA polymerase complex was bound to Magbeads using MagBead Buffer Kit (Pacific Biosciences,USA), according to the manufacturers protocol. The Sequencing Reagent 2.0 (Pacific Biosciences, USA) was used for the sequencing of SMRTbell template and using DNA Internal Control Complex (P6) (Pacific Biosciences, USA) as the internal control. The SMRT sequencing experiment was performed with a total of 240 min per SMRT sample.